Endosperm starch provides prime energy for cereal seedling growth. Cereal endosperm with repression of starch branching enzyme (SBE) has been widely studied for its high resistant starch content and health benefit. However, in barley and maize, the repression of SBE changes starch component and amylopectin structure which affects grain germination and seedling establishment. A high resistant starch rice line (TRS) has been developed through inhibiting SBEI/IIb, and its starch has very high resistance to in vitro hydrolysis and digestion. However, it is unclear whether the starch resists in situ degradation in seed and influences seedling growth after grain germination.In this study, TRS and its wild-type rice cultivar Te-qing (TQ) were used to investigate the seedling growth, starch property changes, and in situ starch degradation during seedling growth. The slow degradation of starch in TRS seed restrained the seedling growth. The starch components including amylose and amylopectin were simultaneously degraded in TQ seeds during seedling growth, but in TRS seeds, the amylose was degraded faster than amylopectin and the amylopectin long branch-chains with B-type crystallinity had high resistance to in situ degradation. TQ starch was gradually degraded from the proximal to distal region of embryo and from the outer to inner in endosperm. However, TRS endosperm contained polygonal, aggregate, elongated and hollow starch from inner to outer. The polygonal starch similar to TQ starch was completely degraded, and the other starches with long branch-chains of amylopectin and B-type crystallinity were degraded faster at the early stage of seedling growth but had high resistance to in situ degradation during TRS seedling growth.The B-type crystallinity and long branch-chains of amylopectin in TRS seed had high resistance to in situ degradation, which inhibited TRS seedling growth.The online version of this article (10.1186/s12870-017-1219-8) contains supplementary material, which is available to authorized users. Cereal endosperm starch is a major source of nourishment for humans, and consists of two main components: linear amylose and highly branched amylopectin. Amylose content and amylopectin structure have important effects on structural and functional properties of starch. The amylose is only synthesized by granule bound starch synthase, and the amylopectin is mainly synthesized by soluble starch synthase, starch branching enzyme (SBE), and debranching enzyme. The SBE in cereal crops has three isoforms: SBEI, SBEIIa, and SBEIIb, and is responsible for the formation of branch points of amylopectin , 2. SuppsbeI mutant starch has an altered branching pattern for amylopectin and amylose. When sbeI mutant kernels are germinated, the mutant is associated with shorter coleoptile length and higher residual starch content, indicating that less efficient starch utilization impairs seedling growth [For plants, seeds provide energy for germination and seedling growth. Starch is the main storage compound in cereal endosperm and serves as the primary source of carbohydrate during germination and seedling growth. Recently, Shaik et al.  report tg growth . Howeverg growth .indica rice cultivar Te-qing (TQ) in our laboratory [A rice line (TRS) with high RS has been developed from an boratory . TRS endboratory . Four diboratory . These hboratory , resultiboratory . The polboratory .In this study, TQ and its derived high RS rice TRS were used to investigate the seedling growth, starch property changes, and starch in situ degradation during seedling growth. Our objective was to further understand whether high RS starch influenced the dynamics of starch in situ degradation in seed and the seedling growth.indica rice cultivar Te-qing (TQ) after inhibition of both SBEI and SBEIIb through an antisense RNA technique [The high RS rice line TRS was generated from an echnique . Mature The rice seeds were imbibed in distilled water (the water was changed three times every day) in the dark at 28 °C for 2 days. The germinated seeds were placed in a well of a 96-well plate with embryo up. The lower two-third of seed was soaked in water (the water was changed every day) in the dark at 28 °C for seedling growth. Samples were taken out at 1, 4, 8, 12 and 16 day after imbibition (DAI).The shoot height was measured from the culm base to the tip of the longest leaf. The shoot and root were separated from the seedling and dried in oven at 110 °C for 3 h and at 80 °C for 2 d. Their dry weight was measured.v/v) ethanol for 30 min at 80 °C and then centrifuged at 5000 g for 10 min. The resulting pellet was further washed two times with 80% ethanol. The combined supernatants were measured for soluble sugar by the colorimetric method of anthrone-H2SO4 [The seeds without embryo, shoot and root were freeze-dried and weighted. The dry seeds were extensively ground into flour. The soluble sugar was extracted from flour in 80%  of 10 mm depth and 7 mm diameter containing 30 mL normal MS medium. The medium was divided into two parts, thus allowing ten TQ and TRS embryos to grow separately in one bottle. The cultured embryos were incubated under 12-h photoperiod at 28 °C. After 6 days, the seedlings were photographed, and their heights were measured.2O. The homogenate was filtered with 100-, 200-, and 400-mesh sieves, successively. The starch was washed three times with H2O and two times with anhydrous ethanol, freeze-dried, ground into powder, and passed through a 100-mesh sieve.The germinated seeds without embryos were homogenized in a mortar and pestle with HThe protein was removed from isolated starch with protease and sodium bisulfite, and debranched with isoamylase following the modified procedures of Lin et al.  accordinThe crystalline structure of isolated starch was analyzed using an X-ray powder diffractometer (XRD) . The sample was exposed to the X-ray beam at 40 mA and 40 kV, and scanned from 3 to 40° 2θ with a step size of 0.02° .−1 before deconvolution was applied using Resolutions Pro. The assumed line shape was Lorentzian with a half-width of 19 cm−1 and a resolution enhancement factor of 1.9 [The short-range ordered structure of isolated starch was analyzed using a Varian 7000 Fourier transform infrared (FTIR) spectrometer with a DTGS detector equipped with an attenuated total reflectance single reflectance cell containing a germanium crystal (45° incidence-angle) . The original spectrum was corrected by subtraction of the baseline in the region from 1200 to 800 cmThe germinated seeds were first fixed in a 2.5% glutaraldehyde fixation solution in 0.1 M Na-phosphate butter for 2 h at room temperature and then held at 4 °C. The seeds were washed 3 times with phosphate buffer, successively dehydrated in gradient ethanol, and embedded in LR White Resin. The semithin sections of 2 μm thickness were cut with a glass knife on a Leica Ultrathin Microtome (EM UC7) following the method of Zhao et al. . The sect test were evaluated using the SPSS 16.0 Statistical Software Program.The one-way ANOVA with Tukey’s test, Pearson’s bivariate correlations, and Student’s 2O. Under this growing condition, the only opportunity of the seed and germinating plantlet system to obtain energy is through respiration of storage compounds in seed [In order to investigate the influence of starch degradation on the seedling growth, the seeds were germinated and grew in the dark only in ddH in seed . The seeIn order to exclude the influence of embryo size and vitality on grain germination and seedling growth, the mature embryos were excised from the surrounding endosperm and in vitro cultured in normal MS medium under 12-h photoperiod at 28 °C. The seed germination and seedling growth did not show significant difference between TQ and TRS Fig. . This reGrain serves as storage material for seed germination and seedling growth. During seedling growth, the weight of remaining seeds without embryos was measured Fig. . Before Starch is the main source of carbon during germination and seedling growth when seed was growth in the dark with water but no nutrients . The staConsidering that TRS had a much lower seed weight and starch content than TQ Fig. , it was Seedling growth is directly supported by soluble sugar. Given the changes in the starch content as carbon sources, the total soluble sugar in remaining endosperm was determined during germination and seedling growth. The soluble sugar content in TRS seed was similar to that in TQ seed at 1 DAI, but the significant difference in soluble sugar content appeared at 4, 8, and 16 DAI. The soluble sugar content in TQ seed was significantly higher than in TRS seed at 4 and 8 DAI, but lower at 16 DAI Fig. . The solStarch is mainly composed of both amylose and amylopectin, and the amylopectin contains short branch-chains (A and short B chains) and long branch-chains (long B chains). The proportions of amylose, amylopectin short branch-chains, and amylopectin long branch-chains in starch can be determined using GPC of isoamylase-debranched starch . In the Starches are classified into A-, B-, and C-type according to their XRD patterns. A-type starch has strong diffraction peaks at about 15° and 23° 2θ, and an unresolved doublet at around 17° and 18° 2θ. B-type starch shows strong peak at 17° 2θ, a few small peaks at around 15°, 20°, 22°, and 24° 2θ, and a characteristic peak at about 5.6° 2θ. Typical C-type starch has strong diffraction peaks at about 17° and 23° 2θ, and a few small peaks at around 5.6° and 15° 2θ . Amylope−1 is linked with order/crystalline regions in starch, the band at 1022 cm−1 arises as a result of absorption by stretching modes in amorphous starch and is sensitive to amorphous structure, and the band at 995 cm−1 results from bonding in hydrated carbohydrate helices [−1 absorbance band gradually decreased during rice seedling growth. The results indicated that the ordered and amorphous structures were simultaneously degraded in TQ starch, but the amorphous structure was degraded faster than the crystalline structure in TRS starch. The result was in agreement with the in