Helicobacter pylori blood group antigen binding adhesin A (BabA) is more common in strains isolated from patients with peptic ulcer disease or gastric cancer, rather than asymptomatic colonization. Here we used mouse models to examine host determinants that affect H. pylori BabA expression. BabA expression was lost by phase variation as frequently in WT mice as in RAG2−/− mice that do not have functional B or T cells, and in MyD88−/−, TLR2−/− and TLR4−/− mice that are defective in toll like receptor signaling. The presence of other bacteria had no effect on BabA expression as shown by infection of germ free mice. Moreover, loss of BabA expression was not dependent on Leb expression or the capacity of BabA to bind Leb. Surprisingly, gender was the host determinant most associated with loss of BabA expression, which was maintained to a greater extent in male mice and was associated with greater bacterial load. These results suggest the possibility that loss of BabA expression is not driven by adaptive immunity or toll-like receptor signaling, and that BabA may have other, unrecognized functions in addition to serving as an adhesin that binds Leb.Expression of the  Helicobacter pylori infects the gastric mucosa of about 50% of the world’s population124H. pylori may be beneficial678H. pylori interaction that influence whether an individual will develop clinical disease or asymptomatic infection. One risk factor associated with more severe disease outcomes is the virulence factor, blood group antigen binding adhesin (BabA), which belongs to a family of H. pylori outer membrane proteins13151617b)2022in vitro evidence that BabA-mediated attachment to host gastric epithelium facilitates translocation of the CagA oncoprotein into host cellscagPAI), itself a well-recognized risk factor for disease25262728b and ABO antigens on gastric epithelial cells or on mucins30313233BabA mediated attachment and development of disease are influenced by host expression of Lewis antigens, which is determined by a number of factors, including ABO blood type and secretor status303132H. pylori expression of BabA is lost during the first 2–12 weeks of infection using animal models such as rhesus macaques, gerbils, and mice3536babA gene and its babB paralog can result in duplication of all or part of the babB gene into the babA locus, resulting in loss of BabA expression and reciprocal overexpression of BabB. Alternatively, in mice and in macaques, slipped strand mispairing of a CT repeat region in the 5′ portion of the babA open reading frame (ORF) can lead to a frame shift, resulting in an early stop codon within the babA ORF and loss of BabA protein expression. That this phenomenon occurs across several animal models, and that loss of BabA expression can also be seen in human clinical isolatesH. pylori-host relationship.Previous studies have shown that b is not required, but that gender- specific physiological differences may be important.Loss of BabA expression might occur as a bacterial mechanism of persistence to adapt to changing levels of inflammation, glycosylation patterns, or a combination of both. Here we used knockout and transgenic mouse models to determine the host factors that affect BabA expression. Surprisingly, the results suggest that adaptive immune responses and toll-like receptor signaling play little if any role in loss of BabA expression, and that the capacity to bind LeH. pylori cultures were maintained on Brucella agar  supplemented with 5% heat inactivated newborn calf serum  and antibiotics , either TVPA  for laboratory adapted cultures or ABPNV  for primary cultures from infected mice. All H. pylori cultures were maintained at 37 °C in a CO2 incubator or in an AnoxomatTM jar  adjusted to contain 5% oxygen, 7.6% carbon dioxide, and 7.6% hydrogenA complete list of bacterial strains used in this study is provided in H. pylori J166 were engineered with 7 (no ORF) or 8 (ORF) CT repeats in the 5′ coding region of babA using a contraselection approach modified from that previously describedbabA translational start site and 39 base pairs of 5′ babA gene sequence were replaced with a CAT_rpsL cassette in a streptomycin resistant strain of H. pylori J166, using the plasmid pBabA as previously describedhypD and intergenic sequence 244 bp upstream of babA in H. pylori J166  or 9 (no ORF) CT isogenic variants of J166 BabACL2, a site directed mutant of J166 in which Cys to Ala replacements at residues 189 and 197 result in a BabA protein that is expressed but cannot bind LebIsogenic mutants of ad libitum as previously describedb transgenic mice)H. pylori strain) throughout the experiment.The local Institutional Animal Care and Use Committee approved all mouse experiments performed at Should read, University of California, Davis and the University of Michigan. Experiments performed at Umeå University, Umeå, Sweden were approved by Umeå Ethical Committee on Animal Research. All methods involving mice were performed according to the locally approved guidelines. Mice  were houH. pylori harvested from agar plates grown overnight (18–24 hr) was used to inoculate liquid cultures to an optical density A600 (OD) of 0.05 to 0.1. Liquid cultures were grown in Brucella broth  supplemented with 5% NCS and TVPA shaking at 60–100 rpm overnight (18–24 hr) to an OD of 0.3 to 0.7. Bacteria from overnight liquid cultures were centrifuged  and suspended in Brucella broth at approximately 1010 CFU/mL. For competition experiments where the inoculum contained equal parts of BabA expressing and deficient H. pylori, liquid cultures were grown to matching OD values prior to centrifugation and resuspension. In a subset of experiments, dilutions of the inoculum mixture were plated and 16 colonies were selected and sequenced at the 5′end of the babA locus to empirically determine the ratio of BabA expressing bacteria in the inoculum. On average the inoculum contained 45% BabA expressing bacteria with a standard deviation of 11%. This variability reflects both variation in the growth and survival of the cultures and technical variation in the detection of BabA expression in the output colonies. Mice were challenged at 10 to 14 weeks of age with 250 μL of suspension by oral gavage with a 20 gauge, 38mm animal feeding needle (Fisher Scientific)36H. pylori culture and calculation of H. pylori colony forming units per gram of stomach tissue (CFU/g). The comparison between male and female mice was performed twice, as indicated in the figure legends and H. pylori J166, 8 CT repeats at the 5′ end of babA yield an ORF, with expression of BabA and attachment to LebH. pylori in each mouse was determined by sequencing the CT repeat region of babA, which was selectively confirmed by RIA analysis using methods previously described36babA ORF is displayed as a single point in each figure describing BabA expression in mice. For RIA analysis, total cultured dilutions from mouse stomach homogenate (sweeps) were collected and 1 mL of an OD600 = 0.1 suspension of the mixture was incubated with a cocktail of 125I radiolabeled Leb conjugated to human serum albumin (Leb-HSA). The ratio of bound Leb-HSA  to free Leb-HSA (radioactivity measured in the supernatant) was used to estimate BabA expression of the H. pylori communityIn H. pylori expression of functional BabA protein was assayed by in vitro attachment to Leb using an ELISA as previously described2235H. pylori cells were applied to Leb-HSA coated wells in a 96 well polystyrene plate. Unbound bacteria were removed by washing and bound bacteria were detected with anti-digoxigenin Fab fragments conjugated to horseradish peroxidase (POD) (Roche Applied Biosciences) followed by incubation with 2,2′-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS). Color change was measured by subtraction of absorbance at 490 nm from 405 nm. Attachment ratio values were reported as an average of readings from two Leb positive wells divided by two Leb negative wells.P < 0.05 was considered statistically significant.Unless otherwise indicated, Mann-Whitney U, Fisher’s exact test or analysis of variance was performed using Graphpad Prism Software. Bacterial colonization (CFU/gram) was log transformed prior to analysis. The proportion of BabA expressing colonies was logit transformed using R softwareH. pylori BabA expression by phase variation might result from adaptive immune pressure directed against BabA. When loss of BabA expression occurs by a gene conversion event in which babB is duplicated into the babA locus35454647H. pylori J166 into wild type (WT) and RAG2−/− mice, which do not develop mature B or T lymphocytesH. pylori colonized RAG2−/− mice at a significantly higher level than WT  at 2 and 8 weeks post infection (PI). Since we previously showed that loss of BabA expression in mice occurs only by phase variation and not by gene conversionbabA open reading frame (ORF) and attachment to LebH. pylori infection, it does not play a role in selection against BabA expressing bacteria in mice.Loss of  than WT , supportH. pylori infection occurs in the context of a gastric microbial community, which is less complex than that in the gut, but is probably autochthonous525354H. pylori infection induces more severe gastrointestinal intraepithelial neoplasia when animals have conventional microbiota compared to germ free counterpartsH. pylori expression of BabA, we infected germ free and conventionally raised C57BL/6 mice with H. pylori J166, and examined BabA expression in output colonies from mice sacrificed 3 weeks PI. Similar to the results 2 weeks PI  with a babA ORF (8 CT repeats) and compared the outcome of infection with J166sc to that with the original J166 inoculum containing a mixture of phase variants. After infection for between 2 and 8 weeks, loss of BabA expression was observed in 6 of 11 (55%) mice infected with the original stock of J166, but only 1 of 12 (8%) mice infected with J166sc . Since J166SC might differ from J166 in other ways, in addition to the number of CT repeats in babA, we used contraselection to generate isogenic clones of J166SC that had either 8 CT repeats (J1668CT), encoding a babA ORF, or 7 CT repeats (J1667CT), which was out of frame and led to loss of BabA expression. J1668CT and J1667CT showed similar growth rates in liquid culture .The marked variability in BabA expression among output colonies  and 2 mi culture , but, asd to Leb . For allH. pylori infection , and demonstrated a similar loss in BabA expression over time , but there was no difference between WT and Leb mice. BabA expression was also similar in WT and Leb mice, but again there was a significant effect of gender (P < 0.0001). Female mice were predominantly colonized by H. pylori lacking BabA expression, similar to what we observed previously . Increased colonization and a small increase in Leb binding were also observed in male Leb relative to female Leb mice 4 weeks PI using H. pylori strain J99  was sufficient to eliminate all Leb binding activity, though the protein was expressed on the cell surface at levels similar to WTb binding on BabA expression in mice, we used contraselection to generate isogenic strains of BabACL2 with either 8 (J166 BabACL28CT) or 9 (BabACL29CT) CT repeats. The BabA expression status predicted by the CT repeat number for each strain was confirmed by western blot  and used to infect female Leb transgenic mice and their WT littermates, which were sacrificed 2 weeks PI. For comparison, WT and Leb mice were also infected with J1668CT:7CT. There were no differences in colonization levels between BabACL28CT:9CT compared to J1668CT:7CT .