Prior studies investigating cortical processing in Deaf signers suggest that life-long experience with sign language and/or auditory deprivation may alter the brain’s anatomical structure and the function of brain regions typically recruited for auditory processing ( We are using the term task-negative to emphasize the absence of task in the research paradigm; however, in the literature, the two terms are used interchangeably. Recent discussions of the task-negative activations, as well as default mode network activity in the human brain , changing the roles of, and the connectivity among, the nodes of TNN. Prior studies of Deaf signers showed increase in right-lateralized processing cf.  comparedThe present study investigated this hypothesis by exploring the functional connectivity among the regions of interest (ROIs) identified within the task-negative network (TNN) in Deaf signers. To this end, we carried out functional connectivity analysis of TNN hubs in Deaf signers and hearing non-signers to explore changes in functional connectivity related to sign language experience.Two participant populations included Deaf signers and hearing non-signers who were part of a larger study that also involved fMRI . SeventeParticipants were presented with dynamic video clip stimuli in a block paradigm . During 3Full details of the task are provided in . The duration of No-task blocks was sufficient to identify TNN activation, since the network has been shown to engage rapidly in the absence of specific task  in the time domain. Data collected from five of the Deaf participants were discarded: two due to equipment malfunction, one due to left-handedness . During the Task condition, participants responded to the stimuli by pressing buttons on an MRI-compatible response box (Current Designs LLC HH-2x4-C) with their left hand, using their index finger.fic task .44See alndedness , one parAll imaging data were collected on a 3 T GE Signa HDx , with 3D FSPGR high-resolution anatomical images  acquired prior to functional scans. Functional scans were collected using a gradient echo-planar imaging sequence . Four runs of this sequence were used to collect functional data for each participant.http://www.fil.ion.ucl.ac.uk/spm). First, the initial 6 acquired volumes were removed to account for scanner stabilization, and each subject’s data were motion corrected to the 7th acquired volume; volumes associated with excessive head movement (more than 1 mm displacement between successive acquisitions) were eliminated. Data were then normalized to the standard Montreal Neurological Institute (MNI) space using the T2-weighted template provided by the SPM5 software and resliced to 2 × 2 × 2 mm3. Image registration was manually tested after the normalization process to verify the validity of this process. Each subject’s T1-weighted whole brain anatomical image was coregistered to the T1 weighted template provided by SPM5, and segmented to extract the gray matter maps. These maps were then optimally thresholded using the Masking toolbox of SPM5 to produce binary masks to be used as explicit masks in subsequent analyses. The last pre-processing step consisted of smoothing the functional data with an isotropic Gaussian filter (FWHM = 8 mm) to compensate for anatomical variability between subjects, and to match the statistical requirements of the general linear model.Preliminary fixed effects analysis of functional imaging data was carried out using SPM5 software  for No Task vs. Task as revealed by random-effects analysis were identified.Individual participant analyses were first performed in all subjects in order to identify the areas of the brain differentially activated during Task and No Task periods. For each subject, 5See  Seed regions of interest (ROIs) - spheres with 5 mm radius—were centered in peak task-independent deactivation coordinates from the Meta-Analysis from 6The full nomenclature of default network nodes in  For each participant the voxel timecourse in the ROIs was regressed against the time series for the motion correction parameters and global signal of the whole brain. Partial correlation analysis (regressing out time series from the other ROIs) was performed on each pair of regions using the first component of independent component analysis (ICA) in the signals from individual ROIs. Z-scores were then computed from the Pearson product-moment correlation coefficients for each ROI pair for each participant using Fisher r-to-Z transformation. Pairwise regional connectivity among TNN hubs in Deaf and hearing participants was then compared using independent-samples t-test in SPSS 15.Functional connectivity analysis was performed on pre-processed fMRI data using partial correlation based on ICA after global signal regression.7Inferior parietal lobe (IPL) includes the portion of the cortex that lies below the horizontal segment of the intraparietal sulcus, and behind the lower part of the postcentral sulcus. In the table for Deaf participants, IPL activation is reported in combined clusters with adjacent activation in the occipital cortex.) and dorsomedial prefrontal (dMPFC) cortices in left and right hemispheres. However, Deaf participants did not exhibit activation of Lateral Temporal cortex in TNN, while hearing ones did ; right IPL and right MTG . The connectivity between the following regions was higher in