vivo and in vitro digestion of isolated TQ and TRS starch [Starch contains amorphous and ordered structure. The amorphous structure is consisted of amylose, and the ordered structure composes of two types of helices from amylopectin branch-chains: the short-range ordered structure, defined as the double-helical order, and the long-range ordered structure, defined as the ordered crystallinity packed by double helices. The former is sensitive to the infrared spectrum, and the latter can be detected by XRD . For inf helices . The FTIS starch , indicatThe in situ degradation of starch in endosperm during seedling growth was investigated using our established method for preparing the whole section of mature cereal seed . The lonIn TRS seed, the starch degradation was complex. The starch in the endosperm close to aleurone layer and embryo was first partly degraded, then the starch in the inner region of endosperm was completely degraded, and the starch in the outer and middle regions of endosperm, especially in the middle region, could not be completely degraded even at 16 DAI Fig. . At 16 DThe starch in TQ seed was gradually degraded from the proximal to distal region of embryo and from the outer to inner of endosperm during seedling growth, and its components of amylose and amylopectin short and long branch-chains were simultaneously degraded. However, in TRS seed, the inner region of endosperm was completely degraded, but the middle and outer regions of endosperm was partly degraded during seedling growth. The B-type crystallinity and long branch-chains of amylopectin in TRS aggregate, elongated and hollow starches had high resistance to in situ degradation, which inhibited TRS seedling growth.Additional file 1: Figure S1.t test . (TIFF 2675 kb)Dry weight of shoot (A) and root (B) on the weight basis of pre-germinated seeds during seedling growth. Values are means ± SD from three replicates. Asterisks (*) highlight significant differences between TQ and TRS by Student’s Additional file 2: Figure S2.t test . For , values are the means of three replicates, and R indicates the regression coefficient. (TIFF 8768 kb)Seed and starch weights and their relationships with seedling weight on the weight basis of pre-germinated seeds during seedling growth. (A), dry weight of seed without embryo; , the relationships between the decreased seed weight and the root (a), shoot (b), and seedling (root + shoot) weight (c) in TQ (B) and TRS (c); (d), dry weight of starch in endosperm; , the relationships between the decreased starch weight and the root (a), shoot (b), and seedling (soot + shoot) weight (c) in TQ (E) and TRS (F). For (a and d), values are means ± SD from three replicates, and asterisks (*) highlight significant differences between TQ and TRS by Student’s


Here we introduce a MIxS extension to facilitate the recording and cataloguing of metadata from samples related to hydrocarbon resources. The proposed MIxS-HCR package incorporates the core features of the MIxS standard for marker gene (MIMARKS) and metagenomic (MIMS) sequences along with a hydrocarbon resources customized environmental package. Adoption of the MIxS-HCR standard will enable the comparison and better contextualization of investigations related to hydrocarbon rich environments. The insights from such standardized way of reporting could be highly beneficial for the successful development and optimization of hydrocarbon recovery processes and management of microbiological issues in petroleum production systems.The online version of this article (doi:10.1186/s40793-016-0203-5) contains supplementary material, which is available to authorized users. Hydrocarbon Occurrences are defined as the natural and artificial environmental features that are rich in hydrocarbons Fig. . Hydroca2S production, an undesirable process better known as reservoir “souring”. Moreover, in terms of microbial monitoring, such methodologies will help us determine to what extent the indigenous microbial community of an HCR is represented by the analysis of readily recoverable produced fluids, and if analysis of produced fluids can reduce the need to analyze material directly recovered from the hydrocarbon rich formation, such as reservoir cores which are much more difficult to obtain. It will also be practically important if the analysis of planktonic populations from produced fluids can provide an alert to potential MIC phenomena caused by sessile populations. A growing inventory of HCR systems will also allow us to determine if specific beneficial or detrimental microbially catalyzed processes in HCRs are the result of the same microbial populations in most instances, or whether a particular process is driven by different organisms in different HCR environments.Restraining or harnessing the potential of these microbes requires a considerable understanding of their metabolisms and role in these environments. Tools and methodologies that allow the enumeration, taxonomic identification, and metabolic prediction of these microbes are therefore essential towards this goal. Such methodologies will enable us to address questions relating to microbial spatiotemporal variability and what drives them within an HCR. This will also help us determine whether particular microorganisms were present in the HCR at the time of deposition or whether they were introduced at a later stage through fluid migration (e.g. during the formation or recovery of the hydrocarbon resource). Other interesting questions pertain to hydrocarbon recovery processes, and especially the effect of water injection (aka “water flooding”) on the indigenous and introduced microbial communities associated with HCRs. One particular operational issue related to this is stimulation of biogenic HIn addition to environmental parameters, the outcome from microbiological surveys of HCRs can be strongly impacted by logistical and technical constraints. These include sample acquisition, transport and preservation; the choice of DNA amplification primers ; enumeration methods ; sequencing platform  and related downstream bioinformatics pipelines.There is a growing list of studies on HCRs, but their collective retrieval (or their corresponding sequences from public repositories) is currently impossible even with complex keyword searches. Moreover, the majority of available datasets lack sufficient contextual data, which would facilitate more comprehensive comparative analysis –6.In order to maximize the knowledge gained from these largely unexplored microbial ecosystems it is important to formalize and standardize environment descriptors for studies of these habitats. It is equally important to define a minimum set of contextual parameters, which should accompany the submission of sequence information from HCR studies to the International Nucleotide Sequence Database Collaboration . The adoThe need for standardization is not novel or unique to HCR studies. Since 2005, the Genomic Standards Consortium , 4 has mbiome only the subclasses aquatic biome, polar biome, and terrestrial biome are currently present. The term subterranean biome , which would include biomes related to certain HCR (e.g. hydrocarbon reservoir) as well as other subterranean biomes [environmental feature branch, additional HCR terms such as gas reservoir, oil sand, and coalbed will need to be included in addition to the existing oil reservoir term. Finally, in the environmental material section of ENVO, HCR terms like formation water, injection water, drilling fluid, tailing pond and many more would supplement the existing oil field production water term. It is therefore apparent that expansion of ENVO to include HCR-related terms will greatly benefit the standardization of a growing number of studies on these environments. An initiative to introduce such HCR-related terms in ENVO is currently underway.Around the same time a separate effort aimed at the development of ENVO, a standardized and semantically controlled representation of environment descriptors was undertaken . ENVO qun biomes –16 is cuIn an effort to assist with the standardization of data acquisition and observations derived from HCR-related environments we introduce the MIxS-HCR minimum information standard. This standard is tailored for HCR-related studies and aims at capturing key environmental parameters influencing microbial activity in these environments and standardizing their method of reporting. This is accomplished by the adoption of terms  from previously reported environmental packages  as well as the introduction of new checklist items specific to these environments. A checklist consisting of 93 fields from several disciplines including geology, geochemistry, petrophysics, reservoir engineering, and production chemistry has been compiled Table . Some ofAmongst the different minimal information standards mentioned above, the MIMS and MIMARKS survey sequence specifications are probably the most relevant ones for HCR-related studies as the majority of these studies involve single gene  or whole metagenome surveys. As such, in addition to the HCR environmental package, the MIxS-HCR extension also includes a subset of the MIxS checklist containing MIMS and/or MIMARKS survey fields (depending on the study)  describing hydrocarbon-rich environments which are not covered under the HCR term. These include anthropogenic hydrocarbon occurrences (e.g. oil and gas production systems) as well as surface and seabed hydrocarbon occurrences  Fig. . Many ofThe newly proposed MIxS-HCR minimum information standard provides the foundation for consistent capture and reporting of valuable contextual information derived from studies pertained to hydrocarbon resources. Its first release has already gained approval by the GSC board and will be incorporated in INSDC’s sequence submission process. A web forum has also been set up to promote MIxS-HCR future improvements and extension to cover a wider range of hydrocarbon occurrences. Active involvement of the research community and adoption of the MIxS-HCR standard are key elements to the success of this initiative.