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Hedgehog (Hh) signaling plays crucial roles in the patterning and morphogenesis of various organs within the bodies of vertebrates and insects. Endochondral ossification is one of the notable developmental events in which Hh signaling acts as a master regulator. Among three Hh proteins in mammals, Indian hedgehog (Ihh) is known to work as a major Hh input that induces biological impact of Hh signaling on the endochondral ossification. Ihh is expressed in prehypertrophic and hypertrophic chondrocytes of developing endochondral bones. Genetic studies so far have demonstrated that the Ihh-mediated activation of Hh signaling synchronizes chondrogenesis and osteogenesis during endochondral ossification by regulating the following processes: (1) chondrocyte differentiation; (2) chondrocyte proliferation; and (3) specification of bone-forming osteoblasts. Ihh not only forms a negative feedback loop with parathyroid hormone-related protein (PTHrP) to maintain the growth plate length, but also directly promotes chondrocyte propagation. Ihh input is required for the specification of progenitors into osteoblast precursors. The combinatorial approaches of genome-wide analyses and mouse genetics will facilitate understanding of the regulatory mechanisms underlying the roles of Hh signaling in endochondral ossification, providing genome-level evidence of the potential of Hh signaling for the treatment of skeletal disorders. Hedgehog (Hh) signaling plays crucial roles in the patterning and morphogenesis of various organs within the bodies of vertebrates and insects . In the Substantial progress has been made in understanding how Hh signaling engages skeletal development and interacts with other signaling factors, mainly based on mouse genetic studies manipulating the Hh signaling molecules mentioned above. This review aims to summarize the roles of Hh and its mode of action in skeletal development, with a particular focus on endochondral ossification.Mammalian skeletons are formed by two distinct processes: intramembranous ossification and endochondral ossification. In intramembranous ossification, mesenchymal cells directly differentiate into the bone-forming cells called osteoblasts. This process forms the frontal bone, parietal bone, maxilla, mandibular bone, part of the clavicle, and so on. In contrast, axial skeletons and the long bones in the limbs develop through endochondral ossification, a process in which cartilage molds are initially formed and replaced by bones. During the endochondral ossification process, cartilage formation (chondrogenesis) and bone formation (osteogenesis) are orchestrated in a spatially and temporally controlled manner.Endochondral ossification starts with the condensation of immature mesenchymal cells at a region where future bone develops. Cells in the center of the condensation give rise to chondrocytes expressing cartilage matrixes, including type II collagen, whereas cells at the periphery of the condensation form the perichondrium. Cartilage tissues grow through the proliferation and maturation of chondrocytes. Chondrocyte maturation is represented by its post-mitotic hypertrophy and the deposition of calcified matrices. Hypertrophic chondrocytes undergo apoptosis, followed by the absorption of residual calcified matrix by chondroclasts that come into the skeleton upon vascular invasion.Cells at a certain region in the perichondrium (see later) are specified into an osteoblast lineage to form the bone collar, a predecessor of cortical bones. Osteoblasts are generated through several precursors. Immature mesenchymal cells initially differentiate into osteoblast precursors expressing runt-related transcription factor 2 (Runx2); the Runx2-positive precursors then express Sp7 (osterix) as well as Runx2, further differentiating into mature osteoblasts. Runx2 and Sp7 are key determinants for the osteoblast lineage. Mature osteoblasts contribute to bone formation by secreting bone matrixes. Bone gla protein  is a bona-fide marker for mature osteoblasts.In the fetal cartilage of endochondral skeletons, referred to as the growth plate hereafter, chondrocytes are sequentially layered and aligned according to their differentiation phases: the proliferating phase, prehypertrophic phase, and hypertrophic phase . Bone coAmong the three Hh proteins in mammals, Ihh is known to work as a major Hh input that induces biological impact of Hh signaling on the endochondral ossification. In developing endochondral bones, prehypertrophic and hypertrophic chondrocytes in the growth plate express the Ihh gene. Target genes of Hh signaling, Ptch1 and Gli1, are expressed in proliferating chondrocytes, perichondrial cells, and primary spongiosa. Cells closer to the Ihh-producing cells express the target genes at higher levels. Genetic studies so far have demonstrated that the Ihh-mediated activation of Hh signaling synchronizes chondrogenesis and osteogenesis during endochondral ossification by regulating the following processes : (1) choet al. and Vortkamp et al. initially proposed possible roles of Hh signaling in skeletal development in relation to the parathyroid hormone-related protein (PTHrP) [In 1996, two papers by Lanske  (PTHrP) ,3. PTHrP (PTHrP) ,5. A ser (PTHrP) ,6,7,8 an (PTHrP) ,11 indicet al., Ihh overexpression in chick limbs decreased hypertrophic chondrocytes while increasing PTHrP expression in the periarticular perichondrium [PTHrP−/− limbs was rescued by the addition of PTHrP, Shh had no effect on the PTHrP−/− limbs. By utilizing mouse limb explant cultures, Lanske et al. also showed that although PTHrP treatment and Shh treatment both induced the elongation of the growth plate with the suppression of chondrocyte hypertrophy in wild-type limbs, neither of them had effects on PPR−/− limbs [et al. and Lanske et al. suggested that the transition from proliferating chondrocytes to hypertrophic ones was regulated by both PTHrP-PPR signaling and Ihh in a common feedback loop, where the PTHrP-PPR signaling mediated the effect of Ihh on the transition.In the paper by Vortkamp hondrium . Althoug/− limbs . Togethe−/−Ihh mice were then reported in 1999 [−/−Ihh mice showed shortening of the proliferating chondrocyte layer and acceleration of chondrocyte hypertrophy, as seen in the loss of function of PTHrP signaling in mice. More importantly, these phenotypes accompanied a loss of PTHrP expression in the periarticular regions. In contrast, when Hh signaling was activated specifically in chondrocytes by the loss of Ptch1 in mice, chondrocyte hypertrophy was delayed and PTHrP expression was upregulated in the periarticular regions [Skeletal phenotypes of  in 1999 . Ihh−/−  regions . These pet al. through the analysis of compound mutant mouse lines [Ihh−/−; PTHrP−/− mutant limbs showed abnormalities similar to Ihh−/− mutant limbs; both mutants exhibited premature chondrocyte hypertrophy and the absence of the growth plate, trabecular bone, and bone collar. Karp and colleagues next activated PTHrP signaling in Ihh−/− chondrocytes in order to clarify which functions of Ihh were mediated by PTHrP. The compound mutants inhibited premature chondrocyte hypertrophy, but did not increase the number of mitotically active chondrocytes compared to Ihh−/− mutants. These results suggest that: (1) Ihh is required for both the differentiation and the proliferation of growth plate chondrocytes; (2) PTHrP mediates part of the function of Ihh to regulate the maintenance of a pool of proliferating chondrocytes; and (3) Ihh can promote the proliferation of these cells in a PTHrP-independent manner.The interaction was genetically determined by Karp se lines . Ihh−/−;PPR−/− cells and WT cells further provided a clue about the interaction between Ihh and PTHrP [PPR-null mutation (PPR−/− cells) were injected into WT blastocysts to produce the chimera [PPR−/− chondrocytes ectopically became hypertrophic in columnar proliferating regions and expressed Ihh. The ectopic hypertrophy of PPR−/− cells was accompanied by the upregulation of PTHrP expression in periarticular WT cells and an elongation of the growth plate. Chimeric mice carrying PPR−/−; −/−Ihh cells and WT cells revealed the role of Ihh in the interaction [PPR−/−; −/−Ihh chondrocytes still exhibited ectopic hypertrophy in the manner of the PPR−/− chondrocytes, the upregulation of PTHrP expression and the elongation of the growth plate were no longer observed in the chimeras.Growth plate phenotypes of chimeric mice carrying nd PTHrP   Ihh has positive effects on chondrocyte proliferation at the postnatal stage, but the effects are unlikely mediated by PPR; and (2) Ihh and PTHrP may functionally interact at postnatal stages, as they do at embryonic stages. However, given the temporal rescue of growth plate phenotypes in the compound mutant, contributions of the interaction to chondrocyte hypertrophy, if any, are likely to be less at postnatal stages than at embryonic stages; other mechanisms or signaling molecules may also mediate the process. Hirai et al. showed that chondrocyte-specific ablation of PPR in postnatal mice led to the acceleration of hypertrophy, followed by premature closure of the growth plate, in association with increased chondrocyte apoptosis [Ihh from Col2a1-expressing cells, because Ihh induces PTHrP.Several studies shed light on postnatal roles of Hh signaling in cartilage and the growth plate. Postnatal deletion of  in mice . When Ih in mice . These d context . The ectpoptosis . They su−/−Ihh mice [i.e., pre- and hypertrophic chondrocytes, in bone collar formation in the perichondrium was further supported by phenotypes of PPR−/−/WT chimeric mice and PPR−/−; −/−Ihh/WT chimeric mice [PPR−/−; −/−Ihh/WT chimeric mice were generated by the injection of PPR−/−; −/−Ihh ES cells into WT blastocysts [PPR−/−/WT chimeras, perichondrial cells adjacent to ectopically hypertrophic PPR−/− cells underwent ectopic calcification. The ectopic calcification was cancelled by the removal of Ihh in the ectopically hypertrophic PPR−/− cells, although ectopic hypertrophy was still observed in the PPR−/−; −/−Ihh cells with robust expressions of bone morphogenetic proteins (BMP) 2 and 6. Thus, Ihh produced by pre-hypertrophic and hypertrophic chondrocytes is necessary for the differentiation of perichondrial cells into osteoblasts.Ihh is a master regulator of osteoblast specification during endochondral ossification. In endochondral ossification, osteoblasts first appear in a region of the perichondrium adjacent to pre-hypertrophic and hypertrophic chondrocytes. The following mouse genetic studies have indicated that direct Ihh input from these chondrocytes to progenitors is required for osteoblast specification in the perichondrium and the primary spongiosa. Both bone collars and the expression of Runx2 and Bglap were absent in the perichondrium of −/− mice ,23. Invoric mice ,16  and type X collagen (Col10a1), and did not undergo osteoblast differentiation; −/−Smo cells in the primary spongiosa did not contribute to bone formation [Ihh or the ablation of Ptch1 from perichondrial cells, which caused supra-physiological activation of Hh signaling in each population, resulted in the accelerated bone collar formation [When Hh signaling activity was removed by the hondrium . In Smo−ormation . Chondroormation ,32.