Akashiwo sanguine bloom using Illumina MiSeq sequencing of 16S rRNA gene amplicons. The bloom was probably stimulated by low salinity and ended after abatement of eutrophication pollution. A total of 658,446 sequence reads and 11,807 OTUs were obtained in both bloom and control samples with Alpha-proteobacteria and Gamma-proteobacteria being the predominant classes detected. The bloom decreased bacterial diversity, increased species evenness, and significantly changed the bacterial community structure. Bacterial communities within the bloom were more homogeneous than those within the control area. The bacteria stimulated by this bloom included the SAR86 and SAR116 clades and the AEGEAN-169 marine group, but a few were suppressed. In addition, many bacteria known to be associated with phytoplankton were detected only in the bloom samples. This study revealed the great influence of an A. sanguinea bloom on free-living bacterial communities, and provided new insights into the relationship between bacteria and A. sanguinea in marine ecosystems.Although phytoplankton are the major source of marine dissolved organic matter (DOM), their blooms are a global problem that can greatly affect marine ecological systems, especially free-living bacteria, which are the primary DOM degraders. In this study, we analyzed free-living bacterial communities from Xiamen sea during an  Phytoplankton blooms are a worldwide ecological problem due to eutrophication pollution123457894616in situ hybridization2124Alteromonas group can greatly influence the flux of organic matter by proliferation during development of diatom blooms, while Bacteroidetes may contribute to bloom decompositionMany molecular tools have been utilized to investigate bacterial communities associated with phytoplankton blooms, such as 16S ribosomal DNA (rDNA) clone libraries11Akashiwo sanguine is an alga that causes blooms world-wide. It is eurythermal and euryhalineChattonella antiqua and A. sanguinea inhibit each other's growthHeterosigma akashiwo inhibits A. sanguinea via allelochemicals and direct cell contact to influence bloom formationet al. studied an A. sanguinea bloom along the central Oregon coast and made speculations that the algal seed came from the Washington coast where a massive bloom of A. sanguinea had been observedA. sanguinea is harmful to birds, fishA. sanguinea blooms frequently occur off the coast of Xiamen. Little research has been reported on the bacterial communities associated with these blooms except for our previous research based on PCR-DGGE, which suggested that bacteria may play an important role in the negative regulation of A. sanguinea-bloomsThe dinoflagellate A. sanguinea bloom in more detail 16S rRNA genes of the free-living bacteria were sequenced using the Illumina MiSeq. The aims of this study were to find out which free-living bacterial taxa were dominant during the bloom, how the free-living bacterial diversity and community structure were influenced by this bloom, and whether there were any bacterial taxa that could negatively regulate this bloom. The results showed that the A. sanguinea bloom significantly changed the free-living bacterial community structure and stimulated most bacterial taxa; however, the bloom decreased bacterial diversity while increasing species evenness.In order to examine the free-living bacterial community during an A. sanguinea bloom had occurred along the Xiamen coast  and the H1 control site  were selected in or near the area where an en coast . Twenty a, dissolved inorganic phosphorus (DIP), nitrate, nitrite, and ammonia, the density of A. sanguinea and bacteria, chemical oxygen demand (COD) and bacterial productivity were determined as previously described23−, NO2− and NH4+. The N:P ratio was calculated by dividing DIN values by DIP values. The silico-molybdenum blue spectrophotometric method was used for the determination of silicate. The eutrophication index (EI) was calculated as follows: Identification of phytoplankton species, phytoplankton cell counts, concentration of chlorophyll Water samples (500 mL) were filtered through 5-μm diameter pore-size filters  to remove particle attached cells. The filtrate was filtered again through 0.22-μm diameter pore-size filters as soon as the samples were taken to the laboratory. Filters were stored at −70°C until analysis (the 5-μm diameter pore-size filters were taken for a comparison study with DGGE). DNA extraction was performed as previously describedPrimers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) targeting the V4 hyper variable regions of bacterial 16S rRNA genes were selectedPCR amplification was performed in triplicate using a Gene Amp PCR-System 9700  in a total volume of 25 μL containing 2.5 μL 10 × PCR bufferII and 0.5 units of AccuPrime™ Taq DNA Polymerase High Fidelity , 0.4 μM of each primer, and 10 ng template DNA. Thermal cycling conditions were as follows: an initial denaturation at 94°C for 1 min, and 35 cycles at 94°C for 20 s, 53°C for 25 s, and 68°C for 45 s, with a final extension at 68°C for 10 min.Following amplification, 2 μL of PCR product was used to confirm successful amplification using agarose gel (1%) electrophoresis. The triplicate PCR reactions for each sample preparation were combined and quantified with PicoGreen: 200 ng of PCR product from each sample was pooled for each sequencing run. The pooled mixture was purified with a QIAquick Gel Extraction Kit  and analyzed on an Agilent 2100 Bioanalyzer using High Sensitivity DNA Chips  for size distribution, and then was re-quantified with PicoGreen.As described in the MiSeq Reagent Kit Preparation Guide , the purified mixture was diluted and denatured to obtain an 8 pM sample DNA library, and mixed with an equal volume of 8 pM PhiX . Finally, 600 μL of the mixture library was loaded with read 1, read 2 and index sequencing primersAfter we obtained the raw sequences, the PhiX sequences were removed. The remaining raw sequences of 16S rRNA gene were sorted and distinguished by unique sample tags. Since each sample had a unique tag, all sequence reads with the same tag were assigned to the same sample. Based on the primer sequences, each sample could be further separated into two (forward and reverse) regions. Finally, the tag, both primers and spacers were trimmed based on Btrimhttp://rdp.cme.msu.edu). All statistical analyses were performed in RForward and reverse sequences were merged by overlapping paired-end reads using FLASHAfter processing, 658,446 high quality sequences remained with an average length of 253 bases. A total of 11,807 OTUs were generated after clustering at a 97% similarity level and 5,672 OTUs were singletons. Species richness estimates of control and bloom samples were quite high  using the Chao estimator, while the rarefaction curves of control and bloom samples  were stiA. sanguinea  occurred in site A1 from 31 July to 4 August 2011 in the sea area near Xiamen. A distinctly colored border line in the water caused by this bloom appeared between A1 and the control area (H1). The water properties and chemical analysis results are shown in A prolonged bloom dominated by All environmental variables showed distinct differences between the bloom and control areas during the sampling periods. Compared to the control area, pH was much higher in A1 although the pH for both A1 and H1 decreased during the bloom. Dissolved oxygen (DO) showed the same pattern except that it was higher in H1 on day 1 and after the bloom. The concentration of suspended particles reached a peak on BP1.A. sanguine density, but recovered dramatically by AB. The much higher COD observed in A1, indicating the bloom outbreak, was associated with the level of water pollution.Nitrite and nitrate dynamics were complex but both declined to their lowest levels on BP2 and then recovered by AB. Ammonia nitrogen showed a generally increasing trend in the bloom area except on BP2. DIP concentration was higher in A1, declined to its lowest level on BP2, and then recovered after the bloom. In general, the bloom area had much higher inorganic N and P concentrations and a lower N/P ratio. The silicate concentration was higher in A1 except on BP2 and AB. Silicate concentrations were negatively correlated with a concentration than H1 during the bloom because of the high algal density. The EI, based on the inorganic nutrient levels, was higher in A1 than in H1 during the bloom (except for BP2) and indicated that the bloom area had a serious eutrophication status (EI > 1). The EI dynamics were in contrast to the algal density dynamics in that EI decreased as the bloom increased. On day 2 the algal density peaked, decreased and then reached a second peak on day 5. During this same time period, the EI declined to its lowest levels. The EI recovered after the bloom, indicating that this bloom was able to reduce the eutrophication pollution of the sea water. Bacterial density was also higher in the bloom area and total algal density coincided well with A. sanguinea cell numbers.In general, A1 had a higher chlorophyll Comparison of the microbial community composition was made between the bloom and control samples for days 1, 3, 4 and 5, these being the only days when the DNA was sufficient in the control area. The 16S rRNA sequences obtained were subjected to random re-sampling at 13,600 sequences for each sample, and sequences appearing in only one of three replicates were removed to minimize errors.Rarefaction curves based on a 97% cluster similarity showed a remarkable difference in the free-living bacterial diversity between A1 and H1. The bloom samples showed a lower bacterial diversity than the control samples, except on day 1 ; otherwiThe bacterial community structure of the bloom and control samples at the genus level is shown in Except on day 1, the bloom area had a much lower percentage of unclassified bacteria than the control area, indicating that the bloom environment had more known bacterial species although the percentage increased along with the bloom . At day Loktanella (P = 0.04) and the NS5 marine group (P = 0.02), but lower Litoricola (P = 0.04). On day 3, A1 had more SAR86 (P < 0.01) and SAR116 (P = 0.03) clades, AEGEAN-169 (P = 0.02) and the NS5 marine group (P < 0.01), and OM60 (NOR5) clade (P < 0.01), but fewer Vibrio (P = 0.02). On day 4, the bloom area also had fewer Vibrio (P = 