Experts representing the International Organization for Standardization (ISO), the International Electrotechnical Commission (IEC), the International Organization of Legal Metrology (OIML), and the International Bureau of Weights and Measures (BIPM), met at the International Bureau of Legal Metrology in Paris, October 1–3, 1986, to initiate the development of a guidance document for the treatment and reporting of measurement uncertainties.The need for such a guidance document has been long felt throughout the international measurement community. In 1980, the BIPM convened a meeting of experts from eleven national measurement laboratories for the purpose of arriving at a uniform and generally acceptable way of assigning uncertainties to measurement data. This BIPM Working Group on the Statement of Uncertainties agreed on a recommendation (Annex 1) which was subsequently adopted by the International Committee of Weights and Measures (CIPM) in October 1981 (Annex 2). The Recommendation consists of five points which provide a general philosophy for reporting uncertainties. In large part, the points are more in the nature of a briefly outlined approach, rather than an explicit specification of algorithms and methods. At the time of the formulation of the Recommendation, it was believed that many further details would have to be addressed and resolved before the recommended approach could be routinely, uniformly, and widely used.In the past year, the CIPM referred this matter to the ISO since it was felt that this was a more logical international body for trying to achieve agreement and uniformity on the statement of uncertainties within international standardization and metrology organizations. Responsibility was assumed by the ISO Technical Advisory Group (TAG) 4 since it serves as a coordinating mechanism for addressing measurement issues of common interest to the two worldwide standardization bodies, the ISO and the IEC, and the two worldwide metrology organizations, BIPM and OIML. The present working group (ISO TAG 4/WG3) was thus constituted under the terms of reference of ISO TAG 4, and consists of 11 experts nominated by the represented organizations. The Chairman of the working group is Dr. R. Collé of the National Bureau of Standards.to develop a document based upon the recommendation of the BIPM Working Group on Uncertainty which provides guidance on the expression of measurement uncertainty for use within standardization, calibration, laboratory accreditation and metrology services. The purpose of such guidance is to promote full information on how uncertainty statements are arrived at and to provide a basis for the international comparisons of measurement results.The terms of reference of the working group, as defined by the ISO TAG 4, is:At the October meeting, the TAG 4 working group concluded that its task is to produce a document which will be firmly based on the BIPM recommendations of 1980, but will be more specific and usable. The document will be directed towards two primary user groups: national primary standards laboratories and secondary level standards and calibration laboratories. The working group meeting resulted in the completion of a detailed outline for the organization and general contents of a guidance document, as well as a schedule and plan for producing a draft of this document. It is envisaged that a first draft of the document will be discussed at the next TAG 4/WG 3 meeting in May, 1987.A complete report of the first meeting may be obtained from Mr. David E. Edgerly, Standards Management Program, Building 101, Room A625, National Bureau of Standards, Gaithersburg, MD 20899.1 The uncertainty in the result of a measurement generally consists of several components which may be grouped into two categories according to the way in which their numerical value is estimated:those which are evaluated by statistical methods,those which are evaluated by other means.There is not always a simple correspondence between the classification into categories A or B and the previously used classification into “random” and “systematic” uncertainties. The term “systematic uncertainty” can be misleading and should be avoided.Any detailed report of the uncertainty should consist of a complete list of the components, specifying for each the method used to obtain its numerical value.2 The components in category A are characterized by the estimated variances, si) and the number of degrees of freedom, vi. Where appropriate, the estimated covariances should be given.3 The components in category B should be characterized by quantities jU like standard deviations. Where appropriate, the covariances should be treated in a similar way.4 The combined uncertainty should be characterized by the numerical value obtained by applying the usual method for the combination of variances. The combined uncertainty and its components should be expressed in the form of “standard deviations.”5 If, for particular applications, it is necessary to multiply the combined uncertainty by a factor to obtain an overall uncertainty, the multiplying factor used must always be stated.The Comité International des Poids et Mesures–the need to find an agreed way of expressing measurement uncertainty in metrology,–the effort that has been devoted to this by many organizations over many years.–the encouraging progress made in finding an acceptable solution, which has resulted from the discussions of the Working Group on the Expression of Uncertainties which met at BIPM in 1980.Considering–that the proposals of the Working Group might form the basis of an eventual agreement on the expression of uncertainties,Recognizes–that the proposals of the Working Group be diffused widely;–that BIPM attempt to apply the principles therein to international comparisons carried out under its auspices in the years to come:–that other interested organizations be encouraged to examine and test these proposals and let their comments be known to BIPM;–that after two or three years BIPM report back on the application of these proposals.Recommends


Editorial on the Research TopicVestibular MigraineVestibular migraine (VM) is now the accepted name for vestibular symptoms that are causally related to migraine. The International Headache Society and the International Bárány Society for Neurootology together have developed a consensus document with diagnostic criteria for VM , but theFor those that acknowledge the existence, VM is considered the most common cause of recurrent spontaneous vertigo attacks. It has a lifetime prevalence of about 1% and a one-year prevalence of 0.9% in the general population  and accoThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Akashiwo sanguinea bloom. The bloom altered the overall community functional structure. Genes that were significantly (p < 0.05) increased during the bloom included carbon degradation genes and genes involved in nitrogen (N) and/or phosphorus (P) limitation stress. Such significantly changed genes were well explained by chosen environmental factors . Overall results suggested that this bloom might enhance the microbial converting of nitrate to N2 and ammonia nitrogen, decrease P removal from seawater, activate the glyoxylate cycle, and reduce infection activity of bacteriophage. This study presents new information on the relationship of algae to other microbes in aquatic systems, and provides new insights into our understanding of ecological impacts of phytoplankton blooms.Phytoplankton blooms are a worldwide problem and can greatly affect ecological processes in aquatic systems, but its impacts on the functional potential of microbial communities are limited. In this study, a high-throughput microarray-based technology (GeoChip) was used to profile the functional potential of free-living microbes from the Xiamen Sea Area in response to a 2011  Phytoplankton are the most important primary producers in marine systems and can accumulate to bloom proportions from eutrophication, especially in coastal waters. Blooms dramatically change seawater systems through their efficient autotrophic processes and the exudation of dissolved organic matter2347In the phytoplankton bloom area, bacterial abundance increases greatly41011in situ hybridization1720Studies of the influence of phytoplankton blooms on marine ecosystems have primarily focused on the changes of other microbial communities as revealed by 16S rDNA clone libraries14seobacterThe influence of phytoplankton bloom on the biochemical process in the water system is limited, except the sulfur cycle. Phytoplankton in ocean plays an important role on global sulfur cycle by producing massive quantities of dimethylsulfoniopropionate (DMSP)To obtain a more comprehensive understanding of changes in community function, functional gene arrays (FGAs), microarrays that contain functional gene probes, can be used to simultaneously detect key genes involved in multiple microbial functional processes. The most comprehensive FGAs are the GeoChip arrays. GeoChip 4.2 contains 107,950 probes for genes involved in cycling of C, N, S, P and metals, virulence and antibiotic resistance, the biodegradation of environmental contaminants, stress responsesA. sanguinea bloom. Specifically, we aimed to address the following questions: (i) does the bloom significantly change the microbial community functional potential? (ii) what functional processes are influenced by the bloom? and (iii) are there specific correlations between the A. sanguine population and the functional potential of the associated microbial communities? Our results showed that this bloom significantly altered overall community functional structure and promoted organic carbon degradation, ammonium accumulation and decreased P removal; there were statistical correlations between A. sanguine abundance and the functional potential of the microbial community. This study provides new insight into our understanding of the ecological impacts of phytoplankton blooms on microbial communities.In this study, we used the GeoChip to functionally profile free-living microbial communities in response to an A. sanguinea bloom  and stored at −70 °C until analysis.Samples were collected along the Xiamen coast at sites within  or near  the ea bloom , during For RNA and proteins, sampling procedure  could easily induce their expression change36GeoChip 4.2 was used in this study and sample preparation and hybridization were performed as previously described3138http://ieg2.ou.edu/NimbleGen/batch_upload.cgi) and the data was processed as previously describedp < 0.05 was adopted for all comparisons. Detrended correspondence analysis (DCA) was used to compare the different functional gene communities, and canonical correlation analysis (CCA) was used to link microbial communities to environmental variables. Variation partitioning analysis (VPA) was conducted to examine the contribution of environmental factors in influencing functional gene in the CCA analysis. R statistics were performed running the Vegan (v. 1.15-1)Hybridization data is available at the Institute for Environmental Genomics, University of Oklahoma  were determined as previously describedThe A1 and H1 were bloom and control site, respectively . The watA total of 14171 probes for 575 genes were detected across all samples, including: 1470 probes (39 genes) for C cycling, 1108 probes (17 genes) for N cycling, 196 probes (3 genes) for P cycling, 492 probes (11 genes) for S cycling, 2578 probes (45 genes) for stress response, 85 probes (32 genes) for bacteria phage and 594 probes (13 genes) for virulence .p > 0.05). However, on BP2, the functional gene diversity in the bloom area was significantly (p < 0.05) higher than that in the control area, while evenness (p < 0.05) lower than in the control area. The greatest percentage of unique genes (18.1%) was found in the day 1 bloom samples. Days 3, 4 and 5 had 10.2, 14.8 and 16.5% unique genes (data not shown).The number and relative abundance of functional genes detected were used to calculate the Shannon–Weiner index (H’) and the Simpson Evenness index (E). For most days, neither the difference in the overall microbial functional diversity  nor the evenness  was signp < 0.01) differences in the functional gene structures of the bloom and control areas based on MRPP analysis on days 1, 3, 4, and 5. And the difference for the total was greater since the p values were all less than 0.05 based on Adonis, Anosim and MRPP analyses. So, the differences were more pronounced between bloom and control areas than over time. The DCA ordination plot (Dissimilarity tests  showed tion plot  shows blp < 0.05) different from control samples (p < 0.05) changed during the bloom. Hierarchical cluster analysis of these 288 genes indicated that samples were well separated by site condition , assimilatory N reduction , dissimilatory N reduction (napA and nrfA), nitrification (amoA) and nitrogen fixation (nifH); the 2 genes were involved in ammonification (gdh and ureC). The signal intensity changing pattern of those genes was shown in the simplified N cycling diagram (dsrB for sulfite reductase.There were more N cycling related genes in Bloom Group (11 genes) than Control Group (2 genes), and the 11 genes were involved in denitrification ( diagram . This suBloom Group had more stress genes related to N limitation (1 to 0) while Control Group had more related to P limitation (1 to 5). Bacteriophage genes involved in lysis and replication were high in Control Group (5 genes) than Bloom Group (2 genes), indicating the bloom might decrease viral infection of bacteria.a (CHLa) and total algal density (TALG).The 288 significantly changed genes represented the main microbial functional responses to this bloom. To test how environmental variables contributed to the functional gene differences, CCA was performed using all 288 genes and measured seawater and phytoplankton properties , includiIn the CCA ordination plot , the firVariation partitioning analysis (VPA) was conducted to test the seawater properties  and phytoplankton (CHLa and TALG) in influencing the significantly changed genes in the CCA analysis . The seap < 0.05) correlations with A. sanguinea density; genes within C, N, S, P cycling and stress categories are shown in AceA-isocitrate lyase, pectinase and phenol oxidase) showed significantly positive correlations with A. sanguinea while a gene for methane monooxygenase showed an inverse correlation. Four genes  for assimilatory N reduction and denitrification were significantly correlated with A. sanguinea. The positive correlation with nirS and nosZ suggest that this bloom enhanced the denitrification potential of the free-living microbes. CysJ and dsrA for sulfite reductase showed higher relative abundances in bloom samples and had significantly positive correlations with A. sanguinea. This suggests that the sulfite reduction potential was enhanced during the bloom. The P cycling gene ppk had a lower relative abundance in the bloom and showed an inverse correlation with A. sanguinea.Among the significantly changed genes, 