−/−Ihh mice by Colnot et al. provide clues regarding the mechanism by which Hh signaling regulates the osteoblast differentiation of perichondrial cells at the cellular level [−/−Ihh mice; the abnormality became evident within 24 h after the physiological onset of Ihh expression at E12.5. In E14 −/−Ihh embryos, Col1a1 expression was diffuse and not restricted to cells adjacent to Col2a1-expressing chondrogenic condensation in the perichondrial region. Given that Runx2 appears at E12.5 in a physiological context, the organization of perichondrial cells may be associated with the cell fate specification of progenitors into Runx2-positive osteoblast precursors.Closer analyses of ar level . They foSmo from Sp7-expressing cells does not affect osteoblast differentiation [Thus, Ihh produced by pre- and hypertrophic chondrocytes is essential for osteoblast development in the perichondrium and the primary spongiosa during endochondral ossification ; more spntiation . Given tntiation , the sigPtch1+/− mice had high bone mass with an acceleration of both bone formation and bone resorption; a similar trend to that in the mouse phenotypes was observed in patients with basal cell carcinoma syndrome (BCCS) caused by an inactivating mutation of one of the Ptch1 alleles [Ptch1 was disrupted in Bglap-expressing mature osteoblasts, they showed low bone mass unlike Ptch1+/− mutants [Gli1 led to decreased bone mass with an uncoupling of bone formation and resorption in mice, suggesting that Gli activators participate in postnatal bone homeostasis upon Hh input [We and others have shown that Hh signaling regulates not only skeletal development, but also postnatal bone homeostasis. Disruption of Ihh in postnatal chondrocytes led to premature vascularization, a delay in the formation of the ossification center, and less trabecular bone , suggest alleles . Althoug mutants ; it is pHh input .Sp7-expressing osteoblast precursors resulted in a lack of Bglap-expressing mature osteoblasts [−/−Ihh mice [Genetic studies may suggest a further conclusion about the hierarchy between Hh signaling and other osteogenic signaling factors such as Wnt and bone morphogenetic proteins (BMPs) during osteoblast differentiation. The differentiation was largely arrested at the Runx2-positive stage, when canonical Wnt signaling activity was removed in the limb bud , the meseoblasts . These d−/− mice , and the−/− mice .ex vivo organ cultures as well as mouse genetics, we analyzed the hierarchy between Hh and BMP signaling in skeletal development [By taking advantage of elopment . The actelopment . In the elopment .Bmp2 and Bmp4 were deleted in Prrx1-expressing skeletal progenitors showed comparable expression of Runx2 to WT mice [Ihh in the ectopically hypertrophic PPR−/− cells as mentioned earlier  was exogenously expressed in Col2a1-expressing cells [A series of studies indicated that Gli1, Gli2, and Gli3 are involved in Hh-mediated osteoblast differentiation. Shimoyama ith Gli2 . In lineng cells . On the Gli1−/− mice; Gli1−/− perichondrial cells expressed Col2a1 and Col10a1, but not Runx2 or Sp7. Gli1−/−; Gli2−/− mice showed more severe skeletal phenotypes than either Gli1−/− or Gli2−/− mice, and osteoblast differentiation was impaired in Gli1−/−; Gli3−/− perichondrial cells compared to that in Gli3−/− cells in vitro [We recently found that bone formation was impaired in in vitro . Indeed,in vitro , and it in vitro . We alsoin vitro . These d−/−Ihh perichondrium did not cancel the abnormality of osteoblast differentiation in −/−Ihh mice [Runx2 to promote osteoblast differentiation. Gli transcription factors are possible candidates for these effectors.Regarding the involvement of Runx2 in the action of Hh, the recovery of Runx2 expression in the −/− mice , suggestet al. found that an abnormal activation of Hh signaling underlay heterotopic ossification, which is caused by the inactivating mutation of Gnas [Recently, Regard  of Gnas . GNAS en of Gnas .As summarized in this review, over the last two decades mouse genetic approaches have unraveled the functions of Hh signaling and their underlying molecular mechanisms in endochondral ossification. Ihh regulates chondrocyte differentiation by constituting a negative feedback loop with PTHrP, which is a central system in the proper growth of endochondral skeletons; Ihh plays a prerequisite role in osteoblast formation. However, the gene regulatory networks underlying these actions are still poorly understood. In particular, what genes are regulated by Gli factors and translate Hh input into the biological actions on skeleton? What transcription factors engage the Gli-mediated network during skeletal development? A combinatorial approach using genome-wide analyses and mouse genetics will facilitate understanding of these regulatory mechanisms, providing genome-level evidence of the potential of Hh signaling for the treatment of skeletal disorders.


Elemental imaging shows that the distributions of Ca, Cu and Zn are almost exclusively controlled by melanin pigment distribution. X-ray absorption spectroscopy demonstrates that the atomic coordination of zinc and sulfur is different within eumelanised regions compared to pheomelanised regions. This not only impacts our fundamental understanding of pigmentation in extant organisms but also provides a significant contribution to the evidence-based colour palette available for reconstructing the appearance of fossil organisms.Melanin is a critical component of biological systems, but the exact chemistry of melanin is still imprecisely known. This is partly due to melanin’s complex heterogeneous nature and partly because many studies use synthetic analogues and/or pigments extracted from their natural biological setting, which may display important differences from endogenous pigments. Here we demonstrate how synchrotron X-ray analyses can non-destructively characterise the elements associated with melanin pigment  Melanin pigment is one of the most widespread mechanisms in the colouration of biological tissues, most familiar as the black/brown (eumelanin) and red/yellow (pheomelanin) hues in keratinous integumentary structures such as skin, hair and feathers12345178131011814310111617However, there are aspects of these interactions that are still unclear. Melanin is notoriously difficult to characterise due to its insolubility and heterogeneous structure2345182021222318et al.in-situ. Large scale imaging showed certain elements  to be constrained within discrete extant and fossil biological structures . XAS showed that in fossil tissues Cu was bonded to organic compounds, with a coordination chemistry very similar to Cu contained within eumelanised extant analogues, eumelanin standards and in theoretical computational models for Cu in eumelanin17A relatively new methodology could provide new insight into melanin and its associated metallome. Wogelius in situ within tissues, and also provide further information that will allow more accurate pigment reconstruction within fossils. In the current study we analysed extant feathers from four genera of birds of prey - Harris hawk (Parabuteo unicinctus), red-tailed hawk (Buteo jamaicensis), kestrel  and barn owl   . These sMelanin identification and quantification was performed according to published proceduresSRS-XRF elemental maps of Harris hawk, kestrel, barn owl and red-tailed hawk feathers were obtained . These i−1 of feathers with a strong eumelanin component and those with predominantly pheomelanin component (Zn K-edge Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy  indicateomponent . These aSulfur X-ray Absorption Near Edge Structure (XANES) spectroscopy was also performed  in order272829SRS-XRF imaging is able to map the distribution of specific oxidation states of an element within a sample by tuning the energy of the incident X-ray beam to specific XANES resonances. This ability allows S speciation and distribution to be better resolved, as data are not restricted to localised spectra from point analyseset al.2+ elicit changes in cellular melanin contents+/Ca2+ exchanger activity of SLC24A52+ signaling by regulating Ca2+ transport from cytosol to melanosome lumen.SRS-XRF unequivocally shows that the distribution of Ca, Cu, and Zn, is controlled by melanin pigment, which is consistent with other studies161718Elemental quantification corroborates the imaging data but shows that quantification can be ambiguous if used in isolation. For example, there are similar levels of Zn in the unpigmented Harris hawk feather (37 ppm) and the pheomelanised red-tailed hawk (32 ppm) and kestrel feather 49 ppm). Additionally, the pheomelanised regions of the barn owl shows higher levels of Zn than its eumelanised speckles, but these differences are only on the order of a few ppm (4 ppm). It is these minute differences in concentration that demonstrate the importance of imaging in being able to resolve element patterns. If point analysis quantification was taken in isolation, one might conclude there is no distinct correlation between pigment patterns and elemental inventory in many of the cases presented above. 9 ppm. Adin situ analysis that the Zn coordination chemistry in pheomelanin is different from that in eumelanin. Interestingly, XAS results of the white region of the Harris hawk feather shows a very similar bonding configuration to pheomelanised feathers  potential influences from contaminants, 2) metal/melanin concentration variability within individual feathers, and 3) different pigment levels within feathers from different species. XAS of both Zn and S revealed that melanin type can be differentiated Parabuteo unicinctus), red-tailed hawk (Buteo jamaicensis), American kestrel  and barn owl  (Parabuteo unicinctus), Eurasian kestrel  and barn owl  were obtained from animals under the care of the non-profit Wild Wings Birds of Prey education and rehabilitation centre located in Bents Home and Garden, Warrington, UK. Red-tailed hawk (Buteo jamaicensis) feathers were obtained from The Falconry Centre, Hagley, UK. The feathers were naturally dropped via moulting and collected by staff from the enclosures. In accordance with Federal regulations, all appropriate United States Department of Agriculture – Animal and Plant Health Inspection Service (USDA-APHIS) permits for keeping birds of prey and transporting feathers across state lines (permit no. 44877) to other research institutions are held by the Live Animal Center (LAC) and the ornithology department at The Academy of Natural Sciences of Drexel University . Appropriate CITES permits from the Animal Health and Veterinary Laboratory Agency (AHVLA) were also acquired for export/import of UK sourced feathers to Japan. Wild Wings Birds of Prey education and rehabilitation centre (UK) also holds appropriate permits for keeping these species.Feathers