0.04) and on day 5, A1 had more of the SAR116 clade (P = 0.04).T-test results for the top 10 genera showed that, on day 1, A1 had a significantly higher relative abundance of The response ratio results in A. sanguinea and bacteria showed that many bacteria had significantly positive correlations with this alga, for example the OCS155 marine group (P = 0.02), the Marinoscillum (P = 0.03), ML602J-37 of Cytophagia (P = 0.4), the NS3a marine group (P = 0.02), AEGEAN-169 marine group of Rhodospirillaceae (P = 0.04), the SAR116 (P < 0.01) and SAR86 clades (P = 0.048) of the Flavobacteriaceae, Piscirickettsia (P = 0.04), and Acholeplasma (P = 0.01). In contrast, Roseibacterium (P = 0.04), Sulfitobacter (P = 0.048), Inhella (P < 0.01) of the Comamon adaceae,Pseudidiomarina (P = 0.04) and Bermanella (P = 0.03) had a negative correlation with A. sanguinea. Candidatus Aquilunai strain, the NS3 a marine group, the OM43 clade, Piscirickettsia and Salinihabitans showed an increase in the bloom. Salinihabitans of the Rhodobacteraceae significantly decreased in A1 compared to H1 but it recovered after the bloom, suggesting that an A. sanguinea bloom could restrain its growth.Spearman's correlation analysis between In general, most bacteria, such as the dominant SAR86 and SAR116 clades, and AEGEAN-169 marine group increased during the bloom. These bacteria could be benefitting from the organic matter released from the algae. A total of 37 genera appeared only in the bloom area ,most fro2 and release O2. As their numbers increase during a bloom, photosynthesis rates increase resulting in higher pH and DO in the bloom area, which in turn promotes the growth of the phytoplankton3−, NO2− and DIP concentrations were negatively related to algal density in A1. While these nutrients were consumed by the bloom, the inorganic nutrient concentration was still higher in the bloom than in the control area. The bloom also had a lower N/P ratio indicating that A. sanguine required more N than P. Unbalanced consumption of N and P also resulted in the N/P ratio of the water changing within the bloom. Low salinity and high nutrient content from eutrophication pollution are probably important requirements for the A. sanguinea bloom formation28A. sanguine since the consumption of silicon was concurrent with A. sanguinea density during this bloom. Information on silicon consumption by A. sanguinea is limited, however, one report notes that silicon concentration decreased alongside an A. sanguinea bloom but recovered after the bloomA. sanguine consumed a large amount of NO3−, DIP and SiO4− but less NH4+. While higher COD and EI were needed for bloom formation, the bloom could reduce COD and relieve eutrophication pollution. The bacterial community increased in density in response to the bloom.Algae performing photosynthesis consume COThe free-living bacterial communities in the bloom and control areas were studied using Illumina sequencing for the first time. Although, in this study, more than 650,000 bacterial sequences and 11,807 OTUs were obtained in the Xiamen sea area, more than half of the OTUs were singletons and the rarefaction curve was unsaturated, indicating the high free bacterial diversity in this coastal area. In addition, 5601 OTUs could not be assigned to any genera with a confidence level higher than 50%, suggesting the presence of abundant novel bacteria in this area. The Proteobacteria was the predominant bacterial phylum while the Alpha-proteobacteria was the predominant class. Alpha-proteobacteria are noted as abundant free-living bacterioplankton both in coastal and open-ocean habitats5051A. sanguine blooms. The AEGEAN-169 marine group was another dominant group with a higher relative abundance in the bloom area, but there is little relevant ecological information available on this group.Compared to the control area, free-living bacterial diversity was lower and evenness much higher in the bloom area, and bacterial composition of the bloom samples was more consistent based on the DCA profile. Since bacterial diversity was relatively stable in the bloom area, this bloom did not change very much in terms of bacterial diversity. In contrast, the bacterial community structure was significantly changed, which is consistent with the results of a study in Norwegian coastal waters where bacterial diversity and community composition were stable during a spring phytoplankton bloom555758Comparison of the bacterial community structures between bloom and control areas showed that the largest difference occurred on day 3 (during the bloom). However, day 1 (during the bloom) was quite different from the other days. Since phytoplankton can release abundant organic matter that would provide bacteria with carbon and energy sources. The bloom would be expected to enhance most bacteria, particularly dominant species which may be adept at using this resource, such as the SAR116 and OM60 (NOR5) clades, and the AEGEAN-169 and NS5 marine groups. Among these, the SAR116 clade is a unique Alpha-proteobacterial group, and its members are widely found in general marine areas60186264Candidatus Aquiluna strain from the Microbacteriaceae, which was first isolated from a lake6970Salinihabitans, Piscirickettsia and the NS3a marine group is limited. Roseobacter had a higher abundance in the bloom area on day 3 (5.6 ± 0.49%), when algal density was lowest, but this is not as high as has been observed in other studies111872Winogradskyella is heterotrophic and many isolates are associated with algae74Aureispira was was found during a Microcystis bloom78Rheinheimera shows anti-algal activityRoseovariusBacillusHoefleaMethylobacteriumDinoroseobacter may have a beneficial relationship with algaeColwellia plays a key role in remineralizing organic matter generated from primary productionAmong the predominant bacteria which increased in this bloom was the A. sanguinea bloom. This bloom stimulated the growth of those bacterial taxa which could take advantage of the organic matter released by the phytoplankton, such as the SAR86 clade, the AEGEAN-169 marine group and some novel bacterial communities from the Rhodobacteraceae. As the A. sanguinea bloom dramatically influenced free-living bacterial communities in many aspects and this study furthered our understanding of its ecological process. Once bacterial communities have changed, so as their ecological functions and which would be studied with functional gene arrays (e.g. GeoChip) in the future.This study found a high bacterial diversity and abundant novel species in the sea area near Xiamen with Alpha-proteobacteria and Gamma-proteobacteria being the predominant classes detected. The low salinity and high eutrophication levels in this area were ideal for the formation of the Decision to publish: T.L.Z. and J.Z.Z. Conceived and designed the experiments: C.Y.Y., Y.L., Y.Y.Z., W.Z., Y.T., J.Z.Z. and T.L.Z. Performed the experiments: C.Y.Y., Y.L., B.J.Z., J.D.N., L.Y.W. and Z.L.H. Analyzed the data: C.Y.Y., Y.L., J.D.N., L.Y.W. and Z.L.H.  Contributed reagents/materials/analysis tools: Y.Y.Z., W.Z., Y.T. and T.L.Z. Wrote the paper: C.Y.Y. and Y.L. All authors reviewed the manuscript.


In this paper, we present the first realization of an elastic metamaterial allowing independent tuning of negative density and stiffness for elastic waves propagating along a designated direction. In electromagnetic (or acoustic) metamaterials, it is now possible to tune permittivity (bulk modulus) and permeability (density) independently. Apparently, the tuning methods seem to be directly applicable for elastic case, but no realization has yet been made due to the unique tensorial physics of elasticity that makes wave motions coupled in a peculiar way. To realize independent tunability, we developed a single-phased elastic metamaterial supported by theoretical analysis and numerical/experimental validations. Interest in negative material properties has initially grown in electromagnetic field124567910111314151718et al.et al.et al.et al.28et al.et al.In that both acoustic and elastic waves propagate by particle motions or oscillations, one may immediately conjecture that the independent tuning method of negativity in material property developed for acoustic waves can be directly used in problems dealing with elastic waves. However, due to the unique tensorial physics of elasticity, complex coupling occurs among various deformation modes such as longitudinal, bending and shear motions. Therefore, the ideas from electromagnetic/acoustic waves cannot be directly used. Earlier investigations20212223242526x direction, i.e., the S0 wave mode the dominant motion of which is in the propagating directionx- and y-resonating parts which have local resonance modes in the x-direction (propagation direction) and in the y-direction (direction perpendicular to the propagation direction), respectively. While more details of the involved physics will be given later, the negative density and stiffness are realized by the x- and y-resonating parts, respectively. Since the resonance frequencies of the two resonators can be independently tuned, independent tuning of negativity in stiffness and density is available and single or double negativity for a selected range of frequencies can also be achieved. Especially, in realizing negative stiffness by the y-resonating parts, the coupling of different deformation modes, longitudinal and bending, is elaborately used; the counterpart of the coupling cannot be found in electromagnetic or acoustic problems. Specifically, the slender members can exhibit both longitudinal and shear-bending motions, unique coupling phenomena in elasticity governed by the tensor field. Later, we will show how the coupling affects negative stiffness behavior. In the present realization of the metamaterial, the inclinations of slender members connected to the central rectangular mass in the y-resonating part make the coupling effect which plays a key role for the stiffness tuning.Here, we present the first realization of an elastic metamaterial allowing independent negativity tuning in stiffness and density. To this end, a single-phased elastic metamaterial in 20212223242526Cmk) of the metamaterial has two resonating parts the physics of which can be better investigated by square unit cells Cm and Ck in Cm and Ck denote the unit cell exhibiting negative