non-signers than signers: PCC and right MTG , and right IPL and left MTG   see . No otheHigher functional connectivity between rIPL and rMTG clusters in Deaf signers vs. hearing non-signers suggests that the parietal cortices in the Deaf might be used to process components of the visual linguistic signal, indicating experience-based difference in processing networks for dealing with systematic input.In Deaf signers, two TNN nodes within the right hemisphere—rIPL and rMTG—showed higher functional connectivity than in hearing non-signers. Although increased right hemisphere activation has been an important issue in studying the neural basis of sign language processing , our anaduring the task (as observed in In Deaf signers, PCC showed stronger correlation with left MTG than in hearing non-signers. Prior analyses of PCC’s role in language processing  suggesteThe contribution of the data on TNN activation and functional connectivity to the current literature on the dorsal/ventral pathway analysis in processing of linguistic and visual information  is the iAdditionally, the observation that lifelong visual language experience leads to changes in TNN that include an increase in connectivity of right IPL and MTG cortices, and PCC and left MTG contributes to the laterality debate surrounding sign language processing , suggestOne possible question that can be raised is whether the findings might be due not to sign language experience, but auditory deprivation instead. One recent study that evaluated participants with varying levels of auditory deprivation and sign language experience  found thAnalysis of the task-negative network activity in Deaf signers demonstrates that visual language experience is associated with increased correlation in the activity of the precuneus/posterior cingulate and left MTG, as well as higher functional connectivity between right IPL and MTG - areas that have been found to show functional recruitment during visual language processing and event schema retrieval. These findings suggest that experience with processing visual language, and subsequent connectivity alterations in the default mode network aimed at predictive/automatized processing of the visual signal, affects organization of the brain at the level of inter-network connectivity. Future studies with hearing signers will be needed to determine with certainty whether the observed differences in functional connectivity between Deaf signers and hearing non-signers are due to sensory deprivation, or sign language experience.


Chloroflexi and Firmicutes in more oxic and nutrient poor post-WWTP sediments. Similarly, quantitative PCR analysis showed 1–3 orders of magnitude reduction in phylogenetic and functional genes of sulphate reducers, denitrifiers, ammonium oxidizers, methanogens and VC-respiring Dehalococcoides mccartyi. In contrast, members of Proteobacteria adapted to nutrient-poor conditions were enriched in post-WWTP samples. This transition in the trophic state of the hyporheic sediments reduced but did not abolish the VC respiration potential in the post-WWTP sediments as an important hyporheic sediment function. Our results highlight effective nutrient load reduction and parallel microbial ecological state restoration of a human-stressed urban river as a result of installation of a WWTP.The impact of the installation of a technologically advanced wastewater treatment plant (WWTP) on the benthic microbial community of a vinyl chloride (VC) impacted eutrophic river was examined two years before, and three and four years after installation of the WWTP. Reduced dissolved organic carbon and increased dissolved oxygen concentrations in surface water and reduced total organic carbon and total nitrogen content in the sediment were recorded in the post-WWTP samples. Pyrosequencing of bacterial 16S rRNA gene fragments in sediment cores showed reduced relative abundance of heterotrophs and fermenters such as  Rivers in urban areas are often heavily impacted by untreated wastewater discharge containing excessive organic inputs that can induce anoxia, eutrophication of freshwater ecosystems and severe reduction in water qualityParallel to reducing nutrient load into downstream receiving ecosystems, complementary measures are mandatory to monitor their ecological state. How will reducing the nutrient concentrations of treated effluent affect the microbial structure and function in the receiving environment ? How will it impact the trophic state of the ecosystem and processes dependent on otherwise high nutrient load ? Allochthonous organic carbon for example fuels river biogeochemical activities101112In spite of intensive studies on microbial community composition and function in hyporheic zones1415161718192021222324262726www.aquiris.be). The biological treatment line of the plant consists of a primary settling stage and a subsequent modern tertiary treatment technology for elimination of nitrogen, phosphorus and carbon-containing pollution by an activated sludge process. The implementation of the WWTP resulted in important chemical changes in the surface water composition such as decrease in organic carbon content and increase in dissolved oxygen concentration. For a conclusive assessment of the impact of the installation of a WWTP on the receiving river ecosystem, we studied the sediment microbial community as indicator