analysed at SSRL - Harris hawk   - were oSepia officinalis melanin (Sigma Aldrich) in 50 ml of 0.1 mol−1 Zn sulfate (ZnSO4.7H20) aqueous solution with a pH of approximately 6.5. This was left to stand for 24 hours then centrifuged and the supernatant removed. The precipitate was washed with deionised water which was then freeze dried for analysis3839All feathers in this study were stored in sealed bags and stored in a freezer until analysis but were subjected to no other treatment or preparation other than being blown with compressed air to remove any surface particulates. Note that the results in 2O and 100 μL (1 mg) aliquots were subjected to Soluene-350 solubilizationMelanin identification and quantification was performed as outlined in10 and 1011 photons s−1) or 3.15 keV (flux ~109 photons s−1) and a beam diameter of 50 microns defined by a pinhole. X-rays were detected using a single element Vortex silicon drift detector.Synchrotron X-ray fluorescence imaging was performed at the Stanford Synchrotron Radiation Lightsource (SSRL) wiggler beam line 6–2 at the Stanford Linear Accelerator Center . Extensive and detailed descriptions of SRS-XRF mapping applied to fossils are provided in recent previous publications41Energy dispersive spectra obtained from SSRL were collected for 50 live seconds and fit using PyMCA414 standards were used to calibrate the energy of the monochromator position, defined at 2482 eV. Data averaging, background subtraction, data normalisation and fitting of the XANES and EXAFS spectra were performed using Demeter software packageSulfur X-ray Absorption Near Edge Structure (XANES) data were recorded at SSRL beam line 6–2 in fluorescence mode using a single element Vortex silicon drift detector set at ~60° scattering angle. S XANES were also collected at Diamond Light Source (DLS) microfocus beamline I18 in fluorescence mode using a four element Si drift Vortex fluorescence detector set at 90° scattering angle. Zn Extended X-ray Absorption Fine Structure (EXAFS) were collected at DLS beamline I18 in fluorescence mode using a four element Si drift Vortex fluorescence detector set at 90° scattering angle. ZnSOSRS-XRF maps from SSRL were processed from the raw detector count raster files using a custom MATLAB computer script that converted the data array into viewable 8 bit tiff images clipped at various contrast percentiles.How to cite this article: Edwards, N. P. et al. Elemental characterisation of melanin in feathers via synchrotron X-ray imaging and absorption spectroscopy. Sci. Rep.6, 34002; doi: 10.1038/srep34002 (2016).


Daphnia magna growth and its apparent dependence on phytoplankton species diversity. We find strong correlative evidence between phytoplankton species diversity, biochemical diversity, and growth. The relationship between species diversity and growth was partially explained by the fact that in many communities Daphnia was co-limited by long chained polyunsaturated fatty acids and sterols, which was driven by different prey taxa. We suggest that biochemical diversity is a good proxy for the presence of high food quality taxa, and a careful consideration of the distribution of the different biochemical traits among species is necessary before concluding about causal links between species diversity and consumer performance.Biodiversity can strongly influence trophic interactions. The nutritional quality of prey communities and how it is related to the prey diversity is suspected to be a major driver of biodiversity effects. As consumer growth can be co-limited by the supply of several biochemical components, biochemically diverse prey communities should promote consumer growth. Yet, there is no clear consensus on how prey specific diversity is linked to community biochemical diversity since previous studies have considered only single nutritional quality traits. Here, we demonstrate that phytoplankton biochemical traits (fatty acids and sterols) can to a large extent explain  For example, a high diversity of primary producers can stabilize primary production4 and is often accompanied by higher resource use efficiency and increased overall biomass production7. These effects at the producer level may also influence higher trophic levels through changes in nutritional quality9. However, there is no clear consensus on how prey specific diversity is linked to nutritional quality. Previous studies have considered only single nutritional quality traits, such as phosphorus (P) or nitrogen (N) to carbon ratios11, polyunsaturated fatty acid (PUFA)13 or sterol concentrations15. However, such consideration might be insufficient because several of these traits can simultaneously limit consumer performance18. In general, the occurrence of such co-limitations could yield strong positive relationships between the biochemical diversity  of the prey community and consumer growth.Biodiversity is a major driver of ecosystem processes and has a wide array of effects on various ecosystem functionsscenario 1, complementarity effect) and a positive correlation between consumer performance and food diversity would be observed. Another scenario could be that one or a few species provide all nutrients required for consumer growth and reproduction21. The presence of such superior nutritional quality species can alter the relationship between producer diversity and consumer growth. In a more diverse community the chance of having the high quality species would increase  and a positive correlation between consumer performance and food diversity would exist. Yet, at the same time, the relative contribution of the high quality species could be diluted in more diverse communities, depending on its competitive strength. The balance between these two counter-acting effects  could either yield null or negative correlations between consumer performance and food diversity (scenario 3).This consideration implies several scenarios of how prey diversity may correlate with nutritional quality and, ultimately, consumer growth. For example, co-limiting nutrients might be heterogeneously distributed among different phytoplankton species. Thus, in a more diverse phytoplankton community, the probability of co-occurring species with complementary nutritional traits increases, thereby promoting biochemical diversity and consumer growth 17, included in all experimental setups, we established different phytoplankton communities differing in species diversity and thus evaluated the growth potential of D. magna on these phytoplankton communities focusing on the underlying mechanism between species and biochemical diversity. The phytoplankton species were selected as representatives of the major taxonomic groups commonly found in natural systems. The members within each taxonomic group share common biochemical traits, in particular their fatty acid and sterol composition27, and the chosen species can thus be considered as representative of the biochemical diversity found in nature. In order to account for the effects of phytoplankton species competition, e.g. for light or phosphorus, on the nutritional quality of the communities28 we allowed the species to develop their own communities rather than to artificially assemble mixtures of given species proportions to obtain a more realistic scenario.To our knowledge, despite the large interest in understanding the effects of biodiversity on trophic interactionsSynechococcus elongatus, which is of low nutritional quality,  modified Woods Hole MBL medium29. To avoid a limitation by potassium (K), the final concentration of K was adjusted to 100 µM using potassium chloride (KCl). Aliquots of these pre-cultures were used to inoculate the experimental phytoplankton monocultures and communities. All experimental cultures started with a total biomass concentration of 1 mg C ∙ L−1; the communities were inoculated with equal carbon shares of each species. Carbon concentrations were estimated using previously determined carbon-extinction equations. All algal cultures were grown semi-continuously (0.15 d−1) in duplicates  at 20 °C and a 16/8 h light/dark cycle (light intensity: 256 µmol photons · m−2 · s−1) in 1 L Erlenmeyer flasks . The cyanobacterium was counted by epifluorescence microscopy  after Acridin-orange staining  under blue light . Cell sizes of phytoplankton were measured and converted into biomass units according to Hillebrand Daphnia magna . A cohort of females was synchronized on a Acutodesmus obliquus diet in artificial Daphnia medium (ADaM)31 to release offspring at day 12 of phytoplankton growth. Shortly before the offspring were released, the females were transferred to ADaM containing S. elongatus (2 mg C ∙ L−1). Neonates born within 24 hours were isolated and randomly transferred to experimental vessels  containing 200 mL food suspensions. The food suspensions were prepared by diluting the removed fractions of the respective phytoplankton communities, whose biomass ranged from 7.6 to 18.2 mg C ∙ L−1, with ADaM to a target concentration of 2 mg C ∙ L−1. Three subsamples, each consisting of 10 randomly selected individuals, were placed into pre-weighed aluminum boats, dried at 50 °C and weighed to determine the offspring dry mass at the beginning of the experiment. During the following 4 days of the experiment daphnids were transferred daily into fresh medium with freshly harvested food suspensions. After 4 days, the animals in each vessel were placed into pre-weighed aluminum boats, dried at 50 °C and weighed. The somatic growth rates were calculated using equation The zooplankton growth experiments were conducted with a clone of 32 after sulphuric acid treatment and oxidative hydrolysation with K2S2O8 at 121 °C and 104 kPa (autoclaving).Each phytoplankton culture was analyzed for particulate organic carbon (C), particulate phosphorus (P), and fatty acids. Only monocultures and a community with the highest inoculated species richness could be analyzed for sterols because the available biomass limited the tests which could be performed (see below). For carbon determination, aliquots of the algal suspensions were filtered onto 25 mm precombusted glass fiber filters , dried at 50 °C and analyzed using an elemental analyzer . To measure particulate P concentrations, aliquots of the phytoplankton cultures were filtered onto polysulfone filters . Phosphorus concentrations were determined using the molybdate blue reduction methodet al.33. Sterols were extracted from GF/F filters loaded with aliquots of the algae cultures (1 mg C) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) as described in Martin-Creuzburg and Merkel27. Fatty acid and sterol concentrations were related to the respective carbon concentrations of the food suspensions.Samples for fatty acid determination were obtained by filtering 0.3 mg algal C onto 25 mm glass fiber filters . Filters were stored at −25 °C in 7 mL of dichloromethane-methanol (2:1 v⁄v) under a nitrogen atmosphere in glass tubes with Teflon seals. Lipids were extracted and transesterified into fatty acid methyl esters and then identified and quantified by gas chromatography  according to Wacker 35, which takes into account how many species were present and how each species was represented in the community.The shares of the