mass and stiffness, respectively, for the S0 wave mode propagating in the x direction. Because the mechanics of the unit cell Cmk can be viewed as a combination of Cm and Ck as illustrated in Cm and Ck will be investigated first. To model the mechanics of Cm and Ck in association with the S0 wave propagating in the x direction, the elastic metamaterial solids forming Cm and Ck will be replaced by the mass-spring system shown in x-y plane will be considered in the mass-spring system. Although the S0 wave mode is a 3-dimensional wave phenomenon, its characteristics can be analyzed with a two-dimensional model at sufficiently low frequencies1415To illustrate our idea clearly, we will consider the metamaterial in To begin with, we review the well-known dispersion equation and expression of characteristic impedance in a simple one-dimensional periodic mass-spring system in ω, k and d denote the angular frequency, the wavevector and the period, respectively.Where To uniquely define the effective mass and stiffness from the dispersion relation, the expression for characteristic impedance is also needed. The characteristic impedance Z of the periodic mass-spring system shown in h is the thickness of the unit cell.Where As can be seen in Cm. As explained, although the S0 wave mode mainly considered in this work is a 3-dimensional wave phenomenon, it can be accurately analyzed with a two- dimensional mass-spring system if the operating frequency range is sufficiently low as in this work1415α denotes the longitudinal spring coefficient of the beam segment connecting masses m1’s and δ denotes the shear-bending spring coefficient of the beam segment connecting mass m1 and m3. To facilitate the analysis, the square unit cell is slightly off-centered but the resulting dispersion curves will not be affected because of periodicity of the unit cells. Following the detailed procedures in the To introduce a method to tune effective mass alone, let us consider the mass-spring model shown in Comparing x-resonating part when m3 oscillates in the x direction. If ω is much lower than ωx, the effective mass simply becomes the total mass, m1 + m3. However, for ω near ωx, the motion of the x-resonating part significantly affects the effective mass, making it negative when ω becomes slightly larger than ωx. The ωx is replaced by its dipole resonance frequency. It is worth remarking that in the present case, the motion of the resonating part involves bending deformation of slender members mainly and interacts with the main motion of m1, thus avoiding a need to use multi-phased materials as used to make the coaxial resonators.where Ck by using the discrete model in Ck should be so designed that we can tune, independently from ωx, the resonance frequency to be associated with negative stiffness. Here, our proposition is to utilize coupling between x and y directional motions by intentionally inclining the slender members connecting m1 and m2. The resulting coupling phenomenon between the longitudinal and shear-bending motions is unique in the tensorial physics of elasticity. The coupled stiffness is denoted by γ in x and y directional motions. Thus, the spring coefficients of the slender members can be expressed in the following relations:Let us now derive the effective mass and stiffness for the metamaterial made of u and v denotes the x- and y-directional displacements applied to the coupled spring. Referring to the original continuum configuration of Ck shown in γ term also appears; the two symmetric members with the opposite inclination angles are used to connect m1 and m2. As a result, not only the x-directional but also the y-directional motions of m2 should be considered in deriving equations of motion.where Following the detailed procedures in the for which the following assumption is used,α is typically one order larger than β as demonstrated in λ is much larger than the unit cell size d . Because the unit cell size is very small compared with the wavelength in the matrix (λmatrix > 6d) in the frequency range below 40 kHz, the use of the assumption can be justified as can be seen in The assumption in Finally, the effective mass and stiffness are identified as, by comparing y-resonating part as it oscillates in the y direction. Equation (8) shows that the y-resonating part affects the effective spring coefficient only while making the effective mass unaffected by ωy. Obviously, the effective stiffness ωy. It is remarked that there were some earlier attempts, without actual realization or experiment, to utilize y-directional motionsx-directional stiffness.Where x- and y- resonating part are identified. Because the metamaterial consisting of the unit cell Cmk in Cm and Ck, the wave dispersion equation and the impedance equation can be written asFrom the previous analytic investigations, the effective mass and stiffness for the metamaterials having only the Accordingly, the effective mass and stiffness for the metamaterial can be written asωx and ωy are independently tunable, one can realize single- or double-negative elastic metamaterials for different ranges of frequencies.Cm, Ck and Cmk.The effective mass and stiffness derived above can be validated directly or indirectly with numerical methods. As an indirect way, one can compute the dispersion curve by using the one-dimensional dispersion x-directional effective density h is the unit cell’s thickness and d, its width and height. As clearly shown in Cm and Ck exhibit single negativities in density and stiffness near fx (=ωx/2π) and fy (=ωy/2π), respectively, which are also confirmed by the dispersion curvesLet us now investigate, in some details, the dispersion curves and frequency dependences of Cm and Ck around or near fx and fy, respectively, show how the negativity in density and stiffness is realized. In m1, the main mass parts through which the unit cells are connected to each other, moves to the right while m3 moves to the left. Thus, the total momentum of Cm becomes negative because of the 180° out-of-phase motion of m3 for a positive velocity of the unit cell, causing the effective density negative. On the other hand, Ck sketched at a frequency just below the resonance frequency fy. Because of the large up- and down-ward y-directional motions of the m2 parts under a force (motion) at the left side of Ck, the right side of Ck moves to the left, the opposite direction to the force (motion) at the left side. Thereby, the effective stiffness becomes negative. This is possible because of the elaborate coupling of x and y directional deformations which cannot be found in electromagnetic or acoustic wave cases.The sketches of the deformation patterns of Cmk, which is the combination of Cm and Ck, exhibit the combined effects of the two independent metamaterials made of Cm and Ck, as clearly demonstrated in Cm and the negative stiffness in Ck. Because fx and fy can be independently tuned in the developed metamaterial, the metamaterials can be tailored to meet specific applications requiring single and/or double negativity.The metamaterials consisting of kd ≪ 1. In this range, one can assume that In this section, the analytic approach on the refractive index and the impedance of the metamaterial will be carried out. One can calculate these parameters directly from vp and the impedance Z:Considering that In the proposed metamaterial, the effective density and stiffness, Ck, Cm and Cmk, which are made of an aluminum only. Since these metamaterials are made of a single-phased low-loss aluminum, adverse effects of loss can be insignificant.Finally, the metamaterials are fabricated and their wave characteristics are experimentally investigated. Cmk has a stop band at around 25 kHz, only the S0 wave mode is measured at the double negative region of the metamaterial made of Cmk. Because we can capture the first arrival pulse of the S0 wave mode only in actual experiments, the resulting data analysis is performed with the S0 mode.In the experiments, the S0 wave was actuated by the actuating piezoelectric transducers  was used to measure the x-directional displacement. For the actuation, the same piezoelectric transducer shown in The experimental results shown in x-directional displacement uA, uB and uC. From Cmk around 25kHz. Also, the experimentally measured displacement fields match well with those obtained with numerical simulation in the We also evaluated the wave dispersion curve of the metamaterial from the experimental measurement. To plot the dispersion curve from the experimental data, the measured displacements were post-processed by the Fourier transform to obtain the phase difference between each measurement point. To minimize statistical errors, measurements were made at various points. Then, the wave vector was calculated for various frequencies around 25 kHz. This study presents the first metamaterial realization with independent tuning of effective negative density and stiffness with a single-phased material. In spite of apparent similarity between elastic waves and electromagnetic/acoustic waves, the independent negativity tuning in elastic metamaterials has not been realized earlier. To realize the independent negativity tuning, a single-phased elastic metamaterial was proposed. Here, among others, the independent-tunable negative stiffness was realized by a locally-resonating part the motion of which is dominant in the direction perpendicular to the wave propagation direction. The theoretical and experimental wave analyses of the metamaterials were carried out to confirm the independent tunability of negativity, with realizations of single negative density/stiffness and simultaneous negativity in density and stiffness. Considering many practical important applications of elastic waves in ultrasonic imaging, vibration shielding, etc., our metamaterials could lead to active explorations in elastic metamaterials, which currently appear to be less active in the community. However, the extensions of the proposed 1-dimensional metamaterial for the realization of 2- or 3-dimensional metamaterials would require further studies because of more complicated coupling of longitudinal and shear waves in higher dimensions.How to cite this article: Oh, J. H. et al. Elastic metamaterials for independent realization of negativity in density and stiffness. Sci. Rep.6, 23630; doi: 10.1038/srep23630 (2016).