67 showed significant  and NO3− (dissimilatory reduction of nitrate to ammonium), nitrogen fixation, and ammonium production. N limitation stress genes were among the bloom enriched genes, suggesting that N consumption in this field was quite large and that extraneous N input and biological N fixation did not meet demand. N limitation could be one reason for the subsequent bloom termination. In addition, the bloom may have increased the transfer potential of nitrate to N2 by enhancing the intensity of denitrification related genes . The total nitrogen dynamic in mixed cultures of algae and other microorganisms hasn’t been reported. One report indicated that denitrification increased and total nitrogen concentration decreased 25.97–28.16% in Microcystis aeruginosa culturesMicrobial communities within the bloom were enriched in N and P cycling genes, suggesting these elements are particularly important in this environment. Changes in many genes suggested the N cycling potential was accelerated in the bloom, especially for utilization of NOA. sanguinea. Raphael et al.3− in single substrate experiments. Similar results were obtained for N-specific uptake rates of natural assemblages dominated by A. sanguinea in CaliforniaA. sanguinea. The production potential of ammonium from N2 and nitrate might be enhanced in the free-living microbial community by enhancing the intensity of nifH, nasA, nir, nirA, napA and nrfA genes, which might have contributed to the duration of this bloom. This potential beneficial effect for the algae from the microbial community is in contrast to other studies that have observed an inhibition or lethality of bacteria and phage towards algae during blooms4250515253Little is known about nutrient utilization by A. sanguinea(ppk) catalyzes the formation of polyphosphate (polyP), a process by which microbes could remove P from waterppk as A. sanguinea increased may be conducive to the bloom duration.The control site contained more P limitation stress genes, suggesting P limitation may have prevented bloom formation in the control site. In addition, there was more DIP in the bloomChanges in some of the functional genes detected in this study suggest that the bloom had surprising influences on the microbial communities.et al.There were fewer lysis and replication related genes of bacterial phage in the bloom suggesting that phytoplankton blooms might reduce viral infections in bacteria. Studies on the infection dynamics of bacteriophage influenced by algal blooms are limited, but Maranger AceA gene which encodes isocitrate lyase showed a significant positive correlation with A. sanguinea, indicating the glyoxylate cycle was more active in bloom-bacteria than in non-bloom bacteria. Rinta-Kanto et al. found bloom bacterioplankton transcribed more copies of genes for organic acid utilization, but transcripts for isocitrate dehydrogenase were less abundant in the bloom transcriptomeThe A. sanguinea bloom did not change the free-living microbial function potential as greatly as expected. Community differences were more pronounced between bloom and control areas than over time. Most significantly changed genes showed an increase in intensity within the bloom, especially for the genes related to carbon degradation. N or P availability appeared to be important for both start and termination of the bloom. The microbes in the bloom appeared to contribute to bloom duration by promoting ammonium accumulation and decreasing the competitive adsorption of biological Pi removal from water. In addition, the bloom may reduce infection activity of bacteriophage by decreasing the intensity of replication and lysis related genes.We applied the GeoChip to the study of a phytoplankton bloom and found that the 2, reduced infection activity of bacteriophage, and that isocitrate was actively shuttled through the glyoxylate cycle in bacteria. These findings provide new research directions for the study of bloom dynamics and provides a deeper insight into the relationship between bloom algae and other microbes in marine systems. Continuing research is focused on RNA studies.Although this study was based on DNA, which provides indirect ecological evidence of functional potential, results were consistent with transcription studies in this field. Changes in gene abundance suggest that the bloom enhanced microbial ammonia accumulation and the transfer of nitrate to NHow to cite this article: Yang, C. et al. A comprehensive insights into functional profiles of free-living microbial community responses to a toxic Akashiwo sanguinea bloom. Sci. Rep.6, 34645; doi: 10.1038/srep34645 (2016).


Educators struggle to develop a journal club format that promotes active participation from all levels of trainees. The explosion of social media compels residencies to incorporate the evaluation and application of these resources into evidence-based practice. We sought to design an innovative “flipped journal club” to achieve greater effectiveness in meeting goals and objectives among residents and faculty.Each journal club is focused on a specific clinical question based on a landmark article, a background article, and a podcast or blog post. With the “flipped” model, residents are assigned to prepare an in-depth discussion of one of these works based on their level of training. At journal club, trainees break into small groups and discuss their assigned readings with faculty facilitation. Following the small-group discussions, all participants convene to summarize key points. In redesigning our journal club, we sought to achieve specific educational outcomes, and improve participant engagement and overall impressions.Sixty-one residents at our emergency medicine program participated in the flipped journal club during the 2015–2016 academic year, with supervision by core faculty. Program evaluation for the flipped journal club was performed using an anonymous survey, with response rates of 70% and 56% for residents and faculty, respectively. Overall, 95% of resident respondents and 100% of faculty respondents preferred the flipped format.The “flipped journal club” hinges upon well-selected articles, incorporation of social media, and small-group discussions. This format engages all residents, holds learners accountable, and encourages greater participation among residents and faculty. Journal club is an essential component of graduate medical education, used to teach trainees how to critically appraise the literature and integrate evidence-based medicine into practice.At our emergency medicine (EM) residency program, we sought input from residents and faculty to identify strengths and weaknesses of our traditional format in order to prioritize goals and objectives for journal club. We then designed a “flipped journal club” to implement for one academic year prior to program evaluation.Our residency consists of a postgraduate-year (PGY) 1–3 EM program, as well as two five-year combined training programs in EM/IM and EM/FM, for a total of 61 residents. Our curriculum includes a monthly journal club held at a restaurant, conference center, or faculty member’s home. All residents on EM rotations are required to attend.Our residents traditionally were assigned in groups of three to lead a journal club. This group would select articles based on the curriculum topic for that block, with approval from a faculty member, and distribute the articles to all participants. On the evening of journal club, the designated residents would each present their chosen article. This was followed by an open discussion among residents and faculty.Feedback from residents and faculty  indicated numerous drawbacks with this format. Some residents were nervous about speaking in front of a large audience. Much of the audience was ill-prepared and disengaged, and the ensuing discussions were generally led by a handful of extroverted residents. Furthermore, the relevance and quality of selected articles was inconsistent. Lastly, we found that residents were using various social media sources rather than reading original research.We developed a new journal club format to effectively engage all learners using three methods: a focus on specific topics associated with landmark articles, incorporation of social media resources, and division into small groups for discussion. These changes were reflective of flipped-classroom models being integrated into other parts of the curriculum.We designated a senior faculty member (an associate program director [APD]) and a chief resident as a leadership team to champion the redesigned format. Each month, they select an important controversial clinical topic with an associated “landmark article.” A group of three residents, comprised of a PGY-1, PGY-2, and PGY-3, 4, or 5, are selected by the chief resident based on schedule availability. The resident group reviews the landmark article and selects an accompanying background article and a social media piece that is relevant to the topic and freely accessible to the residents. These selections require approval by the leadership team.The articles and the social media selection are distributed to all residents and faculty one week prior to each journal club. The three residents must additionally prepare a “facilitator’s guide” with summaries and discussion points . The leaOn the evening of journal club, all residents are divided into small groups , composed of learners from all levels of training and facilitated by a faculty member. Within the small groups, interns are expected to discuss the background article. The PGY-2 residents present their analysis of the landmark article, followed by the senior residents’ critique of the social media piece. Residents truly lead the discussion, while the faculty facilitators pose questions and provide oversight. Following the small-group discussions, everyone reconvenes to openly summarize and debate key points.Following each session, the three designated residents create a summary of the main discussion points to be electronically distributed to all faculty and residents .In redesigning our journal club, we sought to achieve specific educational outcomes, and improve participant engagement and overall impressions. We initiated the flipped journal club in July 2015, and following one full academic year we evaluated our educational outcome measures using an anonymous online survey . The volThe Christiana Care Institutional Review Board determined that the program evaluation survey was exempt under an educational curricula waiver.Of the 61 residents who received the survey, 43 responded (70%). Ten were interns and, thus, unable to compare formats. Of the 16 faculty, nine responded (56%), with all having experienced both formats. Four of the seven faculty who did not respond attended journal club only once or not at all during the study period.We first asked participants to select what they hoped to gain from journal club from a list of optionsOver 90% of the residents reported that they more often arrive prepared for the new format, and greater than 95% responded that the flipped journal club better allowed them to contribute to the discussion. The final question of the survey asked residents if we should continue with the flipped journal club and 95% responded positively. All faculty respondents felt that we should continue to use the new format.In the ever-changing landscape of medical education, innovative techniques for engaging learners of all levels of training must continue to be developed and refined. Our innovation uses small groups, focused clinical topics with a related landmark article, and medical education through social media. This format was preferred by both trainees and faculty in regard to personal goals, educational objectives, and overall impressions. The changes to our format reflect the concepts of the “flipped learning” model in which the four pillars include: a flexible environment, learning culture, intentional content, and a professional educator.10flexibility in the pace and the focus of the discussion and increases the level of active participation. In this learner-centered setting, residents drive the discussion and stimulate insightful conversations that create opportunities for more senior participants to teach and share experiences. An experienced team intentionally chooses impactful articles that merit in-depth review. The works selected by the designated resident teams are vetted to verify relevance and accessibility. Previous literature indicates residents endorse open-access medical education as their most beneficial source of education, yet also reports infrequent review of the references or quality of evidence.professional educators the opportunity to fulfill this duty.The small-group format allows There are potential barriers to implementation of this format. Faculty may need training in small-group facilitation. Continued leadership is necessary to maintain high standards in article selection, review social media resources, and hold residents accountable. In our experience, the designated chief resident and faculty spent 1.5–2 hours per month reviewing articles and coordinating with the assigned teams, generally via email. Our program assigns one chief resident to assist with organization and implementation of our educational curriculum, and journal club leadership naturally fits into that individual’s roles and responsibilities. This structure may not be in place at all programs.This innovation may be feasible for other EM programs, as well as other medical specialties. The similarities to the flipped-classroom model used in conference formats will be familiar to most educators. Some programs may also consider adopting individual components of this format to augment their current journal club. Future research may include evaluation of the feasibility of this model in other programs and studying the effectiveness of each individual intervention (such as a pre-post effectiveness study).Study limitations include a small sample size of participants from a single, large EM residency program as well as a relatively low response rate to the survey, which has potential to bias the results. Validity evidence was not collected for the survey. Potential confounders inherent to the observational nature of our study include changes in core faculty and residents from one year to the next. Furthermore, the study was not designed to assess the impact of the individual components of the flipped journal club model. Lastly, the measured response to our intervention was limited to a post-implementation survey of self-reported goals and objectives obtained after the study intervention.The “flipped journal club” hinges upon well-selected articles, incorporation of social media, and small-group discussions. The format is meant to promote accountability and create an atmosphere that encourages dialogue among all participants. In our study, the modifications to journal club improved the sense of achieving both personal goals and targeted educational objectives, and was strongly favored by residents and faculty.