different species in the respective phytoplankton communities (based on biovolumes) were used to calculate the Shannon-Wiener diversity indexThe diversity of the fatty acid composition within a community was also calculated according to the Shannon-Wiener index. This calculation included the proportions of 17 fatty acids that were most common in phytoplankton species used here  and related to the microscopically determined biovolumes (BVj) of each species (j) and the number of species in the community (I) to calculate the total expected sterol concentration (E) of the whole community , indicating that the total sterol concentrations of the different communities were reliably estimated.The calculated sterol concentration was then converted back to carbon units. To verify the validity of this procedure, we analyzed the total sterol concentration of a community inoculated with the highest species richness and compared these values with total sterol concentrations calculated for this particular community. Both procedures revealed comparable total sterol concentrations  was conducted. Scaled values of the Shannon-Wiener index of phytoplankton diversity were included in the PCA. Additionally, we included the P:C ratio, estimated total sterol concentrations, and concentrations of several prominent fatty acids as indicators of nutritional quality. The following fatty acids and fatty acid sum parameters were considered: the sum of saturated fatty acids (SFA), the unsaturated fatty acid oleic acid , the polyunsaturated fatty acids linoleic acid , α-linolenic acid , arachidonic acid , and eicosapentaenoic acid . Data scores of PC were additionally analyzed for their relationship to Daphnia growth rates using analysis of co-variance (ANCOVA). All calculations and statistical analyses were carried out using the statistical software package R version 3.3.2 .The relationship between The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.2 = 0.6; Fig. 2 = 0.62; Fig. 2 = 0.9) with Daphnia growth rates  of the species in the communities . In the absence of green algae, the communities were dominated by the sterol- and PUFA-free cyanobacterium S. elongatus and, consequently, Daphnia growth rates were low 11.The relationship of Daphnia growth rates with data scores of the principal components . Daphnia growth rates generally increased also with PC2, representing a measure of the Shannon-Wiener index and the concentrations of dietary EPA and ARA . The presence of green algae  led to a parallel shift with higher Daphnia growth rates but no change in the slope . More importantly, however, chlorophytes, which are rich in sterols, presumably saturated the consumer sterol demands, thereby increasing consumer growth further and shifting the limiting nutrient to a mere C20 PUFA limitation.In communities without chlorophytes, the beneficial effect of species diversity on consumer growth can be explained by the higher probability of ingesting C20 PUFA-rich species. By contributing additional fatty acids, these species also increase the fatty acid richness, thereby explaining the positive correlation between species and fatty acid diversity indices. In these communities, C20 PUFA-rich species supply all the known potentially co-limiting nutrients (i.e. sterols and C20 PUFA), since the basal food 41. Overall, there is strong evidence that mixed diets enhance the performance of terrestrial and aquatic herbivores44, thus emphasizing the significance of primary producer diversity for higher trophic levels. Yet in most cases the performance on mixed diets is not better than that on the single most optimal species21. Although we have not considered growth on single species here, our results also indicate that the apparent diversity effects on consumer growth are to a large extent driven by few biochemically superior species i.e. C. meneghiniana, Cryptomonas spp. and N. limnetica. In natural systems, a less diverse community dominated by PUFA- and sterol-rich diatoms and cryptophytes may already be of excellent nutritional value for consumers37. Possibly, in such communities the biochemical diversity is higher since these species increase both the richness and the evenness of the fatty acid pool. Furthermore, overall species diversity based on indices or numbers is likely to change seasonally and annually and depending on species-specific nutritional traits, the availability of essential lipids may change. Even, if the species diversity index does not change, the fatty acid profiles might be significantly altered. While species replacing each other during the season might lead to a relatively constant general species diversity index, the replacement of species with particular species-specific fatty acid composition will generate completely different fatty acid pattern in the communities45.Studies which have investigated the foraging behavior of generalist herbivores suggested that mixed complementary diets are preferably consumed, as they provide all necessary nutrients, may dilute toxic substances, and may even improve digestionS. elongatus they cannot fully saturate all co-limiting nutrient demands of the consumers. In such cases, chlorophytes, which are rich in sterols, can act as complementary food species.However, we also found some evidence for resource complementarity. Nutritionally superior species contribute much more C20 PUFA than sterols and when diluted in a community dominated by 9. However, prey biochemical traits might not be the only drivers of consumer performance. From a more general perspective, the phytoplankton species may also differ in defense traits such as size and cell morphology, directly influencing trophic interactions48. Although the distribution of these traits among species might drive diversity effects in ways we have not considered here, our study clearly underlines the important role of nutritional traits for the understanding of diversity effects.In conclusion, our results provide a possible explanation for the functional link between zooplankton nutrition and phytoplankton diversity found in previous studiesSupplementary information


To help decide when to start and when to stop pre-emptive therapy for cytomegalovirus infection, we conducted two open-label randomized controlled trials in renal, liver and bone marrow transplant recipients in a single centre where pre-emptive therapy is indicated if viraemia exceeds 3000 genomes/ml (2520 IU/ml) of whole blood.Patients with two consecutive viraemia episodes each below 3000 genomes/ml were randomized to continue monitoring or to immediate treatment (Part A). A separate group of patients with viral load greater than 3000 genomes/ml was randomized to stop pre-emptive therapy when two consecutive levels less than 200 genomes/ml (168 IU/ml) or less than 3000 genomes/ml were obtained (Part B). For both parts, the primary endpoint was the occurrence of a separate episode of viraemia requiring treatment because it was greater than 3000 genomes/ml.In Part A, the primary endpoint was not significantly different between the two arms; 18/32 (56%) in the monitor arm had viraemia greater than 3000 genomes/ml compared to 10/27 (37%) in the immediate treatment arm (p = 0.193). However, the time to developing an episode of viraemia greater than 3000 genomes/ml was significantly delayed among those randomized to immediate treatment (p = 0.022). In Part B, the primary endpoint was not significantly different between the two arms; 19/55 (35%) in the less than 200 genomes/ml arm subsequently had viraemia greater than 3000 genomes/ml compared to 23/51 (45%) among those randomized to stop treatment in the less than 3000 genomes/ml arm (p = 0.322). However, the duration of antiviral treatment was significantly shorter (p = 0.0012) in those randomized to stop treatment when viraemia was less than 3000 genomes/ml.The results illustrate that patients have continuing risks for CMV infection with limited time available for intervention. We see no need to alter current rules for stopping or starting pre-emptive therapy. Opportunistic infection represents a significant problem after transplantation. Cytomegalovirus (CMV) is the most common opportunist and may produce serious end organ disease if not controlled by deploying antiviral drugs for prophylaxis or pre-emptive therapy.–3 In traOur natural history data showed that patients with CMV end organ disease had CMV PCR viraemia ranging from 14,000 to 203 million  genomes/ml blood. The loweThese randomized controlled trials were designed from the perspective of a diagnostic virology laboratory aiming to provide advice to our clinical colleagues on when to start and stop pre-emptive therapy. The viral load parameters for the three transplant patient groups studied here  are slightly different in that the bone marrow patients get CMV end-organ disease at a lower level than do the solid organ transplant recipients. However,Patients undergoing CMV surveillance in a single institution because of allogeneic transplantation of kidney, liver or haematopoietic stem cells were studied.,11 ExcluOur strategy for CMV surveillance is described elsewhere. Briefly,The real time PCR method used a Taqman probe and the ABI7700 thermal cycler as described elsewhere. The valuDoubling times of CMV viraemia and decline rates following therapy were calculated using a standard exponential function. The nadiRandomization employed random number tables administered by means of sequentially numbered sealed envelopes prepared by the first author, one set for each patient group, to allow stratification by patient type. The randomization did not use pre-specified block sizes and the allocation ratio was 1:1. The study was implemented by medical staff in the Centre for Virology. As soon as a PCR result indicated that a patient was potentially eligible, the result was discussed with their attending physician. If the patient was considered well enough to be approached, the study objectives were explained and they were offered randomization into the appropriate study by ER, MR, SW, TD, FM, GN, SA or AS. For those who agreed, the next numbered sealed envelope for their patient group was opened and the indicated treatment policy followed. The drug and dose of antiviral drug to be given to an individual patient was at the discretion of their attending physician , but normally consisted of ganciclovir 5 mg per kilogram twice a day or valganciclovir 900 mg twice a day. As alternatives, foscarnet at 60 mg per kilogram three times a day, foscarnet at 90 mg per kilogram twice a day or half dose foscarnet plus half dose ganciclovir were permitted, having previously been shown to control CMV viraemia to a similar extent as full-dose ganciclovir. All drugPatients with a single viral load measurement between 200 and 3,000 genomes/ml were monitored but were not treated. Those who developed a second, consecutive viral load between 200 and 3,000 genomes/ml were offered randomization to be treated immediately until two consecutive blood samples without detectable CMV DNA were obtained or to only start treatment if the viral load increased above 3000 genomes/ml. Because the availability of real-time PCR results was novel, there was no available data to guide the sample size required. Assuming