Brachypodium distachyon as a model.A thorough study of starch biosynthesis and deposition in a non-domesticated wild grass was performed using  Brachypodium distachyon is a non-domesticated cereal. Nonetheless, Brachypodium was recently introduced as a model plant for temperate cereals. This study compares grain starch metabolism in Brachypodium and barley (Hordeum vulgare). In Brachypodium, we identified and annotated 28 genes involved in starch metabolism and identified important motifs including transit peptides and putative carbohydrate-binding modules (CBMs) of the families CBM20, CBM45, CBM48, and CBM53. Starch content was markedly lower in Brachypodium grains (12%) compared to barley grains (47%). Brachypodium starch granules were doughnut shaped and bimodally distributed into distinct small B-type (2.5–10 µm) and very small C-type (0.5–2.5 µm) granules. Large A-type granules, typical of cereals, were absent. Starch-bound phosphate, important for starch degradation, was 2-fold lower in Brachypodium compared with barley indicating different requirements for starch mobilization. The amylopectin branch profiles were similar and the amylose content was only slightly higher compared with barley cv. Golden Promise. The crystallinity of Brachypodium starch granules was low (10%) compared to barley (20%) as determined by wide-angle X-ray scattering (WAXS) and molecular disorder was confirmed by differential scanning calorimetry (DSC). The expression profiles in grain for most genes were distinctly different for Brachypodium compared to barley, typically showing earlier decline during the course of development, which can explain the low starch content and differences in starch molecular structure and granule characteristics. High transitory starch levels were observed in leaves of Brachypodium (2.8% after 14h of light) compared to barley (1.9% after 14h of light). The data suggest important pre-domesticated features of cereals. Worldwide, starch produced by cereal crops has provided the most important human dietary energy for millennia. In 2012, world production of cereals amounted to 2.3 billion tons  P. Beauv. is a wild grass that has recently been established as a model system for temperate grasses. Brachypodium remnants have been recovered from a ground stone artefact from the Upper Palaeolithic site of Ohalo II in Israel (1–4)-β-glucan (BG) content . They indicate both important conserved molecular features of starch and its metabolism as well as innovations related to domestication of cereal grain from wild grasses. Specifically, Brachypodium grain displayed low starch content, small granular and amorphous starch granules, and high cell wall and protein content. Distinct features could be linked to specific differences in starch biosynthesis enzyme expression profiles.Here we present the first investigation on diversity of starch in grasses. We focussed on the biosynthetic capacity, structure, and composition of starch in the http://www.ncbi.nlm.nih.gov/). Sequence similarity searches were performed by BLASTP with standard settings in the Brachypodium genome database (http://www.brachypodium.org/) and in Phytozome (http://www.phytozome.org). Sequences with high percentage identity were selected to represent putative Brachypodium starch enzymes. Correct members of each of the gene classes were confirmed by BLASTP using the NCBI non-redundant database. Additional alignments were done using ClustalW Omega . Alignment and phylogeny relationships were done with MEGA 5 (http://pfam.sanger.ac.uk/). The tandem domain CBM45 was identified manually, using available sequences .Sequences were collected from the National Centre for Biotechnology Information database  to allow identification of expression values, and expression trends for starch-related genes in both species were analysed individually.Transcriptome data for starch biosynthetic genes for Hordeum vulgare ssp. vulgare cv. Golden Promise (spring barley) and Brachypodium distachyon (L.) P. Beauv.  lines 21 (Bd21) and 21–3 (Bd21-3) were used throughout this study. Bd21 had the first sequenced genome of Brachypodium and Bd21-3 is superior for genetic transformation. Plants were grown in a temperature-controlled greenhouse (22°C day/16°C night) at ambient light levels. Seed samples of B. distachyon were a gift from Dr John Vogel, USDA-ARS, USA.Grains were harvested from plants and ground to a fine powder with a tissue lyser . Starch was isolated as described in Brachypodium and barley leaves (3g) were collected after a 10h light period, immediately frozen in liquid nitrogen and stored at –80°C. Frozen leaves were homogenized using a Polytron PT 3000 Blade-type Homogenizer in 35ml of 100mM 3-(N-morpholino)propane sulfonic acid (MOPS), pH 7.2, 5mM EDTA, and 10% (v/v) glycerol. The homogenate was filtered through two layers of cloth and centrifuged for 10min, at 4°C (3000 g). The pellet was suspended and washed twice in 30ml of the same medium including 0.5% (w/v) SDS. A layer on the top of a dense debris pellet was identified as starch granules, which were collected with a pipette, and washed twice with SDS-containing medium and six times with ddH2O to completely remove debris. Sedimented starch was inspected visually for purity and granule quality, and then air dried at room temperature.Extraction of starch granules from leaves was performed as described by g for 5min. The powder was suspended in 500 µl of 100mM Na citrate buffer (pH 4.4) and incubated for 5min at 80 °C to gelatinize the starch. Heat-stable α-amylase  was added and the samples were incubated for 6h at 80°C. The tubes were cooled to 60°C and 0.3 AGU units of amyloglucosidase  were added to samples. Samples were incubated at 60°C overnight, with constant mixing. After centrifugation, the supernatant was analysed for glucose using high-performance anion-exchange chromatography, with a DX 500 system  equipped with an S-3500 auto sampler, GP40 pump, ED40 PAD (HPAEC-PAD), and a CarboPac PA-1 column as previously described  ethanol at 80°C, with intermittent centrifugation at 10 000 et al., 2009). Brachypodium line 21-3 was used for sampling. Leaf samples were collected over a 14-h light period every 2h, immediately frozen in liquid nitrogen and stored at –80°C. The tissue was ground to fine powder using a tissue lyser. Samples of 20mg were used for analysis. Soluble sugars were removed by extraction four subsequent times in 80% (v/v) ethanol at 80°C for 1h followed with intermittent centrifugations at 13 000 g for 5min. The powder was suspended in 200 µl of 0.2M NaOH, incubated for 30min at 80°C to gelatinize the starch, and neutralized with 15 µl of 1M HCl. Na acetate buffer  containing α-amylase  and amyloglucosidase  was added and the samples incubated at 50°C overnight with constant mixing. The supernatants were analysed as for the grain samples.Starch measurements in leaves were performed, with modifications, as described in (Scofield Brachypodium (line 21-3) and barley were collected after a 10-h light period or a 14-h dark period. Pigments were removed by extraction in 80% (v/v) ethanol at 80°C and the destained leaves were transferred to petri dishes and stained with iodine solution.Leaves from 2O was added to each sample and 10 µl aliquots applied in wells of a 96-well microtiter plate in technical triplicates. 200 µl of diluted iodine solution  was added, the absorbance measured at 550nm and 620nm and amylose concentration calculated by plotting the A620/A550 ratio against standards with known amylose contents  was used as a control. Each sample (1mg) was incubated with 1M NaOH at 80°C for 5min and the pH was neutralized with 10 µl 1M HCl. Citrate buffer  was added containing 1 µl of isoamylase , samples were incubated for 1h at 40°C, shortly centrifuged, and supernatants were analysed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a CarboPac PA-100 column as described previously , peak temperature (TP), conclusion temperature (TC), and enthalpy transition energy (ΔH).Starch granule thermal characteristics in water were analysed using differential scanning calorimetry (DSC) as described previously (X-ray diffraction (XRD) analyses were performed as described previously (The degree of starch phosphorylation was determined as the content of Glucose-6-phosphate (Glc-6-P) and glucose-3-phosphate (Glc-3-P) after complete starch hydrolysis as described previously , 2000b.Brachypodium and barley grains were mounted on carbon tabs on aluminium stubs. Samples were sputter coated with gold/palladium for 120 seconds and observed with a secondary detector at an accelerating voltage of 10kV in Quanta 200 scanning electron microscopy .Grains were sectioned (400 µm) and dry starch granules previously purified from Brachypodium and barley grains were produced using a microtome with vibrating blade Microm HM 650 V-Thermo Scientific. The slices were stained with iodine solution (as above) and observed with light microscopy using a Leica DMR HC fluorescence microscope combined with digital camera Leica DC 300F. Shape and size of extracted starch granules from seeds and leaves were investigated using bright field (BF) microscopy after suspending powder in iodine solution. Granule birefringence was investigated using a cross-polarizer.Sections of 400 µm thickness of both Native starch granules were stained with 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) and afterwards confocal laser scanning microscopy (CLSM) was performed as described previously (1–4)-β-glucan analysis was performed according to Total nitrogen and carbon contents of the grains were determined on finely ground grains using an ANCA-SL/GSL elemental analyser  attached to a stable isotope mass spectrometer. A nitrogen-to-protein conversion factor of 5.7 was used for protein content .Brachypodium and barley was investigated in silico and the enzyme functional gene structure deduced. We identified 23 unique genes and annotated them in the groups starch synthases (SSs), starch-branching enzymes (SBEs), debranching enzymes (DBEs), α-glucan phosphorylase (PHOs), and glucan water dikinases (GWDs) according to their catalytic domains. Recent annotations . Each clade turned out to constitute a cluster with specific enzymatic function, experimentally documented for many members. The putative Brachypodium BdGBSSIb sequence is an outlier. Its sequence codes for a catalytic domain, but the protein sequence is possibly truncated indicating that it might be a pseudogene.In order to identify the main starch biosynthetic relationships in et al., 2009a, b), CBM45  with affinity to starch and plastid-targeting signal peptides . Four di), CBM45 , and CBMBrachypodium and barley was carried out based on available data . Distinct gene expression was noted for most of the genes.Comparative expression profiling of genes involved in starch biosynthesis in Brachypodium grain takes less time than of that of barley. Cellularization starts at 8 DAP for Brachypodium and 6 DAP for barley; the post-cellularization period ends with maturity at 20 DAP and 38 DAP, respectively  or remained at the same high level . The sbeI gene was the only one in Brachypodium having constant, slightly increasing expression, while in barley it increased extensively after 10 DAP to peak at 15 DAP, followed by a decrease to half maximum expression level.Development of Brachypodium and barley .Total grain starch and BG contents were determined in d barley . Brachypn Bd21-3 . Hence, Brachypodium, amylose, starch-phosphate  was higher compared to the level found in barley starch (29%). On the other hand, Brachypodium starch contained 2-fold less phosphate (0.35–0.40 nmole Glc-6-P mg–1 and 0.12–0.15 nmole Glc-3-P mg–1) as compared to barley (0.80 nmole Glc-6-P mg–1 and 0.32 nmole Glc-3-P mg–1).The amount of amylose in Brachypodium and barley were found in the DP 14–20 region where both Brachypodium and barley had a distinct peak at DP 19–20, which was lacking in the rice distribution.The amylopectin chain-length distribution, as compared to rice and barley  showed aBrachypodium starch granules as studied by light microscopy and SEM were examined and compared to barley granules ; even if the outer shape was different, barley granules were more oval and filled, while Brachypodium granules kept the doughnut-shape.The internal structures of the ing CLSM –F givingBrachypodium endosperm starch granules ranged between 0.5 and 12 µm in Brachypodium, displaying a bimodal size distribution , which is typical for cereal storage starch. However, the wide-angle X-ray scattering (WAXS) diagram of Brachypodium contains traces of V-type crystalline polymorph . The degree of crystallinity was low and estimated to be 10%, which is two times lower than that observed for barley starch (20%)  than in Brachypodium after a 14-h light regime . The Brachypodium leaf starch granule shape, size, and topography were very similar to that of barley leaf starch, having small, flattened, and round-shaped morphology as deduced from SEM , where a genetic marker for B-type granules was identified , and The Danish Ministry of Science, Innovation and Higher Education (SLJ).