Two decades ago, professors Saleh M. Al-Deeb, Khalaf Al-Moutaery and Basim Yaqub launched the neurosciences bulletin as the specialized journal in the kingdom and the Arab world due to the growing interest in neurosciences research and publication. The journal started as the official bulletin of the Neurosciences Department, Prince Sultan Military Medical City (PSMMC) as a twice-yearly publication including articles, and abstracts from related meetings. After that, it became a quarterly publication, and continues to be well-respected peer review journal and services its mission as a mean to disseminate accessible, up to date medical information for clinical neurosciences and promote neurosciences research locally and internationally.With our devoted and hardworking editorial team the journal achieves successful steps and improvements. In 2000, the journal established the journal website which now had 124,813 hits, where readers can view, read and download the articles PDF. In 2006 the journal received an approval for indexing by the Thomson Scientific (formerly known as Thomson ISI) and following that the journal indexed and included in MEDLINE and that the English abstracts will be included using PubMed in July 2010. In 2016, we start including the journal in the Pub Med Central, and increasing the journal visibility in many databases.After years of dedicated editorship, the journal associate-editors Dr. Sonia khan and Dr. Mohammed Al-Zawahmah left the journal in 2016 after many accomplishments. In this regard, we would like to introduce ourselves to the readers as associate editors of neurosciences journal. Dr. Waleed Khoja is Deputy Director of the Neurology at the PSMMC; founder and past president of Saudi Stroke Association. Dr. Khaled hundallah is pediatric neurology consultant at the PSMMC and assistant professor at Al-Imam Muhammad Ibn Saud Islamic University.To set our strategic plan, we decided to involve our target audience. An electronic survey was distributed and posted in the journal website. We received a total of 120 respondents. More than 60% of our respondents spend 2 hours and more weekly reading the medical journal and 65.83% find the journal important in their field. For the journal website, 39.47% of the respondents rate the journal website 3 out of 4. For the journal logo, 55% find out the logo represents the journal. However, improving the journal website, decreasing the processing time and establishing an electronic submission system, and increasing the journal visibility are the key points our audience look for.To satisfy the needs, demands and the expectations of our target audience the following steps were initiated. To increase the visibility of the journal, we invite all the local societies related to the neurosciences to sign an agreement and the journal will be the official journal of the society. In return, the society will contribute to the development of the journal scientifically. We also focus on the student corner and encourage the students to submit and publish an abstract of their research. The students’ abstracts will be published in the journal website and indexed in the pub med central. In addition to that, e-journal, the journal website, old issues archives, online submissions systems are all areas in our agenda.As per to a local study conducted by JamjoomIn 2016, we received a total number of 135 manuscripts, with an average rejection rate of 37.8%. Reasons for rejection included papers out the scope of the journal, of low scientific quality, not meeting the requirements of the journal, authors failing to submit the revisions and other necessary requirements, and duplicate publication. However, over 4 issues of 2016, we published a total number of 70 articles which include: 22 originals, 1 editorials, 9 reviews, 11 case reports, 10 clinical note, 3 brief communications, 2 brief report, 4 correspondences, 1meeting highlight, and 4 MCQs. The average processing time from received to acceptance was 2.6 (1-6) months and 3.1 (1-9) months from acceptance to publication and we are pleased to decrease the average processing time frame this year.We extend our thanks to the Editorial and Advisory Board Members for their significant contributions to maintaining the standards of the journal, and we look forward to their important continued role in achieving our goals for 2017. We would like to thank the outgoing Advisory Board member  who had now finished his term and would like to welcome Nader Honjol to the Advisory Board. We also extend our thanks to the outgoing Editorial Board members . We are honored they have agreed to join the board, and their individual contributions will be of great value to the journal. We continue the international diversity of members that the current board offers. We hope all our readers continue to benefit from the published material, and we extend our sincerest thanks to our authors, readers, reviewers, and board members, and wish all a successful year.Waleed KhojaAssociate EditorKhalid HundallahAssociate EditorOur thanks also go to the following reviewers, who have participated in the excellent review of manuscripts and books for the year 2016.Angela VincentAdria ArboixAdria ArboixAhmed H El BeltagiAhmed Osman Abdel-ZaherAjay PanwarAlberto VerrottiAlberto VerrottiAli Hassan RajputAlim P. MithaAnnalisa PastoreBoyd KoffmanBurak KaraaslanCarlotta SpagnoliCelia GodfreyChetan G KChristian BrandtChristian PagnouxChristian UrbanekClement DarlingDan HarrisonDanielle RiceElena Erro AguirreElena Zapata-ArriazaElisa TamburoEmiel CraccoEmilio PeruccaErling TronvikErnane Torres UchoaEzequiel UribeFabrizio SchifanoFahad Abdu BashiriFatih ÇelikelFeng ZhangFlávia de Lima OsórioGro SolbakkenHadeel Al KhameesHai ZhangHermann StefanHiroshi KataokaHiroyuki KobayashiHsiuying WangHung-Yu ChanIoannis KarakisJaime Gállego CulleréJames SieglerJaume RoquerJavier Pablo HrybJermaine dambiJian-min LiuJinn-Rung KuoJosé Roberto WajmanJoseph JankovicJun ZhangJunshi WangKatrin HahnKenneth LiuKonark MalhotraLeonie Walkerluca GallelliLuigi Maria CavalloLuisa Escobar SánchezLynette M. SilvaMajed AlHameedManonita GhoshMaría Frenzi Rabito AlcónMarjo van der KnaapMartha Eva Viveros-SandovalMartha ShiellMasahito MiharaMatias NollMatti IsokangasMeheroz H. RabadiMichael BerginMichael G. YoungMichael SsonkoMiguel Angel Palomero RodríguezMirjana Babic LekoMohammad TariqMohammed Hasan BangashMohammed JanMyoung Soo KimNadia El KadmiriNisa Cem ÖrenOktay AlginOve SonessonPasquale StrianoPatricia Rzezakpuneet mittalR.Van den BergRaidah Saleem AlbaradieRaúl PelayoRayaz MalikRichard BrightRiyadh alokailiRobert DumaineRohan.R.MahaleRoland ThijsSamir R. BelagajeSandeep TalariSandra Orozco–SuárezSanja PekovicSeyed Ali NabavizadehShanshan JiangSlaven PikijaSotirios GiannopoulosSushil SharmaSuzannah IadarolaTayfun HakanThomas HaysTracy MelzerWolfgang HerrmannWouter VanderplasschenYuBao JiangYun HwangZaida Chinchilla-RodríguezZhongheng Zhang


Medicine and innovation: these terms have always been inseparable. In each phase of medical history, the continuous expansion of medical knowledge and the development of technical solutions went hand in hand. Interdisciplinarity also played an important role from the beginning. Integrative Medicine has become the driving force behind the latest innovations in medicine over recent years.Medicines, launched in 2014, is being published by a Swiss publisher from Basel. Its objective is the publication and wide electronic dissemination of innovative and consequential research in all types of medicine. Medicines will publish refereed articles of high quality for a professional audience. The journal thereby aims to promote communication between medical researchers. The Swiss publisher will host the journal’s website, which will be accessible to readers free of charge. The main topics covered include, but are not limited to:History of medicine; medicine and philosophy.Proven or evidence-based non-conventional  medicines or ethno-medicines, natural product-based medicines, nutraceuticals.The chemistry, medicinal chemistry, structure–activity relationships and computer aided modeling based on naturally occurring agents, either therapeutic or potentially therapeutic.Conventional medicine: both established and emerging natural product-based therapeutic agents and treatments.The journal vice versa.Therefore, we invite investigators and researchers to submit original research articles as well as review articles associated with medicine. We are particularly interested in articles dealing with eastern/oriental medicine and its possible contributions to western medicine—and Medicines, this annual World Congress [A very successful example of such a cross-over approach is high-tech acupuncture . Only a Congress  shall seContributors to the journal should be experts and scientists from all over the world. They are welcome to send their most recent research and describe current scientific medical trends.This is a cordial invitation to all of you to submit high-level articles to our new journal.


Results of the demographic health surveys since 2004 show that the rate of stunting decreased by only 1.5 percentage points per year  , Canada; Swedish International Development Cooperation Agency (Sida) and the Department for International Development (UKAid). We gratefully acknowledge these donors for their support and commitment to icddr,b's research efforts.The authors declare that they have no conflicts of interest.TA conceptualized and wrote the first draft of the paper. MH, MM, NC and SA substantially contributed by revising the paper.