that only 1% of those randomized to receive immediate treatment would develop a viral load greater than 3000 genomes/ml, a study size of 72 patients had 90% power to detect an increase to 25% among those randomized to monitor without treatment.Any patient with a viral load measurement above 3,000 genomes/ml blood was treated as part of our standard clinical management protocol. Patients were then offered randomization to stop therapy when they had two consecutive viral load measurements below 200 genomes/ml or two consecutive viral load measurements below 3,000 genomes/ml. All patients were monitored to determine if the viral load subsequently increased above 3000 genomes/ml. The sample size calculation was based on the estimate that a second episode of replication requiring pre-emptive therapy (greater than 3000 genomes/ml) was expected in 30% of patients randomized to be treated until they had two consecutive viral load measurements below 200 genomes/ml. A study size of 106 patients had 90% power to detect a doubling of this incidence to 60%To define the number of patients with CMV viraemia who subsequently develop a viral load greater than 3000 genomes/ml in each part of the study.The primary end-point of Part A compared statistically the proportion in each arm who developed viraemia greater than 3000 genomes/ml. The primary end-point of Part B compared statistically the proportion in each arm who subsequently developed viraemia greater than 3000 genomes/ml.These objectives and primary end-points did not change during the study.The studies (protocol 6077) were approved by the Royal Free Hospital and Medical School Research Ethics Committee  on 20 November 2002. All patients gave informed consent for the study which was documented in writing. No children, or adults unable to give informed consent, were recruited into the study. The sponsor was University College London. The first patient was randomized in February 2003 and the last in December 2011. The study was registered in June 2009 as ClinicalTrials.gov NCT00947141 when registration of non-industry clinical trials became required, but patients had already been recruited by then. The authors confirm that all ongoing and related trials for cytomegalovirus have been registered before recruitment of the first patient. Patients who were excluded after randomization were replaced. Once a patient had been randomized into Part A or Part B, they could not subsequently be entered into the other part. Patients taking part in a randomized controlled trial of a prototype CMV vaccine at our institution post-transplant day of randomization was 44.5 [10–122] in the monitor arm and 44 [6–78] in the immediate treatment arm. The median post-transplant day of last sample collection was 102 [13–595] in the monitor arm and 93 [39–651] in the immediate treatment arm.The primary endpoint  was observed in 28 patients (48%); 18/32 (56%) in the monitor arm versus 10/27 (37%) in the immediate treatment arm (p = 0.1925). The time from transplant to developing the primary endpoint was significantly longer  p = 0.022) among those randomized to immediate treatment . Three pAlthough the study was not powered to provide results for each patient group separately, we performed exploratory analyses to determine if there were marked differences for patients in the high risk groups  or for bOnly one patient in Part A developed CMV end-organ disease. A bone marrow transplant patient became PCR positive on post-transplant days 71 and 77 (both less than 3000 genomes/ml) and so was recruited into the monitor arm of Part A. On day 102, symptoms of vomiting prompted endoscopy. Gastric and duodenal biopsies had high CMV viral loads by PCR, 135,618 and 35,476 genomes/microgram of DNA respectively, but no inclusion bodies were seen by histopathology or by immunostaining so a diagnosis of presumptive gastrointestinal disease was made. None of six blood samples taken between recruitment and biopsy had a viral load greater than 3000 genomes/ml. His viral load became undetectable after a three week course of intravenous ganciclovir after which repeat gastric and duodenal biopsies contained no CMV DNA detectable by PCR. He was then given prophylaxis with valaciclovir to avoid the bone marrow toxicity of valganciclovir. By day 200, his viral load became detectable above 3000 genomes/ml and valganciclovir, limited by bone marrow toxicity, failed to bring this to undetectable levels. After another 100 days of continuing viraemia he developed retinitis which was confirmed by PCR from a vitreous sample. Thus, this patient had proven CMV end organ disease almost one year post-transplant and approximately 7.5 months after randomization into the study.A total of 193 eligible patients were identified but 31 were considered by their clinicians to be too ill to discuss the study while 50 declined to take part . 112/112The patients were well matched, because there was no significant difference (p = 0.8) in the peak viral load of the initial episode among patients randomized to two levels less than 200 genomes/ml (mean peak = 4.01± 0.52 log10 genomes/ml) compared to those randomized to two levels less than 3000 genomes/ml (mean peak = 4.04 ± 0.57 log10 genomes/ml). There was also no significant difference in the doubling time  or the half-life of decline  between the two groups.The primary endpoint of a subsequent episode of viral replication greater than 3000 genomes/ml blood was reached in 42 patients; 19/55 (35%) randomized to two levels less than 200 genomes/ml and 23/51 (45%) randomized to two levels less than 3000 genomes/ml ; this diWhen the number of days of treatment for subsequent episodes were included, the overall duration of pre-emptive therapy given to the two sets of patients was no longer significant . Because each median value had increased by 8, we suspected this was because of the expanded range of values seen, but explored the results to ensure that patients stopping therapy earlier had not been predisposed to have additional episodes of viraemia. The median days of treatment amongst those who needed any subsequent treatment was 27 [range 12–308] versus 33 [10–336]; p = 0.22). Several patients developed more than one subsequent episode of viraemia, some with viral loads greater than 3000 genomes/ml. The number of patients who had subsequent episodes of high level viraemia, low level viraemia, both or neither are shown in We explored potential clinico-pathological links between viral load measurements and outcomes. The median nadir viral load was 1.25 log10 genomes/ml for those randomized to two levels less than 200 genomes/ml, which was significantly (p = 0.03) lower than that (1.60 log10 genomes/ml) in those randomized to two levels less than 3000 genomes/ml. Among those with second episodes above 3000 genomes/ml, there was no significant difference (p = 0.9) in the viral load of the initial peak of those randomized to two levels less than 200 genomes/ml (4.01 log10 genomes/ml) compared to those randomized to two levels less than 3000 genomes/ml (4.06 log10 genomes/ml). There was also no significant difference in doubling time  or half-life of decline  in the two groups. However, within each group, the median viral load of the initial peak was significantly greater than that of the subsequent peak . All the treated patients received ganciclovir or valganciclovir except for six (2 in the less than 3000 arm and 4 in the less than 200 arm) who received foscarnet. There was no obvious difference in the viral load parameters of those who received foscarnet.Although the study was not powered to provide results for each patient group separately, we performed exploratory analyses to determine if there were marked differences for patients in the high risk groups  or for bNo patients in Part B developed CMV end organ disease.This manuscript describes the first assessment of a policy of when to start and when to stop pre-emptive therapy using a randomized clinical trial design. For Part A of the study, it is clear from In Part B of the study, patients randomized to stop treatment before viraemia became undetectable (below 200 genomes/ml blood) needed a significantly shorter duration of pre-emptive therapy, so the objective of reducing exposure to antiviral drugs was achieved. However, this potentially beneficial effect was attenuated on follow-up. Based on these results, we cannot recommend that the current criteria for stopping pre-emptive therapy are changed. Indeed, the significantly higher nadir viral load in those given pre-emptive therapy until they had two blood samples with CMV DNA below 3000 genomes/ml suggests that the initial infection may have been inadequately treated to represent a focus from which viraemia could recur. Thus, despite the significantly shorter initial episode of treatment, we cannot recommend that treatment is stopped, as a matter of routine, at the earlier time point chosen here. However, if an individual patient experiences side-effects from pre-emptive therapy, then our results suggest that stopping this once the viral load falls below 3000 genomes/ml on two occasions is likely to have only minimal effects on the overall outcome.All of the other measurements of viral load described here are consistent with those we have reported previously, emphasizing that these represent robust viral biomarkers of the natural history of CMV infection post transplantation in both solid organ and stem cell transplant recipients.,8,11,12 The strengths of this single centre study include the use of validated assays of CMV viral load and detailed biomathematical analyses. Weaknesses include the relatively small sample size and the long time required to recruit patients. Nevertheless, in the absence of any licensed new antiviral drug with activity against CMV, we believe the results are relevant to contemporary medical practice. We also show that, despite differences in CMV natural history,,19 patieS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Glioblastoma is the most malignant tumor and has high mortality rate. The methylated prompter of MGMT results in chemotherapy sensitivity for these patients. However, there are still other factors that affected the prognosis for the glioblastoma patients with similar MGMT methylation status. We developed a signature with three genes screened from the whole genome mRNA expression profile from Chinese Glioma Genome Atlas (CGGA) and RNAseq data from The Cancer Genome Atlas (TCGA). Patients with MGMT methylation in low risk group had longer survival than those in high risk group . Moreover, the prognostic value of the signature was significant difference in cohorts stratified by MGMT methylation and chemotherapy (P=0.0473), while there is no significant difference between low and high risk group or unmethylated MGMT patients without chemotherapy. Multivariate analysis indicated that the risk score was an independent prognosis factor (P = 0.004). In conclusion, our results showed that the signature has prognostic value for patients with MGMT promoter-methylated glioblastomas based on bioinformatics analysis. Gliomas are the most common and lethal primary tumors of central nervous system . GlioblaMany biomarkers, such as mutations of