To minimize cold ischemia time, transplantations with kidneys from deceased donors are frequently performed during the night.However, sleep deprivation of those who perform the transplantation may have adverse effects on cognitive and psychomotor performance and may cause reduced cognitive flexibility. We hypothesize that renal transplantations performed during the night are associated with an increased incidence of pure technical graft failure.A retrospective analysis of data of the Dutch Organ Transplant Registry concerning all transplants from deceased donors between 2000 and 2013 was performed. Nighttime surgery was defined as the start of the procedure between 8 p.m. and 8 a.m. The primary outcome measure was technical graft failure, defined as graft loss within 10 days after surgery without signs of (hyper)acute rejection.versus 2.6 % for daytime surgery (p = .001). In a multivariable model, correcting for relevant donor, recipient and graft factors, daytime surgery was an independent predictor of pure technical graft failure (p < .001).Of 4.519 renal transplantations in adult recipients, 1.480 were performed during the night. The incidence of pure technical graft failure was 1.0 % for procedures started during the night Limitation of this study is mainly to its retrospective design, and the influence of some relevant variables, such as the experience level of the surgeon, could not be assessed. We conclude that nighttime surgery is associated with less pure technical graft failures. Further research is required to explore factors that may positively influence the performance of the surgical team during the night. As the diagnosis of brain death and decisions around potential multiorgan donors are usually made during office hours, multiorgan donor surgery is usually started during the evening or night . As alloHowever, younger surgeons are becoming more critical regarding nighttime work and their own quality of life. Also evidence exists showing that sleep deprivation may have adverse effects on cognitive and psychomotor performance –9. MoreoTherefore, we performed a cohort study with data obtained from the Dutch Organ Transplant Registry to evaluate whether kidney transplantation performed during the night is associated with technical graft failure.We used the data of a consecutive series of transplantations with deceased donor kidneys in adult recipients performed from January 1, 2000, to December 31, 2013. Data were obtained from a prospectively maintained electronic database by the Dutch Organ Transplant Registry .We identified the following factors as possible confounders: donor gender, age and body mass index (BMI), donation after brain death (DBD) or circulatory death (DCD), hypertension of the donor, kidney side, number of renal arteries, start cold perfusion time, recipient gender, age and BMI, smoker/nonsmoker, vascular status of the recipient, number of dialysis days, previous renal transplants, first warm ischemia time (WIT1), WIT2 and CIT and center of transplantation.WIT1 was defined as the time from cessation of the blood circulation to the renal allograft to start of cold perfusion. CIT was defined as time between start of cold perfusion and removal of the renal allograft from ice. Second warm ischemia time (WIT2) was defined the time between placement of the renal allograft into the iliac fossa of the recipient, until revascularization of the kidney.e.g., ultrasound proven), while nonviable kidneys (NVK) were defined as never-functioning renal allografts with poor perfusion from the beginning. In many cases of graft failure due to PNF and NVK, it was difficult to differentiate between pure technical graft failures and graft failures due to poor organ quality. Therefore, the primary outcome measure was pure technical graft failure, defined as graft loss within 10 days after the transplantation without signs of (hyper)acute rejection, excluding cases of PNF and NVK. Nonimmunological graft failure was defined as graft loss within 10 days without signs of (hyper)acute rejection, including cases of PNF and NVK, this was a secondary outcome measure. In cases with vascular or urological problems in which the renal allograft could be saved by reintervention, the cases were not considered as technical failures. (hyper)Acute rejection with graft loss within 10 postoperative days was referred to as graft failure due to immunological causes.In line with existing literature, daytime surgery was defined as start of surgery between 8 a.m. and 8 p.m. and nighttime surgery was started between 8 p.m. and 8 a.m. , 14. Surt test was used to compare normally distributed variables, and χ2 for categorical variables. For all possible confounding factors, logistic regression was performed to identify factors associated with time of start of the procedure and with pure technical graft failure. All factors associated with time of start of the procedure and pure technical graft failure (defined as p ≤ 0.05) were included in the multiple regression analysis. Graft survival was estimated using the Kaplan–Meier method, and groups were compared with log-rank tests. For all analyses, statistical significance was set at α = 0.05.Statistical analyses were performed using SPSS . Demographic data were reported as means and standard deviation (SD) for continuous variables; categorical variables were reported as number and percentages. Student’s In total, 4.519 consecutive renal transplantations from deceased donors in adult recipients were included in the analysis. Baseline characteristics for donor and recipients, and surgical parameters are shown in Table versus 2.6 % during daytime (p < .01).The overall incidence of pure technical graft failure was 2.1 % , Table A significant association between daytime surgery and pure technical graft failure was observed (p ≤ 0.05).For each center, the incidence of pure technical graft failure was (slightly) higher for daytime surgery with a significant difference for two centers acute rejection with graft loss within 10 postoperative days was, respectively, .2 % for daytime and .3 % for nighttime surgery, which was not a significant difference (p. 51).p = .320).Long-term graft survival for procedures performed during the day or night are shown in Fig. So far, four studies have been performed addressing the impact of nighttime surgery on complications and allograft function in renal transplantation –15. FechData regarding the influence of overnight surgical activity on complications of next-day surgery and conversion to open surgery are not univocal; some authors could not demonstrate any impact, while Rothschild et al. observed an increased complication rate when next-day surgical procedures were performed by physicians with sleep duration below 6 h , 20, 21.In other professions, the influence of fatigue is more pronounced: less psychomotor vigilance and more lane deviation in a driving simulator for police officers , more pre.g., due to septic shock or hypovolemia). In contrast, renal transplantations are performed during the night in order to limit CIT. Our observation that nighttime surgery is associated with less pure technical graft failures is remarkable. A possible explanation may be that recipients with a poorer physical condition were postponed to the next morning; however, this is not supported by our data as recipient characteristics  did not differ significantly. Moreover, significantly more transplants with kidneys from DBD donors and retransplants have been performed during the night. Altogether, our data suggest that other factors than those related to the patient and kidney  may play an important role. Since organ preservation pumps are only scarcely used in the Netherlands, this could not have had a relevant impact on our data.Gastrointestinal or trauma surgery is usually performed during the night to prevent deterioration of the patients’ condition  surgery was performed. Secondly, the definition of pure technical graft failure is challenging. However, early graft failure is a devastating complication for the recipient and therefore a highly relevant primary endpoint. Early graft failure is associated with poor patient survival [There are also some limitations. First, data regarding possible confounders, survival , 32 and survival . To overAnother possible factor which could have influenced sleep deprivation is the centers’ policy regarding cold ischemia time. In center 5, which performed 52 % of the kidney transplantations during the night, the shortest mean cold ischemia time was observed (740 min).Another limitation is the fact that the registration of postoperative complications was insufficient and there could not be analyzed reliably.In conclusion, renal transplantations during nightly hours (8 p.m.–8 a.m.) may have a beneficial impact on the pure technical graft failure rate. This supports the current practice in many transplant centers, performing nightly procedures to minimize cold ischemia time. Further research is required to explore factors that may positively influence the performance of the surgeon and his/her team during the night. This knowledge may help to improve the performance of the surgical team during daytime surgery.