Fusobacterium most associated with inflammatory influences and Bacteroides most associated with tolerogenic influences. We validated these effects using PBMCs stimulated with various sets of microbiome peptides. “Tolerogenic” microbiome peptides elicited IL-10 production, “inflammatory” peptides elicited mixed IL-10/IFNγ production, while microbiome epitopes homologous to self were completely unreactive for both cytokines. We also tested the sequence similarity of cockroach epitopes to specific microbiome sequences derived from households of cockroach allergic individuals and non-allergic controls. Microbiomes from cockroach allergic households were less likely to contain sequences homologous to previously defined cockroach allergens. These results are compatible with the hypothesis that microbiome sequences may contribute to the tolerization of T cells for allergen epitopes, and lack of these sequences might conversely be associated with increased likelihood of T cell reactivity against the cockroach epitopes. Taken together this study suggests that microbiome sequence similarity influences immune reactivity to homologous epitopes encoded by pathogens, allergens and auto-antigens.The microbiome influences adaptive immunity and molecular mimicry influences T cell reactivity. Here, we evaluated whether the sequence similarity of various antigens to the microbiota dampens or increases immunogenicity of T cell epitopes. Sets of epitopes and control sequences derived from 38 antigenic categories  were retrieved from the Immune Epitope Database (IEDB). Their similarity to microbiome sequences was calculated using the BLOSUM62 matrix. We found that sequence similarity was associated with either dampened  or increased  likelihood of a peptide being immunogenic as a function of epitope source category. Ten-fold cross-validation and validation using sets of manually curated epitopes and non-epitopes derived from allergens were used to confirm these initial observations. Furthermore, the genus from which the microbiome homologous sequences were derived influenced whether a tolerogenic versus inflammatory modulatory effect was observed, with  Mycobacterium tuberculosis [Several studies indicate that similarities between related and even phylogenetically distant protein sequences can influence host immune reactivity. This phenomenon, termed molecular mimicry, was originally described in Fujinami et al.  and is hrculosis  certain A large body of literature indicates that the host microbiome has a profound influence in shaping and modulating host reactivity and immune functioning, primarily at the level of innate and adaptive immunity –9. In thPrevious analyses  suggesteHere we expanded this analysis to a broader set of epitope sources, to consider 11 different allergens, 18 viral, 4 bacterial, 1 eukaryote, and 4 autoimmune and inflammatory antigens. We found that while microbial exposure might be, in certain cases, involved in blunting potentially harmful antigen/allergen-specific immune responses, in other cases microbial sequence similarity actually increased the likelihood of immunogenicity for pathogen-derived sequences.www.iedb.org). The IEDB harvests and curates epitope data from published literature. The database does not have any consistent information related to the vaccination status of subjects. To ensure consistency in our study, all peptide sequences analyzed were 15 residues long, and any epitope longer than 15 residues was represented by all possible 15-mer components, which were then utilized to calculate their similarity to the 15-mers derived from human microbiota sequences , negative peptides were derived using the sequences of the antigens found in the category’s epitope sets. The 15-mers derived from an antigen were compared to epitope sequences from the same antigen, and in order to be considered a negative peptide it was required that the 15-mer did not share more than 9 continuous residues with a given 15-mer epitope sequence. Further, an additional set of 15-mer epitopes found in the IEDB  that werex vivo or in vitro), using ELISPOT assays measuring various cytokines (primarily IFNγ and IL-5). In all cases, the identification of these epitopes is described in peer reviewed reports [We also studied sets of dominant epitopes, derived from antigens and allergens from various target indications . Namely, reports –16; in shttp://www.hmpdacc.org/HMRGD/) [Human microbiome sequences were retrieved from the Human Microbiome Project (/HMRGD/) , 18, wheTo estimate the similarity between two given sequences we used a BLOSUM matrix, which is based on the relative frequency of amino acids and their substitution probability between sets of aligned proteins, and contains log-odds scores for each of the possible substitutions of the 20 amino acids. In this study we used the BLOSUM62 matrix , as prevFor each sequence in the epitope and non-epitope sets, similarity scores of the best 15-mer for each epitope and non-epitope sequence were recorded. To ensure the consistency of our results, we performed a ten-fold cross-validation for each category. Epitopes were randomly divided into ten groups with their associated non-epitopes and a cross-validation was performed using the ten groups.16S rRNA-based microbiota profiles derived from house dust from residences in which children developed atopy  or did not develop atopy  were useThe predicted genomic content of both pro-allergic and non-allergic house dust microbiota largely represented overlapping KOs. Therefore, KOs were separated into three groups: 1) those unique to pro-allergic house dust microbiota (41), 2) those unique to non-allergic house dust microbiota (150), and 3) the remaining KO shared between pro-allergic and non-allergic microbiota. Next, we derived all the possible 15-mers from the protein sequences found in each of these groups of KOs, and a list of 15-mers was generated for each of the main groups. As peptides from the pro-allergic and non-allergic sets could potentially be found in both sets, we decided to compare the 15-mers per KO from each group against the other two remaining groups, to ensure the uniqueness of the sets. As an example, each 15-mer of the KOs from the non-allergic group that was found common in the shared or allergic group was discarded from the analysis.As the similarity scores of epitopes and non-epitopes per category were not normally distributed, a Kruskall-Wallis test was used to identify significant differences in the median similarity scores between epitopes and non-epitopes. Standard Chi-square testing was used to assess significance of difference in frequency distribution of different peptides in different genera, with appropriate Bonferroni corrections. Statistical analyses were performed in R studio and Graph Pad Prism  software.th percentile were excluded (979 peptides were predicted as binders). Similarity to the microbiome was estimated by BLOSUM score calculations as described above. Peptides were purchased from Mimotopes  and/or A and A  as crude material on a small (1 mg) scale. Individual peptides were resuspended in DMSO to a final concentration of 20 mg/ml. Peptides were pooled (10 peptides per pool) according to their category. Each pool was at a final concentration of 2mg/ml per peptide.The hits per category were sorted according to descending BLOSUM scores. From the 14 inflammatory categories, defined as instances where epitopes were less similar than non-epitopes to the microbiome, the top ten hits for each category were selected for analysis. In the case of nine tolerogenic categories, defined as instances where epitopes were more similar than non-epitopes to the microbiome, the top ten hits from their respective epitope sets were likewise also selected, as non-epitopes will inherently produce negative results in immunogenicity assays. Hits from the Rheumatoid Arthritis tolerogenic category were not included in this analysis because too few hits were available. The set of peptides selected for experimental testing can be found in We recruited 10 healthy adults from San Diego, USA. All participants provided written informed consent for participation in the study. This study was performed with approvals from the Institutional Review Board at La Jolla Institute for Allergy and Immunology (protocols VD-101 and VD-071). No detailed clinical history including vaccination records is available for the participants. PBMC  were isolated from collected blood or leukapheresis by density gradient centrifugation, according to manufacturer’s instructions. Cells were cryopreserved in liquid nitrogen suspended in FBS containing 10% (vol/vol) DMSO.5 cells/well with peptide pools (2μg/ml). PHA (10μg/ml) was used as a positive control and medium containing 0.1% DMSO (corresponding to the percentage of DMSO in the pools) was used as a negative control. Plates were cultured for 24h at 37°C and developed using anti-human IFNγ  and anti-human IL-10 . Fluorescent spots were counted by computer-assisted image analysis using an AID iSPOT ELISPOT reader . To be considered positive a peptide pool response had to match all of three different criteria: 1) elicit at least 20 net spot-forming cells (SFC) per 106 PBMC, 2) p≤0.05 by Student’s t-test or by a Poisson distribution test, and 3) stimulation index ≥2. No cells producing both IL-10 and IFNγ were detected.After overnight resting of the PBMCs, the response to peptide pools was measured by IFNγ and IL-10 dual Fluorospot, according to manufacturer’s instructions. Plates were coated with 5μg/ml anti-human IFNγ , and 10μg/ml anti-human IL-10 . Cells were plated at a density of 2x10https://github.com/LJI-Bioinformatics/HLATyphon).To ensure that the donors represented a diverse set of HLAs four-digit HLA typing  was perfPlasmodium derived epitopes.We previously developed a method to select reference epitope sets  from IEDThe list of epitope sets is shown in To investigate the potential impact of microbiome sequence similarity on epitope reactivity, we next estimated the similarity of each epitope and non-epitope to published microbiome sequences by calculating the maximum BLOSUM score for each of the sets, following the methodology described by Bresciani et al .Aspergillus is represented; the median score is 0.599 for the epitopes, and 0.626 for non-epitopes .Accordingly, we calculated for each epitope in each set the maximum degree of BLOSUM similarity to any sequence contained in the human microbiome sequence set. We then calculated the p-values associated with the epitope set being less similar to the human microbiome than the matching non-epitope. In the context of the present study, this could indicate a tolerogenic influence of the microbiome. An example distribution of the epitope and non-epitope BLOSUM scores can be found in more similar to microbiome sequences . In the remaining fourteen instances, epitopes were significantly more similar than non-epitopes to microbiome sequences . In conclusion, while significant differences between epitope and non-epitopes sets are frequently detected, the differences can be either reflective of a positive or negative influence. In general, allergen epitopes tended to be less similar, with the important exception of allergens of mammalian origin. In the autoimmune sets, diabetes and multiple sclerosis epitopes were less similar, while rheumatoid arthritis and gluten were more similar. In these cases, the microbiome sequences might have an overall “tolerogenic” or “blunting” effect on T cell responses.Clostridiales, Alpha- and Beta-Proteobacteria, and less similar for Plasmodium.RNA viral epitopes, with the exception of Yellow Fever, were more similar to microbiome sequences. In the cases where epitopes are more similar to microbiome sequences the microbiome might exert an “inflammatory” or “priming” influence. Contrasting patterns were seen in the case of DNA viruses, with epitopes from papilloma and Hepatitis B virus epitopes more similar to the microbiome, while epitopes from Adenoviruses and Betaherpes viruses were less similar. Contrasting patterns were also seen in the remaining sets, with epitopes more similar in the case of To further support our results, we performed ten-fold cross-validation experiments. In these analyses, the epitope sets were partitioned in different subsets as described in the methods section. If the significant difference observed was due to sampling biases, we expected that significance would be lost in the partitioned sets. The results in To further validate these observations, we analyzed manually curated sets of dominant epitopes, derived from allergens from Timothy Grass (TG), Ragweed (RAG), house dust mites (HDM), and cockroaches (CR). In general, the epitope sets were selected to encompass the majority of the responses with a limited number of epitopes. We also included manually curated non-epitopes, defined on the basis that these peptides did not induce responses in any of donors tested in the studies in which the positive epitopes were defined. In most cases the more dominant epitopes had more significant p-values. In the case of the IEDB-derived allergen epitope sets, the Ragweed and Cockroach sets did not achieve significance, whereas the dominant sets did. Therefore, we identified ten categories for which a “tolerogenic” influence was detected.The above results indicate that in certain instances microbiome similarity might exert an “inflammatory” influence, while in other instances microbiome similarity might be linked to a “tolerogenic” influence. To investigate this issue further we selected the 14 and 10 categories, for which the significant p-values were