PTEN, IDH1, TP53 and methylation of O6)-methylguanine DNA methyltransferase (MGMT) promoter have been identified for GBM. PTEN is a tumor suppressor and mutation of PTEN could up-regulated the AKT pathway which play a critical role in cell cycle regulation, apoptosis, and cell migration in GBM , may ser-methylguIn our study, we obtained whole genome mRNA expression profiling microarray data from Chinese Glioma Genome Atlas (CGGA) as training set and two validation datasets, CGGA mRNA-sequencing data and The Cancer Genome Atlas (TCGA) mRNA sequencing data. By applying significance analysis of microarray (SAM) and Cox regression analysis, we screened three target genes and thus generated a signature based on these genes. Patients with MGMT promoter-methylated were divided into low risk and high risk group based on the cutoff (median risk score) and the clinical outcomes and molecular features were quite different between two groups.A total of 41 GBM with MGMT promoter methylation, 60 GBM with MGMT promoter unmethylation and 5 non-cancerous brain tissue (NBT) samples were included in the comparison, respectively. Moreover, we compared NBT and methylated or unmethylated patients using SAM analysis and 688 mRNAs (760 probes) were differentially expressed in these samples  . We evalWith the three genes, we developed a risk score for each patient which was calculated based on a linear combination of the mRNA expression level weighted by the regression coefficient (β) derived from the univariate Cox regression analysis. Patients with MGMT promoter methylated (41 patients) in the training set were divided into low risk and high risk group based on the cutoff (median risk score) and patients in low risk group had longer survival time than high risk group  and TCGA (55 patients) RNAseq datasets showed similar results  in OS as shown in Figure The expression levels of the three genes showed significant difference between patients with MGMT promoter methylation and non-cancerous brain tissue samples Figure . MoreoveWe assessed the independence of the three-gene signature in the CGGA microarray dataset. It showed that the signature was significant associated with the OS (P = 0.004) along with radiotherapy and chemotherapy in the univariate cox regression analysis. On multivariate analysis, it also showed the signature was an independent prognosis factor (P = 0.004) Table . In TCGAWe observed that GBM patients in the high risk group had shorter OS than low risk group Figure . The relWe performed SAM (FDR < 0.05) between low and high risk group in the CGGA microarray dataset and we screened top 500 positively 599 probes) and 500 negatively 588 probes) correlated expression genes with the risk score, respectively. The expression patterns of genes were showed in Figure 8 probes 9 probes Overall, the results of GSEA analysis were consistent with GO analysis' and functional annotation of the signature mainly enriched in immune response, apoptosis, cell adhesion pathways. It might partially explain the poor OS of patients with MGMT promoter methylation in high risk group.It is reported that the status of MGMT promoter methylation is closely associated with chemotherapy sensitivity , 7. HoweThe three genes have different expression between GBMs with non-cancerous tissues, and they all have prognosis values. The risk score based on the three genes divided patients into low and high risk groups and patients in low risk group had longer OS than high risk group. Further, low risk group also showed better OS than high risk groups and unmethylated group in patients with chemotherapy, but no significant difference between low and high or unmethylated MGMT patients without chemotherapy. The signature can specifically predict the prognosis of MGMT promoter methylation patients with chemotherapy treated. It also showed the signature was an independent prognostic factor along with age, gender, IDH1, radiotherapy and chemotherapy.All of the 3 genes were remarkably associated with prognosis in GBM with MGMT methylation status. FPR3 is a member of the human formyl peptide receptors, which plays an important functional role in the regulation of immune responses and host defense mechanisms , 15. FPRThere are limitations in our manuscript, only 5 NBT samples were included into SAM analysis and only 41 patients  collected into our analysis which could cause bias of data analysis. The three-gene signature were selected based on the bioinformatics analysis and it is a totally observational study which may just provide clues for further study of GBM patients with MGMT promoter methylation. We will continue to collect samples and validated these findings experimentally in our future work. However, we further validated screened target genes in CGGA and TCGA RNAseq datasets.In conclusion, our results showed that the three-gene signature has prognosis value for patients with MGMT promoter-methylated glioblastomas. Due to the poor prognosis in high risk group, clinicians should pay more attention to new treatment therapies. Further study need to validate these findings experimentally in future work.http://www.cgga.org.cn/) were included in our analysis. All patients' clinical information was download from this website. The RNAseq data were normalized by log2 transformed before analysis. 119 patients from The Cancer Genome Atlas  were included as validation dataset.145 patients  from the Chinese Glioma Genome Atlas  weighted by the regression coefficient (β) derived from the univariate Cox regression analysis as previously reported –28. The gene1 × exprgene1 + βgene2 × exprgene2+ ··· + βgenen × exprgenenRisk score = βThe high risk score group presented shorter OS than low risk score group. The significance analysis of microarray (SAM) and Cox analysis was calculated using R software (version 3.2.3) with the samr and survival packages. Moreover, the univariate and multivariate cox regression analysis was performed by SPSS software . The Kaplan-Meier analysis was used to evaluate the OS distributions by using GraphPad Prism 6 . A two-sided P value of < 0.05 was regarded as statistically significant.


Advancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines.Chicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150–199 for GS FLX+ and bases 90–99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME.The three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.The online version of this article (10.1186/s12866-017-1101-8) contains supplementary material, which is available to authorized users. Sequencing of 16S rRNA amplicons is now a well-established and robust method used in compositional studies of the gut microbiome of humans, animals, and insects. These studies have generated a wealth of information regarding the impact of diet , diseaseCollective concerns regarding methodological biases and the ability to compare studies from different research groups originated a collaborative effort designed to comprehensively evaluate methods employed in the study of the human microbiome, the Microbiome Quality Control project (MBQC). Inspired by earlier projects like the Microarray Quality Control project (MAQC), the MBQC focused on the analyses of the impacts of sample collection, DNA extraction, sequencing protocol, and bioinformatics data analysis pipelines on amplicon profiling of the human fecal microbiome. Although the coalition is still in early stages, the MBQC has identified the DNA isolation method (and the lab performing the DNA isolation), as well as 16S rRNA amplification primers used, as major sources of variation, while sequencing depth and sample storage had small but detectable effects on the generated data .A number of recent studies have attempted to identify errors or bias generated by the intrinsic characteristics of sequencing platforms , 27, 28.Further comparative studies between Ion Torrent PGM, Illumina MiSeq, Illumina HiSeq, and Roche GS FLX+ confirmed that the later generated the longest reads among these platforms (up to 600 bp) but had a relatively high error rate with poly-bases of more than 6 base pairs . AdditioTogether, the previously discussed data indicate that technical protocols and sequencing platforms have a variable impact on output. In this study, we report a comparison of the three most widely utilized sequencing platforms: Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX+, using the most current library preparation protocols and sequencing kits available for 16S rRNA amplicon sequencing. Although Roche discontinued support for the 454 GS FLX+ sequencing platform in 2016, the platform still has relevance to studies that have used this technology in the past or any ongoing studies that may utilize a sequencing provider whom is still running the 454 GS FLX+ sequencer. More importantly, this study couples the platform comparison with a systematic assessment of bioinformatics pipelines commonly used for amplicon data analysis: Quantitative Insights Into Microbial Ecology (QIIME)  and UPARSalmonella and prebiotic supplementation on the chicken gut microbiome and immune responses were used in this study. We have previously shown that prebiotics have a modulatory effect on the gut microbiome [Bifidobacterium, Lactobacillus) were impacted by this additional step. Clearly, the utility of chimera removal has been widely demonstrated in the analysis of 16S rRNA amplicon sequencing data [Fourteen cecum samples randomly selected from an ongoing study aiming to investigate the impact of vaccination against crobiome , 39, noting data , 41.The aim of the present study was to explore influences of sequencing platforms and bioinformatics pipelines on diversity and relative abundance of bacterial taxa in 16S rRNA amplicon data. Additionally, each platform and analysis pipeline was compared for their abilities to discriminate between samples from various treatment groups in order to validate their functionality in microbiome studies., 200 mg of intestinal content were added to a tube containing 540 μl of SLB buffer and 200 mg of glass beads. Samples were homogenized using a TissueLyser  for 5 min at 30 Hz in 1 min intervals between bead beating and ice incubation cycles. DS buffer and proteinase K were added according to the manufacturer’s instruction. The mix was incubated at 70 °C for 10 min, followed by another incubation at 95 °C for 5 min. Quality of the isolated DNA was assessed by agarose gel electrophoresis and purity verified using 260/280 and 260/230 ratios measured by NanoDrop 1000 instrument . DNA concentration was quantified using Quant-iT™ PicoGreen dsDNA Reagent .The same physical samples were used for all sequencing experiments across platforms and bioinformatics pipelines. Total genomic DNA was extracted using E.Z.N.A. Stool DNA Kit  according to manufacturer’s instructions with minor modifications. BrieflyProtocols for preparation of libraries for sequencing are represented in Fig. ) to the amplicon target. The final libraries were again purified using AMPure XP reagent (Beckman Coulter), quantified and normalized prior to pooling. The DNA library pool was then denatured with NaOH, diluted with hybridization buffer and heat denatured before loading on the MiSeq reagent cartridge (Illumina) and on the MiSeq