Journal of Medical −Radiation Sciences in January – September 2013. Thank you for your passion towards improving and maintaining the quality of publications in Journal of Medical Radiation Sciences.Publishing a quality peer-reviewed journal relies on the contribution of many peer reviewers. The members of the Editorial Review Board – especially the editors – acknowledge the −contribution of the following colleagues who worked behind the scenes of Mohd Hanafi AliBerry AllenElizabeth BaileyLuke BarclayCharlotte BeardmoreRobin BellLisa BoothScott BowmanJulie BurberyDavid ChristieAndrew CousinsJenny CoxAgnella CraigRob DavidsonKaren DobeliEdgar EstoestaCraig EverittMark FarnworthRhys FitzgeraldDiana GentilcoreShaun GradyKareen GrimshawHazel Harries-JonesJill HarrisRobin HartSuzanne HenwoodPeter HoggDaphne JamesPaul KanePeter KenchTracy KirkbrideKaren KnappPeter LaveryJoerg LehmannSarah LewisAndrew MallittKristie MatthewsClare MonkLaura MullaneyMartin NecasHazel NeserCurtis NgRebecca OwenNick PhillipsSuresh RanaAndrew RennerTaryn RobinsonJenny SimTony SmithPauline SohZhonghua SunJonathan SykesKerry ThoirsHannah ThompsonChristine VanderleyGillian WhalleyKate WilkinsonStephanie WrightsonBosco Yu


Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. Morbillivirus in the family Paramyxoviridae. Two types of glycoprotein spikes, the hemagglutinin (H) and fusion (F) proteins, protrude from the viral envelope. Although both the H and F proteins are the targets of neutralizing antibodies, the humoral immune response is mainly directed against the H protein ..22]. WheAmino acids 379–400 form an immunodominant epitope. This epitope is known as HNE because three cysteines in the epitope form a surface-exposed loop  ,31. SimiMV escaped neutralization by MAb-48 when it acquired mutations at amino acid positions 395 and 398 . HoweverMAbs that recognize HNE neutralize MV infection via SLAM, nectin-4, and CD46. MAb-B5 shows very high neutralizing titers, regardless of the receptor used by the virus  28]. MA. MA28]. RBS is located on the lateral side of the H protein propeller fold structure  18]. A . A 18]. One study has suggested that the epitope recognized by MAb-BH26 is the most important immunodominant epitope because MAb-BH26 inhibits the binding of ~60% of human serum antibodies in convalescent measles patients and vaccinees . Both thThere is a linear epitope in the region defined by residues 188–199 and a potential glycosylation site at position 187 (Asn-Cys-Ser at positions 187–189) . VariantVarious MAbs recognize RBE . SeveralThe H protein is glycosylated at four amino acid positions, 168, 187, 200, and 215 . PositioNeutralizing antibodies often recognize conformational epitopes, but linear epitopes can also be targeted. MAb-BH47, -BH59, -BH103, and -BH129 recognize a linear epitope consisting of amino acids 244–250 blue in C,D 53],,53], whiMAb-I-29 recognizes the amino acid region 309–318 and neutralizes MV . A largeII-18 and B95a cells were maintained in RPMI medium  supplemented with 7.5% fetal calf serum (FCS). Vero and Vero/hNectin4 cells   have been reported previously . Sera frThe details of the method have been reported previously . BrieflyMV antigen (4 hemagglutinating units in a volume of 25 µL) was added to 25 µL of serially twofold-diluted MAbs in U-bottom 96-well microplates. Phosphate-buffered saline  containing 0.1% bovine serum albumin and 0.01% gelatin was used for dilution. Each well received 50 µL of a 0.5% suspension of African green monkey erythrocytes. Plates were shaken and incubated for 1.5 h at 37 °C. The highest dilution of each MAb that completely inhibited hemagglutination was considered the HI titer of that MAb.http://www.glycosciences.de/modeling/glyprot/php/main.php). Figures were generated with PyMOL (http://www.pymol.org).The crystal structures of the H protein unbound to its receptors  and whenIn this review, we have reorganized large amounts of data from various papers concerning the MV-H protein epitopes, together with additional data. The MV-H protein epitopes can be roughly clustered into five distinct epitopes. The first is HNE; the second comprises NE and the BH1-binding epitope; the third is LE and is located on the loop protruding from the β2 sheet; and the fourth is RBE. The exact location of the fifth epitope, SSE, is still unclear, but it is located near RBS and is shielded by the N416-sugar in the specific genotype strains currently circulating.Among these epitopes, the function of RBE is very clear. The epitope is part of a region that must bind to two proteinaceous receptors, SLAM and nectin-4. Therefore, it is not shielded by sugars and must be subjected to strong structural constraints. Significantly, amino acid changes that allow MV to escape neutralization cause a loss of its receptor-binding activity . The antCurrently used MV vaccines were generated from MV strains isolated more than 50 years ago. Nonetheless, they are still highly effective against the MV strains circulating at present. Sera obtained from convalescent measles patients and vaccinees neutralize different MV strains, including vaccine strains and currently circulating strains, with similar efficiencies. This fact and our knowledge of the H protein epitopes strongly suggest that MV cannot undergo large antigenic changes, so the global elimination of MV should be feasible with the tools at hand. This comprehensive review of the epitopes of the MV-H protein will contribute to the global efforts to this end.


Endovascular stent grafting with different commercially available stent graft systems is widely applied for the treatment of abdominal aortic aneurysms with high success rates in the current era. Various types of endoleaks are potential complications of the procedure. They usually occur in the early period. In this report, we present type 3b endoleak occurring 14 months after a successful endovascular abdominal aortic aneurysm repair with a Medtronic Endurant stent graft.  Endovascular abdominal aortic aneurysm repair (EVAR) is a safe and efficient method, which has been widely used for treatment at abdominal aortic aneurysms (AAA). New generation endografts make it possible to treat more complex AAA with less perioperative mortality and systemic complications –4. HowevWe report a case of late type 3b endoleak from a tear in the main body of an Endurant  endograft 14 months after deployment and treatment with coil embolization. Consent from the patient and his family for possible academic activities including publication acts regarding his medical history was obtained. To the best of our knowledge, this is the first report of a late type 3b endoleak with an Endurant endograft.An 81-year-old man underwent EVAR for the treatment of a 70 mm AAA with deployment of 36 × 16 × 166 mm main body Endurant bifurcated endograft, 16 × 20 × 120 mm contralateral limb, and extensions of 16 × 16 × 95 mm to the right and to 20 × 20 × 80 mm to the left. His medical history included heart failure with an ejection fraction of 25%, hypertension, chronic obstructive pulmonary disease, and chronic renal failure requiring hemodialysis. He has been followed up regularly with Doppler ultrasonography and computerized tomography (CT) angiography at 3, 6, and 12 months showed successful exclusion of the aneurysm sac with no leak. Bilateral internal iliac arteries were patent Figures .About 14 months after EVAR, the patient was admitted to the emergency clinic with sudden onset back pain. Clinical examination revealed a pulsatile mass at the epigastrium. Computerized tomography showed enlarged AAA bounded with hematoma and an endoleak originating from distal main body of the EVAR graft .The patient was taken to the angiography unit in the emergency night conditions. Several angiography runs were performed to determine the location of the endoleak; type 1a endoleak was excluded. The pigtail catheter was pulled into the main body. Angiogram showed endoleak at the level of the bifurcation endograft . This tiWe discussed the treatment options with the cardiovascular surgeons and we decided to treat the patient endovascularly because of the high mortality risk of the surgery. Endovascular options for the treatment included placement of a new aorto-uni-iliac or bi-iliac stent graft or coil embolization of the sac through translumbar or transarterial approach. We decided to deploy an aorto-uni-iliac stent graft, as there was not enough distance to deploy an aorto-bi-iliac graft between the bifurcation of the previous endograft and ostia of the renal arteries. In addition, we lacked enough and appropriate sized coils for the embolization of the sac in the night conditions. We deployed an Endurant uni-iliac stent graft with a body diameter of 36 mm . The control angiogram showed persistent endoleak despite additional ballooning. The antegrade flow was still present into the right limb decoding incomplete apposition of the latter stent graft to the previous one. The patient was clinically stable and there was no marked filling into the aneurysm sac so we decided to end the procedure and evaluate the filling of the right limb with a postoperative CT examination.The second day CT angiography showed inaccurate apposition of the new stent graft into the previous stent graft because of the inadequate diameter of the aorto-uni-iliac stent graft; 36 mm is the largest diameter in the market. The gap between the stent grafts allowed persistent blood flow into the right limb and into the aneurysm sac through the fabric tear.We decided to coil-embolize the fabric tear and aneurysm sac through the gap between the two-stent grafts after providing the appropriate coils sizes. The gap was catheterized with 5 F vertebral catheter rather than a 2.7 F microcatheter inserted into the aneurysm sac through the fabric tear . We coilThe procedure was terminated and the patient was transferred to the intensive care unit. Unfortunately, the patient was lost due to severe heart failure 72 hours after the second procedure.Type 3 endoleak is a rare complication but potentially has a high risk of aneurismal rupture and always warrants urgent intervention . Type 3 Type 3 endoleak can be treated endovascularly or surgically. Endovascular options include repairing the defect with aortic cuff extension or placement of a new aorto-bi-iliac graft or placement of an aorto-uni-iliac graft with cross-femoral bypass. Our patient came with ruptured aneurysm and he was unstable clinically. Then we evaluated his CT angiogram; we suggested a type 3a endoleak from contralateral leg disconnection. Later type 3b endoleak from a fabric tear was proved.We tried to close the defect with an aorto-uni-iliac graft but there was bad apposition between the new and the previous grafts despite several balloon dilatations. Therefore, we closed the endoleak with coiling the aneurismal sac through the defect. However, the patient died, 3 days after the procedure.In conclusion, type 3b endoleak can occur on new generation endografts and it is associated with high-risk aneurysm rupture morbidity and mortality. It is difficult to diagnose the type of the endoleak only with CT angiogram without catheter angiography.


Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing (CBP) applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes toward the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermocellum's role in this transitory process by highlighting recent discoveries relating to its genomic, transcriptomic, proteomic, and metabolomic responses to varying biomass sources, with a special emphasis placed on providing an overview of its unique, multivariate enzyme cellulosome complex and the role that this structure performs during biomass degradation. Both naturally evolved and genetically engineered strains are examined in light of their unique attributes and responses to various biomass treatment conditions, and the genetic tools that have been employed for their creation are presented. Several future routes for potential industrial usage are presented, and it is concluded that, although there have been many advances to significantly improve C. thermocellum's amenability to industrial use, several hurdles still remain to be overcome as this unique organism enjoys increased attention within the scientific community.First isolated in 1926,  The current, non-renewable fossil fuels that supply the vast majority of energy needed for transportation will inevitably increase in cost as their supplies are depleted, and already present significant concerns relating to their longevity and sustainability, energy security, and environmental impact. For these reasons, renewable energy sources are attracting considerable attention as alternatives to their non-renewable counterparts. However, in the search for an alternative replacement, any new fuel compound must first meet three primary considerations in order to be regarded as a viable candidate: it must have the potential to supply the world's energy demands, it must be able to reduce negative environmental effects relative to current fossil fuels, and it must be cost-competitive. With the current state of the art, ethanol derived from lignocellulosic biomass addresses two of these considerations, however, its production in a cost-effective manner is currently lacking due to the difficulties in breaking down and converting the sugars locked within the lignocellulosic feedstocks.These feedstocks consist primarily of cellulose, hemicellulose, and lignin, collectively referred to as lignocellulose Figures , with sm6 and C5 sugars. In most process schemes, this conversion of biomass into sugars typically requires an initial pretreatment step to increase plant polysaccharide accessibility, followed by the hydrolytic production of glucose from cellulose, fermentation of the pentose and hexose monomeric sugar streams to ethanol, and distillation of the ethanol from the fermentation mixture . CBP technologies combine the enzyme production, hydrolysis, and fermentation stages into a single step, improving processing efficiencies, eliminating the need for added exogenous hydrolytic enzymes, and reducing the sugar inhibition of cellulases , and one strain, DSM 1313, has both a finished genome sequence and a developed genetic transformation system that allows for the construction of mutant strains  preferentially digests high degree of polymerization xylan, a hemicellulose common to birch wood, with degradation becoming increasingly efficient as the number of monomer units in xylan exceeds six. In contrast, degradation of lower, 2–5 unit, degree of polymerization xylan did not occur until 240 h later and, after 300 h, only xylose remained, as these monomers are not imported by the cell  in 120 h, compared with free Trichoderma reesei cellulases that can only achieve less than 20% conversion in the same time frame  that was attributed to the simultaneous hydrolysis of amorphous and crystalline cellulose. Cellulose experienced an increase in its weight-average degree of polymerization, while the polydispersity remained the same following microbial treatment, indicating the preferential consumption of low degree of polymerization cellulose. These findings ran contrary to those obtained using free cellulases from Neocallimastix frontalis, Trichoderma koningii, and Penicillium pinophilum, providing one of the first indications that the organization and ultrastructure of C. thermocellum's cellulosome contained unusual properties  corn stover. While Avicel displayed high conversion rates (>95%) after 24 h when treated with C. thermocellum, AFEX pretreated corn stover glucan experienced lower conversion rates (60–70%), even after extended incubation times of 4 days. While the reason for this discrepancy in efficiencies was not elucidated during this study, initial enzyme concentrations and restricted cell growth on AFEX pretreated corn stover were ruled out as possibilities , and wheat straw (Triticum vulgare) with C. thermocellum strain NCIB 10682 using either unpretreated, organosolv (methanol/water), or hydrothermolysis pretreated biomass, and used pH to indicate the extent of cellulose metabolism via acetic acid production. Under this experimental design, hydrothermally-treated poplar produced lower pHs (~6.0–7.0) than unpretreated poplar (~7.4) after 150 min of processing, signifying an increased efficiency in the presence of the hydrothermal substrate  in a high pressure bioreactor, resulting in a ~100-fold rise in the ethanol:acetate ratio compared to batch cultures at atmospheric pressure. These results were attributed to the enhanced solubility of gaseous fermentation products under their reaction conditions  content of the genome is a moderate 38.9%, and 3173 candidate protein encoding genes have been identified via automated analysis  ethanol and several analysis were performed to determine the basis of this increased tolerance in the mutant strain, which was designated strain C. thermocellum EA. Proteomic analysis of this strain by Williams et al. showed changes in membrane-associated proteins, leading them to hypothesize that the increased tolerance was the result of lower quantities and/or lower incorporation rates of proteins into the membrane, preventing increased fluidity upon ethanol exposure  and pH (9.0) than the wild type while consuming a broader range of substrates including sorbitol. However, as of yet, CS7, CS8, and S14 do not have draft genome sequences, which will be crucial for the development of genetic or metabolic engineering approaches in these strains.Isolation of additional C. thermocellum strains including the wild type (ATCC 27405), YS, LQRI, JW20, BC1, and DSM 1313 , however, once subsequently evolved, they produced contrasting results in their effect on ethanol yield. In one case, no increase in ethanol yield was observed following mutation  and has been optimized specifically for strain DSM 1313  but, in general, the thermophilic RepB origin of replication is the most prevalent for use in C. thermocellum. This origin, which works via rolling circle replication, has also been synthetically modified to generate a temperature sensitive variant that cannot function above 55°C. This provides an additional layer of flexibility that can be utilized for controllable expression of the novel DNA sequence being added  and a third region, which is referred to as the “int region” and is homologous to a 500–1000 bp region of the gene of interest, is placed downstream of the selection markers. In this multi-step process, an initial selection is performed to isolate strains that have achieved homologous recombination at the 5′ flanking and int regions, which successfully replaces a portion of the gene of interest with the remaining 3′ flanking region and cat and hpt selection markers. A second selection is then made to remove these markers (and the remaining portion of the gene of interest) and isolate the subset of strains that have performed a second homologous recombination event between the two 3′ flaking regions that are now present on the chromosome. This second recombination will successfully remove all exogenous material, leaving only the 5′ and 3′ flanking regions on the chromosome, with no genetic material between them  or Hygromycin phospotransferase (hpt) markers may be used to provide resistance against 5-fluorodeoxyuridine and hygromycin, respectively. Of these, tkd is often preferred since C. thermocellum has an endogenous hpt homolog, and thus requires an hpt deficient genetic background for proper function  and aminoglycoside phosphotransferase (neo) markers can be similarly employed for positive selection, however, the former is preferred as the latter has been demonstrated to inhibit growth at the expression levels required for selection  can also act as either a positive or negative selection maker. On the one hand, expressing the pyrF gene in a pyrF deficient host makes it possible to compliment a uracil auxotroph and select only for strains actively expressing the maker. On the other hand, treatment of pyrF-expressing strains with 5-fluoroorotic acid will lead to cellular death as those harboring the gene will incorporate it as a toxic uracil analog  have already been evaluated to assess their efficiencies in hydrogen production after using microcrystalline cellulose as a feedstock. Under these conditions, yields ranged between 0.7 and 1.2 mol of hydrogen per mol of glucose (Cheng and Liu, C. thermocellum's hydrogen synthesis pathways (Carere et al., Although ethanol has been the focus of this review, C. thermocellum's natural fermentation pathways. By mimicking the action of existing lactic acid bacteria or the fungus Rhizopus oryzae, which have previously been demonstrated to produce lactate using corn starch biomass (Hou and Shaw, C. thermocellum under the appropriate CBP conditions. Regardless, the success of these processes will rely heavily on several factors, such as the existing limitation regarding lactic acid (Cheng and Liu, In addition to hydrogen, lignocellulosic biomass remains an attractive starting material for the production of lactic acid, formic acid, and acetic acid using Clostridium spp that naturally produce butanol are non-cellulolytic, and only two, Clostridium acetobutylicum and Clostridium beijerinckii, have been studied in detail (Gheshlaghi et al., C. thermocellum, and it has recently been reported that a co-culture of C. thermocellum and Clostridium saccharoperbutylacetonicum N1-4 can produce up to 7.9 g/L butanol in 9 days using crystalline cellulose as a carbon source (Nakayama et al., C. thermocellum has led to research efforts aimed at engineering C. thermocellum with new pathways to produce butanol as well (Kastelowitz et al., C. thermocellum's natural array of products toward industrial scale production.Similarly, the production of butanol from lignocellulosic biomass using a CBP platform is another attractive option because butanol, which is more similar to gasoline than ethanol, has a higher energy density, and can be mixed with gasoline at higher ratios. Unfortunately, all The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Currently a large number of atypical antipsychotics available in the market are endorsed as better option for treating schizophrenia than the typical antipsychotics. Information regarding the utilization pattern of antipsychotic drugs is lacking in Nepalese population particularly in Western Nepal. By means of this study one is expected to acquire an