noted, to be associated with enhanced “inflammatory” immunogenicity and decreased “tolerogenic” immunogenicity, respectively.Next, we selected for each category the top 10% highest BLOSUM score epitopes for the inflammatory categories and non-epitopes for the tolerogenic categories. This was done considering highest BLOSUM scores of the epitopes in which the epitopes were more similar than non-epitopes; conversely, in the case where the non-epitope were more similar than the epitopes the highest BLOSUM scores of the non-epitopes was considered. For each of the epitope or non-epitope sequences we selected the hits (homologous sequences in the human microbiome) with a score above the threshold determined by the epitope or non-epitope median of max BLOSUM scores for the respective category. We next counted how frequently each microbiome microorganism was represented in these hits. The hits were then mapped back to the genus of origin from the microbiome sequence sets. Finally, we tabulated for each indication the frequency of hits mapped to each microbiome genus and compared them to the frequency of all 15-mers in the whole microbiome dataset.An example of this type of analysis for the “Mammal Allergens” category is shown in We next compiled the results of the analysis outlined above for each of the various epitope categories. Fusobacterium, Streptococcus and Clostridium were enriched in more than 90% of the non-epitopes categories (9/10 or more of the instances). Conversely, the genus Bacteroides was associated with the “inflammatory categories” with Bonferroni corrected significant values in at least 60% of the epitope categories (8/14 or more of the instances). The most remarkable finding was, however, that the same genus tended to be enriched in either “inflammatory” or “tolerogenic” categories, suggesting that no clear inflammatory vs. tolerogenic categories can be established at the genus level. Indeed, the genus Fusobacterium, Streptococcus, Bacteroides, Clostridium, Bifidobacterium and Propionibacterium were enriched in hits from both tolerogenic and inflammatory categories. A complete checkerboard analysis showing the genus and each of the 14 epitope and 10 non-epitope categories, respectively, the frequencies in epitopes or non-epitopes, control set, and associated p-values after Bonferroni correction is shown in In instances where the non-epitopes were most similar to the human microbiome (“tolerogenic” categories), the genera We next reasoned that the fact that similar microbiome categories might be associated with inflammatory or tolerogenic influence is consistent with microbiome-epitope specific T cells existing in the host in a tolerant and quiescent state. Exposure to the cross-reactive pathogen-derived epitopes might result in an inflammatory or tolerogenic outcome depending on the milieu and the co-stimulation associated with each exposure, and the particular microbiome niche occupied by the microbes that encode the homologous epitopes.ex vivo Fluorospot assay measuring IFNγ and IL-10. The microbiome derived epitopes produced a significant amount of IL-10 for both inflammatory and tolerogenic categories . Conversely, significant IFNγ production was only detected in the case of the inflammatory, but not the tolerogenic category (p = 0.006)  were next tested for immunogenicity in human PBMC representing a diverse set of HLAs. From the 10 “tolerogenic”  and 14 “inflammatory” categories, the top ten hits for each category were selected for analysis. Since ten high BLOSUM score peptide hits were selected for each of the 23 categories , this co= 0.006) .These data is consistent with microbiome-epitope specific T cells existing in the host in a tolerant state, associated with constitutive IL-10 production. We further hypothesized that this mode of recognition is distinct from the tolerance induced by self-sequences at the level of thymus education . To address this point, we tested three sets of peptides: 1) The microbiome sequences most homologous to dominant epitopes , 2) the dominant epitopes themselves (n = 230 as above), and 3) self-sequences most homologous to the microbiome  and also predicted to be high affinity promiscuous HLA binders . As showWe hypothesized that in cockroach allergies, where the microbiome is potentially blunting allergen-specific immune responses, differences at the level of the microbiome associated with allergic versus non-allergic individuals might result in a broader allergen-specific T cell repertoire, increasing the potential for allergen T cell recognition and associated immunopathology. To test this, we analyzed 16S rRNA-based microbiota data derived from dust collected from households of cockroach allergic and non-allergic children .The collection of 16S rRNA sequences derived from allergic and non-allergic microbiomes were analyzed according to Okuda et al. . The phyWe next analyzed the set of epitope and non-epitope sequences  describeTo verify this result with a different approach, taxonomy maps, obtained as described by Okuda et al. , for theOur results show that microbial sequence similarity is a relevant variable to be considered when assessing immunogenicity. Sequence similarity might influence and explain downregulation and tolerogenic immune responses versus "inflammatory" immune reactions to potentially cross-reactive antigens/allergens. The data presented herein provides an extension of the concept of how self/non-self cross-reactivity shapes the repertoire of an individual’s reactivity. First, it provides an extension of the concept of immunological tolerance beyond the sequences encoded by the individual host genome. It is well appreciated that T cells recognizing self-sequences capable of binding self MHC are largely eliminated by central tolerance, and those that do escape the process are actually often associated with regulatory activity, leading to the concept of Tregitopes , 28. OurWe emphasize that the mechanism for microbiome-based modulation of immune responses described herein is based on sequence homology between specific microbiome sequences and potential epitope sequences from allergens, pathogens or autoantigens. As such, this is different and distinct from other general means by which the microbiome has been described to modulate immune responses. Bacteria present in the microbiome secrete soluble factors and metabolites and these bioactive compounds affect the function of intestinal epithelium and mucosal immune cells, resulting in production of cytokines and related factors , 30. MicIn addition, and in parallel to general immunomodulatory effects, we propose an analogous mechanism based on sequence similarity and molecular cross-reactivity. Indeed, microbiome antigens would be expected, like any other antigen (self or pathogen), to be processed by host APCs which will result in the generation of HLA bound peptides. Furthermore, these peptides are not expected to be presented in the thymus during early ontogeny and would therefore not be subject to central tolerance, and would thus have the potential of being recognized as non-self epitopes. Yet, under normal conditions these microbiome-derived epitopes are not associated with effector type responses, presumably because of peripheral tolerance mechanisms. We propose herein that these mechanisms controlling and regulating potential responses to HLA class II bound and microbiome-derived peptides generated by antigen processing of the microbiome microorganism must necessarily extend to highly homologous epitopes encoded in other protein sequences of pathogen, allergen or autoantigen origin. We propose that the microbiome influences the host immune reactivity both by general immunomodulatory mechanisms and antigen-specific modulation of immune responses, and that both mechanisms act in parallel and in a potentially synergistic fashion. Microbial sequence similarities in subsequent heterologous T cell responses may have profound clinical implications on several levels: (i) heterologous T cell immunity between microbes and self-antigens may extend the concept that (early life) exposure to certain microbes favors the development of clinical immunological tolerance. In addition to unspecific effects promoted by microbial compounds and metabolites , here weIn our study, we did not assay for IL-4 or IL-5. However, most of the curated allergen epitope sets from the IEDB , and in particular the cockroach epitopes analyzed in more detail here, have been defined as allergen-specific epitopes on the basis Th2 cytokine production (among them IL-4 and IL-5). Assaying for a broader set of cytokines would be of interest, an in this context, also assaying for IL-17 would be of significant interest.One additional point to clarify is whether the distribution of HLA class II influence the responses to antigen peptides. The HLA class II alleles are extremely diverse . The samWe also examined whether microbiome modulation of antigen specific responses would be HLA class II-specific, or would also extend to HLA class I. Preliminary analysis did not detect significant effect in the case of known class I epitopes (data not shown). This result is in agreement with the hypothesis that the effects detected are based on the internalization and presentation of microbiome derived epitopes through the exogenous HLA class II presentation pathway.In the present study, no non-inflammatory, non-tolerogenic peptides were tested. These peptides could represent a useful control since they would be expected to be associated with an intermediate response pattern.Mycobacterium tuberculosis specific T cells appears to be regulated by microbiome sequences, and that antibiotic treatment with the capacity to alter microbiome composition alters most profoundly T cell reactivity against those mycobacterial sequences highly homologous to the microbiome [The fact that microbiome homology might alter and modulate the reactivity of pathogen or allergen specific T cells was at first surprising. However, several studies have demonstrated that sequence homology can modulate T cell responses, in instances such as epitope sequences conserved across different herpes viruses or allergen genera and several others , 34–36. One interesting aspect of the results is that some pathogens were in the “tolerogenic” category. These results were at first unexpected, but it should be noted that the “tolerogenic” label refers to the microbiome homology. In those instances, apparently, the microbiome contains sequences that are highly homologous to the pathogen’s genome, and T cells reactive against those crossreactive epitopes are inactivated potentially by peripheral tolerance. As a result, the epitopes recognized by the host immune system are by default those that do not share as much sequence homology with the microbiome.In our studies, we relied on a rather stringent definition of sequence homology/similarity, utilizing the BLOSUM matrix . It is lThe degree of similarities detected in our studies are certainly influenced by the breadth of metagenomic databases, which have only just started to be developed. Indeed, the Human Microbiome Project database currently represents samples from relatively few, primarily American, individuals and therefore likely underestimates the true diversity of microbial genes. However, our study reports relative differences in degrees of similarity between epitopes and non-epitopes from a given origin. The fact that epitopes are more or less similar to the microbiome than non-epitopes is not likely to be affected by the number of microbiome sequences considered.Fusobacterium, Streptococcus and Clostridium were enriched in more than 90% of the non-epitope categories from “tolerogenic” categories. Conversely, the genus Bacteroides was more frequently associated with “inflammatory categories”. The most remarkable finding was however that the same genera tended to be enriched in both “inflammatory” and “tolerogenic” categories. Indeed, the genus Fusobacterium, Streptococcus, Bacteroides, Clostridium, Bifidobacterium and Propionibacterium were enriched in hits from both tolerogenic and inflammatory categories. These results are compatible with the notion formulated above that microbiome–specific T cells normally found in the peripheral T cell repertoire, depending on the additional milieu and context, might exert either a tolerogenic or inhibitory effect.The genera In conclusion, microbial epitopes may influence the immune responses to antigens of similar sequences, no matter whether the antigens are derived from pathogens, allergens or hosts. This is a key point of interest of our study, however, more studies are necessary to further elaborate on this conclusion, and in particular to clarify the mechanisms involved. These observations have implications for different disease manifestations in humans. The above-mentioned study in the context of antibiotic treatment in tuberculosis illustrates an example of how microbiome homology can alter the pattern of immune responses. The data presented in this study related to microbiome homology and cockroach allergen T cell epitope repertoire in allergic and non-allergic children suggest that exposure to different microbiome compositions can lead to repertoire differences associated with differential disease susceptibility. Whether this type of observation will be expanded to additional disease settings remains to be established, but it is nevertheless possible that differential repertoire compositions influenced by microbiome homology could be exploited for diagnostic or therapeutic applications.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.