instrument (Illumina). Automated cluster generation and paired-end sequencing with dual reads were performed according to the manufacturer’s instructions.DNA was amplified using primers targeting the V1-V2 region of the bacterial 16S rRNA gene , 43 and For amplicon library preparation the V1-V2 hypervariable region of the 16S rRNA gene was amplified from total bacterial DNA using the forward primer composed of Ion Torrent adapter A, a 10 bp IonXpress ™ barcode , unique to each sample and the universal bacterial primer 8F. The reverse primer consisted of Ion Torrent trP1 adapter followed by reverse bacterial primer 338R. PCR reactions contained 50 ng of DNA template, 2.5 units of HotStar Hi-fidelity DNA polymerase , 1× HotStar Hi-Fidelity PCR buffer containing dNTPs, and 0.6 μM of each primer. Negative controls, not containing template, were amplified for all barcode-primer sets. The PCR products were gel purified individually using the E-Gel Electrophoresis System. DNA concentrations were quantified using Quant-iT™ PicoGreen® dsDNA Reagent  and mixed at equimolar concentrations. Template-Positive Ion OneTouch™ 200 Ion Sphere™ Particles were prepared from library pool using the Ion OneTouch™ 2 system . The prepared templates were sequenced on the Ion Torrent PGM instrument (Life Technologies) in the Microbiome Core Facility using the Ion PGM 400 sequencing reagents. All kits were used according to the manufacturer’s instructions. Initial data analysis, base pair calling and trimming of each sequence was performed on Ion Torrent browser to yield high quality reads.The library preparation protocol for the three evaluated sequencing platforms is depicted in Fig. -dimensional principal coordinate analysis plots. QIIME was also used to calculate alpha diversity with a sub-sampling depth of 1000 using observed species, Shannon and phylogenetic diversity (PD) metrics. To evaluate the similarities between bacterial communities a principal coordinate analysis (PCoA) using Unweighted and Weighted Fast Unifrac [Roche 454 sequencing results were initially processed using GS Data Analysis Software package . The two Unifrac , 53 was In addition to the primary bioinformatics pipeline, additional pipelines were applied. The first pipeline was based on the de novo OTU picking algorithm as implemented in QIIME 1.8.0  as the pRestricting consideration to the two Illumina MiSeq platform results, two pipelines intended to assess microbial communities at a finer resolution than the traditional 97% similarity threshold were considered. The software package DADA2  was usedPhyloToAST  analysisAnalysis of variance (ANOVA) was used to identify significant differences in phylogenetic diversity (PD) and species richness (S) indexes. Analysis of Similarities (ANOSIM) and Permutational Multivariate Analysis of Variance (PERMANOVA) analyses were applied to Unweighted Unifrac similarity matrices to compute similarities between groups. The Kruskal Wallis test for multiple pairwise comparisons was performed to evaluate significant differences in relative abundances of bacterial taxa using JMP genomics . In all analyses alpha was set at 0.05.Salmonella challenge study conducted in poultry. The parent study aimed to evaluate the impact of an attenuated Salmonella strain on the gut microbiota of poultry, and to determine its effect on the gut microbiome and efficacy on Salmonella infection clearance after challenge. A secondary objective was to determine how prebiotics modify the structure of the gut microbiome, and the impact of this modification on Salmonella infection clearance . In the current study, analysis of microbiome composition was conducted on 14 chicken cecum samples randomly selected from 3 different groups: 1) control group, 2) Salmonella-vaccinated group, and 3) prebiotics-fed group. Each of the groups was assigned also to two subgroups according to a Salmonella challenge. Table Samples from this study are from a The bioinformatics analysis pipelines used in our study to determine the taxonomic composition and bacterial diversity of 14 chicken cecum samples using three different platforms are depicted in Fig. Analysis of Similarities (ANOSIM) and Permutational Multivariate Analysis of Variance (PERMANOVA) analyses were performed on Unweighted Unifrac data to evaluate differences between the 6 runs and bioinformatics pipelines. We did not observe clustering by sequencing platform   analyzed using the QIIME2 pipeline (de novo OTU picking plus chimera depletion) showed substantial differences (Monte Carlo es) Fig. , these dOperational Taxonomic Units (OTUs) were identified at a 97% similarity cut-off in the QIIME and UPARSE pipelines. The number of sequences assigned to OTUs for GS FLX+ was similar in all bioinformatics pipelines (99%). De novo (QIIME1 and QIIME2) versus open-reference OTU picking impacted the number of assigned sequences for Illumina MiSeq runs but not for GS FLX+ runs. QIIME1 and QIIME2, both using UCLUST for de novo OTU picking, and UPARSE1 and UPARSE2, both using USEARCH for open reference OTU picking, resulted in a higher number of unassigned reads from the Illumina MiSeq generated data (ranging from 3.6 to 4.1%). Marginal differences were observed in the OTU assignment between the three PGM runs in all bioinformatics pipelines. PGM3  showed a Firmicutes abundance of almost 99%, while PGM1 and PGM2 ranged from 96.8% to 98.7%. However, this difference was exaggerated when comparing Vaccinated and Vaccinated-Challenged samples, with Firmicutes dropping as low as 72.8% for PGM3 vs 90.3% for PGM2.  over represented in the MiSeq generated data compared to PGM and GS FLX+.Firmicutes and Bacteroidetes were the most represented phyla in QIIME and UPARSE pipelines. GS FLX+ generated data showed the highest abundance of Firmicutes with an average of 96.2% followed by the MiSeq platform with an average of 93.2%, and PGM with 91.8%. Bacteroidetes were detected at 3% by GS FLX+, 2.3% by the MiSeq platform, and 6.4% by PGM. Proteobacteria and Tenericutes were represented in low abundance in all platforms and pipelines; however, they were significantly . The open reference OTU picking pipelines (QIIME3 and QIIME4) generated a differential representation of unclassified Erysipelotrichaceae , Turicibacter , and potential Ruminococcus species. Taxa differentially represented by platform in the UPARSE pipelines included Turicibacter, unclassified Clostridiaceae, Clostridiales , Eubacterium biforme, Lactobacillus spp., unclassified Erysipelotrichaceae, Ruminococcus, Anaerotruncus, and Lachnospiraceae.Distinct differences were observed between platforms and pipelines in the relative abundance of specific genera Fig. . Overall effects  and OsciBifidobacterium spp. (absent in the open reference and UPARSE OTU picking approaches) and Bifidobacterium adolescentis (not identified by UPARSE). Differences in the detection profiles were also observed for lactobacilli. Bifidobacteria and lactobacilli are of significance for the samples analyzed in this study since these populations are expected to be impacted by prebiotic feeding, one of the conditions analyzed. Other taxa differentially identified by the different pipelines included species of Tepidimicrobium, Thermacetogenium, Tindallia_Anoxynatronum, and Tissierella_Soehngenia. These groups correspond to poorly characterized soil bacteria.Further analysis using PhyloToAST  Fig. 6)6) showedFinally, comparing the results of the pipelines intended to assess finer resolution features of the microbial communities, we found that the conclusions obtained in the previous analyses largely hold. For both pipelines, samples clustered in beta diversity PCoA plots according to treatment and sequencing run. Procrustes analysis showed that the primary difference in taxa assessments occurred due to the OTU/variant selection rather than the mechanism of assigning taxa to individual sequence representatives Fig. . The numNext Generation Sequencing (NGS) has revolutionized the field of microbiome research –63. OverPCR-based high throughput amplicon sequencing introduces biases to the results obtained, regardless of the sequencing platform , 50. In Lactobacillus reuteri has a GC content of 35.9%–38.9% [Bifidobacterium longum has GC content of 60% [Different sequencing platforms/pipelines generate results with differing phylogenetic distributions Fig. . As the 9%–38.9%  while Bit of 60% . This dit of 60% . The impt of 60% . The stuProcrustes analysis of data showed clear differences between data generated from each platform Fig.  and 7a. Determining the most accurate and appropriate approaches to generate and analyze sequencing data from complex microbial communities remains an important goal of researchers focused on microbiota studies. Unfortunately without a standardized set of protocols from sample preparation and handling to final data analysis (and every step in between), it is impossible to eliminate biases from these studies entirely. However, making an effort to choose the correct sequencing platform for a given study as well as the most appropriate data analysis tools will drastically reduce errors in data acquisition and processing between studies. In this context, emerging analysis methods (methods that attempt to differentiate between sequences with similarity greater than the 97% convention) such as DADA2  and OligIn our study, we compared DADA2  and QIIMSalmonella infection study, despite these differences, samples from variable treatment groups were differentiated from one another regardless of the sequencing platform and/or bioinformatics pipeline used allowing us to draw similar conclusions. This suggested that the same biological conclusions could be drawn from data, as long as the data is collected and analyzed consistently throughout the course of the experiment. It is critical for researchers to take into consideration the limitations of each sequencing platform, and choose a system appropriate for their experimental design.Our study confirmed differences between sequencing platforms and library preparation protocols in the determination of microbial diversity and species richness. Moreover, we showed that bioinformatics pipelines used to analyze sequence data yielded results that differ depending on specific parameters. However, we concluded that, specifically for our chicken-Additional file 1:Table S1. Phylogenetic diversity (PD) and species richness (S) values by platform, experimental group, and bioinformatics pipeline. Table S2. Most abundant phyla identified by platform, experimental group, and bioinformatics pipeline. (DOCX 159 kb)Additional file 2:Figure S1. Principal Coordinates Analysis PCoA (Unweighted UniFrac) plots of data generated by the three different platforms, analyzed by different bioinformatics pipelines and colored according to sequencing platform. PERMANOVA F and P values and ANOSIM R and P values are indicated. Figure S2. Differences in relative abundances of the most impacted taxa according to data generated by different platforms (indicated by different colors) and bioniformatic analysis pipelines (indicated across the top). (PPTX 4115 kb)Additional file 3: Table S3.Relative abundance of taxonomic groups by treatment and bioinformatics pipeline. (XLSX 74 kb)