idea concerning clinician’s preference to the antipsychotic drugs in actual clinical setup. The main objective of the study was to find the commonest antipsychotics prescribed in a tertiary care center among hospitalized patients in Western Nepal.This cross sectional study was carried out between 1st January 2009 and 31th December 2010 at Manipal Teaching Hospital, Nepal. The diagnosis of schizophrenia was based on ICD-10 (Tenth revision).The main outcome variables of the study was commonest antipsychotic drug prescribed. Z test, Chi square test and logistic regression were used for analytical purpose. P-value < 0.05 was considered to be statistically significant. This is the first study done on the utilization pattern of antipsychotics drugs among hospitalized patients in Nepal.Out of 210 cases of schizophrenia, most of the patients were less than 40 yrs. 78.6%, male 61.9%, unemployed 86.7% and having their monthly income less than NPR 10000 /month 80.5%. As far as religion, 78.1% patients were the Hindus and ethnically schizophrenia was common among the Dalit 26.2%. The study revealed that 46.2% of patients were students followed by 25.2% of housewives. Olanzapine was the commonest antipsychotic drug to be prescribed 34.3%. It was observed that the psychiatrists had a tendency of using antipsychotic drugs by trade names [OR 3.3 ] in male patients as compared to female patients.According to the utilization pattern of antipsychotics, it is concluded that atypical antipsychotics were used relatively more commonly than that of typical antipsychotics. Among the atypical antipsychotic drugs, there is a trend of using Olanzapine during Schizophrenia as compared to other atypical antipsychotic drugs in Western Nepal. According to WHO, Drug utilization study is defined as a study of marketing, distribution, prescription and uses of drugs in a society highlighting on the resulting medical, social and economic consequences . MedicalGlobally the prevalence of schizophrenia shows a wide range of differences . It is eEvidently, there is a dearth need of relevant literatures concerning schizophrenia in Nepal as there is no adequate research about it. A recent study conducted at psychiatric clinic in Janakpur, Nepal showed high prevalence of schizophrenia (30%) . SimilarThe main objective of the study was to find out the commonest antipsychotics prescribed for patient with schizophrenia among hospitalized patients in western Nepal. The specific objectives were to find out whether the antipsychotics were prescribed by generic or trade names, essential or nonessential drugs, drug therapy or combination of drugs and psychotherapy, groups of antipsychotics prescribed, socio demographic details of schizophrenic patients in Nepalese context. This is the first study undertaken in the utilization pattern of antipsychotics among the hospitalized patients in Nepal.The present study was a cross sectional study done at Manipal teaching hospital, Pokhara, Nepal. It is a tertiary care hospital of Western Nepal and has been providing services to patients from 1998 onwards. It was expected that all the critically ill psychiatric cases with schizophrenia will report at this hospital Western Nepal.st October 2009 and 30th September 2010 at Psychiatric ward in Manipal Teaching hospital. The collected data include socio demographic details such as age (<40 years and >40 years), gender , occupation , religion , ethnicity , employment (employed and unemployed), monthly income NPR  Treatment , trade/generic, commonest antipsychotic drug. Anatomical Therapeutic Chemical Code was also used for different antipsychotics prescribed  more tendency of prescribing medicines not from the essential drug list Nepal if the patient is from Gurung community as compared to Magar and Pun. Psychiatrist has 6.0 and 4.6 times more tendency of prescribing medicines not from the essential drug list of Nepal among students [OR 6.0] and housewives [OR 4.6] respectively. It is also observed that the tendency of using antipsychotic drugs by trade names were [OR 3.3 , Managing Editor of International Journal of Brain Research (IJBR), Section Editor of Nepal Journal of Epidemiology (NJE). He is an Organizing Committee member of International Epidemiological Association Conference 2013 and Confederation of Epidemiological Associations (CEA) Conference December 2013 Organized by Mahatma Gandhi University. He is also Assistant Secretary General of CEA. Mr. Bedanta Roy MSc Physiology. Currently working as Assistant Professor in the Department of Physiology, Manipal College of Medical Sciences, and Pokhara, Nepal. He is at present pursuing PhD in Neurophysiology. He is the Chief Editor of International Journal of Brain Research (IJBR), Managing editor of International Journal of Physiology and Pharmacology (IJPP), Section Editor of Nepal Journal of Epidemiology (NJE). He is an Organizing Committee member of International Epidemiological Association Conference 2013 and Confederation of Epidemiological Associations (CEA) Conference December 2013. He is also Assistant Secretary General of CEA.Dr. Brijesh Sathian MD(AM), PhD. working as Assistant Professor and Bio-statistics Chief in the Department of Community Medicine, Manipal College of Medical Sciences, Pokhara, Nepal. He is the editorial board member of NJE, IJBS, AIIJMS, GMJ, IJPP, IJBR and Joint Organizing Secretory and treasurer of International Epidemiological Association Conference 2013. Joint Organizing Secretary CEA  Conference December 2013 Organized by Mahatma Gandhi University. He is also the President of CEA.Dr. P.K. Chakraborty MBBS, MD Psychiatry. Currently working as Professor in the Department of Psychiatry and Hospital Director Manipal Teaching Hospital, Pokhara, Nepal. He is an organizing committee member of International Epidemiological Association Conference 2013.Dr. Archana Saha MBBS, MD Pharmacology. Currently working as Professor and Head in the Department of Pharmacology Manipal College of Medical Sciences, Pokhara, Nepal. She is the editorial board member of NJMS. She is organizing committee member of International Epidemiological Association Conference 2013.Dr. Indraneel Banerjee, MBBS, MS, MRCS. Currently working as a Senior Resident, R. G. Kar Medical College, Kolkata, West Bengal, India.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/13/96/prepub


A 67-year-old man underwent full-thickness skin graft reconstruction of an ear defect following skin cancer excision. The graft was dropped on the floor during preparation. Following decontamination, it was subsequently used to complete the reconstruction, with full graft take and no evidence of infection after 4 months of follow-up.How can one prevent graft contamination?What is the preferred method for management of a contaminated graft?Is there an antibacterial solution with superior efficacy?How should one approach medical error disclosure?Although likely underreported, graft contamination appears relatively a commonplace. In a survey, 70% 157/223) of responding plastic surgeons reported a graft contamination experience, and more than 70% of these surgeons reported multiple occurences.57/223 ofSimilar to our case, the majority of reported graft contaminations are a result of falling on the floor. Unfortunately, there is no consensus on a management protocol for dropped grafts, but the preferred method among plastic surgeons appears to be decontamination and completion of grafting, with povidone-iodine solution being the most popular decontaminant.,4,The majority of available literature regarding graft decontamination focuses on bone and tendon grafts, with relatively few studies looking specifically at contaminated skin grafts. Results are largely inconsistent, but many studies recommend decontamination with either chlorhexidine gluconate1After accidental contamination, full disclosure to the patient is an ethical imperative. Surprisingly, in the survey by Centeno et al, 60% of the surveyed surgeons did not disclose the incident and only 20% informed the patient/family postoperatively. In addition, full disclosure is also likely in the best interest of the physician, as nondisclosure is a frequently cited reason for malpractice law suits.When deciding whether or not to salvage a contaminated graft, the surgeon must take into consideration the individual risk factors of the patient, blood supply to the graft, donor tissue type, and possible alternatives reconstructive options. While there is a dearth of literature on the management of contaminated autologous skin grafts, survey data suggests that it can be salvaged without significant morbidity.


There are three errors in the Abstract section of the manuscript, specifically lines 29–34. The correct text should be: Absolute RT-qPCR on 29 all 45 animals identified RORAv5 as being significantly differentially-expressed (p = 0.038) 30 between resistant, intermediate and susceptible groups; RORAv2 was not differentially- 31 expressed (p = 0.77). Spearman’s rank analysis showed that RORAv5 transcript copy number 32 was significantly negatively correlated with parameters of susceptibility, AWC and FEC; and 33 was positively correlated with BW. RORAv2 was not correlated with AWC, FEC or BW but 34 was significantly negatively correlated with IgA antibody levels.


The cushioning of running shoes and leg stiffness influence tibial impact shock . This knTwenty-eighty physically active males , with similar left and right leg stiffness, participated in this study. Two pairs of identical custom-made running shoes, representing harder-cushioned  and softer-cushioned (10.2g) footwear, were used. The four single shoes were combined into four experimental conditions (left foot-right foot): hard-hard, hard-soft, soft-hard, soft-soft). In each condition, subjects ran 200m on a concrete track at self-selected pace. Conditions were blinded, the order randomized and a 100m run was performed in a neutral running shoe between conditions. Directly following each condition, subjects rated the cushioning of the left and right shoe separately on a visual analogue scale . A mobile 3D accelerometer  strapped to the lower back at L5-S1 recorded vertical acceleration. As a measure of running symmetry [In both of the mixed conditions, subjects perceived the soft shoe to be significantly softer than the hard shoe (p=.031), according to their actual mechanical impact hardness. Vertical impact peaks at the lower back did not differ between any of the tested conditions and were symmetrical for the mixed conditions.Despite the well described effects of shoe cushioning on tibial impact shock, impact at the lower back was not influenced by differently cushioned running shoes. Thus, runners adapted their ankle, knee and/or hip stiffness, reducing the impact shock on its way upward. Interestingly, as runners perceived different cushioning of shoes correctly, this adaptation was controlled for each leg individually, so that also in the mixed cushioning conditions the shock at the lower back remained symmetrical.Maintaining low and symmetrical impacts at the lower back seems to be important during running, and is achieved by adjusting the leg stiffness, which can even be controlled for each leg individually. In further research, the mechanism of this individual leg stiffness control should be investigated.