The scarcity of human donor organs in terms of availability for transplants is a renowned problem. The high request of organs moves toward an increased use of marginal donors, including organs from old or very old donors usually transplanted into younger recipients. Within the context of orthotopic liver transplants, clinical evidence suggests that livers from aged donors (≥ 70 years) do have function and duration comparable to those achievable with livers from younger donors. Paradigmatic are the cases of 26 octogenarians livers being transplanted between 1998 and 2006, 15 patients out of 26 are currently alive and 2 of those organs being centenarians [i.e. miR‐31‐5p; miR‐141‐3p; miR‐200c‐3p [ a relatively older recipient (Δ age-mismatch average: + 27 years) the expression of these miRs significantly increased in the organ (follow up after graft at 15 ± 7 months). It is interesting that we were not able to document the reverse. Indeed, when a relatively old liver was transplanted into a relatively young recipient (Δ age-mismatch average: - 17 years), the expression of the three above-mentioned miRs did not change (follow up after graft at 10 ± 2 months). On the whole, these observations suggest that in the setting of liver transplantation the aging phenotype can be “transmitted/propagated” more easily than the young phenotype via the body (micro)-environment. Recently, we studied the above mentioned miRs using single-miR real time-RT qPCR on blood serum samples from 34 recipients stratified on the basis of donor liver chronological age. No difference was observed , thus suggesting that the phenomenon previously found was tightly related to the organ itself without miR-specific exocytosis and changes at circulating level, at least for the identified miRs. Thus, many other questions emerge, such as: i) the potentiality of the younger recipient on “rejuvenating” a liver from an old donor, a phenomenon that we did not observe but cannot be excluded in a larger samples and with other more informative biomarkers likely developed in the future; ii) the complexity of the interaction between the biological ages of donors and recipients, where a systemic rejuvenation effect of a young liver on the recipient should be further pursued with adequate tools. Indeed, biological and chronological age can differ substantially and chronological age is becoming an inadequate parameter to describe the health and clinical status of individuals and likely organs [During last years, our team was deeply involved in an Italian national project (PRIN08) to collect biological data to answer the question - why livers from old donors may be successfully used for transplants. The first evidence was a relative low grade of aging signs of liver donors at histological and cytological level, also including the three major proteolytic activities of proteasome, comparing young and old livers . FurtherActually, the biological effect of donor and recipient age‐mismatch is a topic rather neglected despite its great potential, biological and clinical interest, as depicted in In the future the use of bio-engineered organs is expected, but not in a short time and not with cost accessible to everyone. In the meantime, the idea that biological age-mismatch between donor and recipient could modulate the duration of the graft at least until the complete engraftment and eventually the weaning of immunosuppressive therapy or operational tolerance seems extremely challenging. The use of donor-recipient biomarkers of biological ages and their modelling in time series studies could help the prediction of engraftment successful.


Eurosurveillance articles are automatically deposited with PubMed Central (PMC). This will facilitate for authors who have hitherto had to make an author deposit in order for their manuscripts to appear in PMC. Eurosurveillance is a fully participating journal to PMC, described as ‘a free full-text archive of biomedical and life sciences journal literature at the US. National Institutes of Health's National Library of Medicine’ on their website.Since early August 2016, the final version of all Eurosurveillance further supports scientists in complying with public access polices by funders that include peer-reviewed journal manuscript deposition in Open Access archives.With this,


The development of methods to produce nanoscale features with tailored chemical functionalities is fundamental for applications such as nanoelectronics and sensor fabrication. The molecular-ruler process shows great utility for this purpose as it combines top-down lithography for the creation of complex architectures over large areas in conjunction with molecular self-assembly, which enables precise control over the physical and chemical properties of small local features. The molecular-ruler process, which most commonly uses mercaptoalkanoic acids and metal ions to generate metal-ligated multilayers, can be employed to produce registered nanogaps between metal features. Expansion of this methodology to include molecules with other chemical functionalities could greatly expand the overall versatility, and thus the utility, of this process. Herein, we explore the use of alkanethiol molecules as the terminating layer of metal-ligated multilayers. During this study, it was discovered that the solution deposition of alkanethiol molecules resulted in low overall surface coverage with features that varied in height. Because features with varied heights are not conducive to the production of uniform nanogaps via the molecular-ruler process, the vapor-phase deposition of alkanethiol molecules was explored. Unlike the solution-phase deposition, alkanethiol islands produced by vapor-phase deposition exhibited markedly higher surface coverages of uniform heights. To illustrate the applicability of this method, metal-ligated multilayers, both with and without an alkanethiol capping layer, were utilized to create nanogaps between Au features using the molecular-ruler process. In a time when many technological advances are driven by the miniaturization of fabrication methods, much effort has been placed on the development of novel methods to produce nanoscale features with chemical functionalities that go beyond traditional semiconductors –3. RecenThe molecular-ruler process can be employed to form nanogaps between registered metal surface features that have been generated using conventional lithographic techniques such as photolithography or electron-beam lithography  14–24].–24.14–2418214)2·6H2O for 5 min, and C16 for 1 h. 29Although mercaptoalkanoic acid molecules, such as 16-mercaptohexadecanoic acid (MHDA), are the most widely studied molecules used in the molecular-ruler process, this method is inherently versatile through the use of molecules with alternate functionalities –31. Towa4)2·6H2O for 5 min followed by vapor deposition of C16 for 1 h at 80 °C. To overcome this limitation, the vapor deposition of C16 is explored. Interestingly, when C16 is deposited from the vapor phase onto MHDA monolayers to produce Cu-ligated MHDA-C16 bilayers, protruding islands with uniform thickness are observed across the Au{111} substrate . Figure 4)2·6H2O followed by the solution deposition of MHDA for 1 h. The higher-intensity region corresponds to the first Au deposition (100 nm thick) before multilayer growth, and the lower-intensity region corresponds to the second Au deposition (30 nm thick) after multilayer growth. The lowest-intensity region between the two Au regions corresponds to the nanogap where the Si substrate is exposed. This nanogap measures 26.0 ± 4.3 nm and is consistent with the thickness of the Cu-ligated MHDA decalayer measured via spectroscopic ellipsometry (24.8 ± 0.1 nm) and the thickness of Cu-ligated MHDA decalayers from previous studies [To illustrate the applicability of the vapor-phase deposition of C16 in the molecular ruler process, Cu-ligated MHDA multilayers with and without a C16 capping layer are utilized to create nanogaps via the molecular-ruler process.  studies .4)2·6H2O followed by the vapor deposition of C16 at 80 °C for 1 h. Similar higher and lower intensity regions are observed and correspond to the first and second Au deposition steps. The nanogap between the two Au regions measures 31.0 ± 9.4 nm, which is both larger and exhibits greater variability than the nanogap without the C16 capping layer. The width is consistent with the thickness of a Cu-ligated MHDA decalayer with the C16 capping (31.0 ± 1.0 nm) measured via spectroscopic ellipsometry.The standard deviations of the nanogap widths, thus the quality of the nanogaps, result from the morphologies of the Cu-ligated multilayers of MHDA only and MHDA with vapor-phase deposition of C16 (Figure S2, In conclusion, Cu-ligated MHDA-C16 bilayers formed from the solution and vapor deposition of C16 have been characterized with AFM revealing varied surface morphologies. The solution deposition of C16 results in structures that exhibit protruding islands of varying heights with relatively low surface coverages. These results agree with previous AFM topographic images of solution deposited Cu-ligated MHDA-C16 bilayers . The vapSupporting Information features additional AFM data and experimental details.File 1Additional experimental data.


The Brazilian Journal of Cardiovascular Surgery (BJCVS) is grateful for the reviewers,listed below, which collaborated in this edition. Without their work would be impossibleto keep the high scientific standard of our journal.Aubyn MarathBeatriz FurlanettoBruno RochaCarla TanamatiFabio NavarroFrancisco CostaGabriel LiguoriLuciano AlbuquerqueMagaly Arrais dos SantosMarcos Aurelio Barboza de OliveiraNelson HossneNoedir StolfOmar MejíaPaulo Roberto EvoraRachel NinaRenato ArnoniRicardo BenfattiRicardo LimaRodrigo JaenischVictor Ferreira