for system recovery and impact on particular ecosystem function i.e. vinyl chloride (VC) respiration. In contrast to former studies based on differences in upstream and downstream sediment microbial community where no information was available about the pre-WWTP conditions2627In the present study, we investigated microbial community composition and function across sediment depth in a CAH impacted hyporheic zone of a short stretch of the Belgian Zenne River, as a response to an upstream WWTP installation. The Zenne river was notorious for being one of Belgium’s most polluted rivers receiving untreated municipal effluents from the Brussels Capital Region leading to severe eutrophication of the river. In previous studies performed at that river stretch between 2004 and 2007, during which the Zenne surface water was eutrophic, the highly reducing and organic rich sediments were shown to be conducive to natural attenuation of CAHs present in the discharging groundwater mainly by organohalide respiration (OHR)1213ρ correlation was found significant at the 0.25 level after Benjamini-Hochberg correction for multiple testing , oxygen , pH , Fe  and conductivity  showed significant differences. In particular we observed that the dissolved oxygen content in the surface water increased to above 3 mg/L in 2007 and on average above 4 mg/L after 2008 . DOC concentrations in the surface water decreased from an average of 51 ± 27 mg/L in the period Dec 2005-May 2006 to an average of 7 ± 1 mg/L in May-Sept 2011 . The DOC and sulphate values in interstitial water (ISW) collected by means of permanent pore water samplers were not significantly different  and core-B in 2011 . The total nitrogen (TN) content on the other hand decreased with depth only for core-B in 2005  and core-A in 2010 . A significant decrease over time for both TOC and TN values was observed only in the top 20 cm sediment horizon   in the surface water during the experimental period : none of testing . Howeverifferent . The conifferent  in most ctively) .7 copies per gram of sediment , methanogens (mcrA), bacterial/archaeal ammonium oxidizers  and denitrifiers  significantly decreased over time in post-WWTP . In contrast, Nitrospira had higher relative abundance in deeper sediment layers . No significant depth-related stratification of bacterial community composition was noted in post-WWTP samples at the phylum level (data not shown). However, significant differences were observed between pre- and post-WWTP samples. The relative abundance of Chloroflexi and Firmicutes significantly decreased in post-WWTP samples while those of Nitrospirae and Proteobacteria increased significantly , Clostridiales (Clostridia class), Rhodocyclales (Betaproteobacteria class) and Desulfobacterales (Deltaproteobacteria class) showed a significant reduction in relative abundance in post-WWTP samples  and Xanthomonadales (Gammaproteobacteria class) significantly increased in relative abundance over time in post-WWTP samples  and richness (Chao1 and OTU count) indices did not show significant difference between two cores . Similar2 concentration in surface water. The samples of 2010 were distinctly grouped in the centre and along the second canonical axis  to study the relationship of environmental parameters and bacterial community composition at the order level . The 200sediment .The objective of this study was to assess the long-term impact of the implementation of a WWTP on the size, structure and function of microbial communities residing in the hyporheic zone of a stretch of the Belgian Zenne River impacted by a VC-contaminated groundwater plume. Previous studies on riverbed sediments impacted by WWTP effluent showed decreased microbial diversity262 increase in the Zenne surface water. This translated into effective reduction of TOC and TN in post-WWTP sediments in particular in the surficial sediment horizons (dsrB), denitrifiers  and methanogens (mcrA) in pre-WWTP sediments that in most cases significantly decreased in post-WWTP sediments (amoA) in pre-WWTP sediments could be due to ample presence of nitrogen resources  in the pre-WWTP hyporheic sediments favored other bacterial community members that are active under anoxic and sub-oxic conditions. Accordingly, bacterial fermenters, such as members of the Lactobacillales and Clostridiales, sulphate-reducing Desulfobacterales and denitrifying members of Rhodocyclales showed strong positive correlations with organic carbon content of the surface water and sediment  that are commonly found to coexist with OHRB, and have been suggested to drive OHR by syntrophic interactions and exchange of essential metabolites32333334The low-organic carbon concentrations after the installation of the WWTP also correlated with reduced abundance of Burkholderiales sequences showed a significant increase over time especially in the deeper sediment horizons  and richness  between the pre- and post-WWTP sediment samples, one of the 2010 cores showed significantly higher values compared to the remaining cores, whereas one of the cores of 2011 had significantly lower richness and diversity. Conflicting results have been reported for the impact of WWTP on sediment bacterial diversity with some showing an increase in bacterial diversity downstream of a WWTP effluent input2826151621151615Our study showed that operation of a technologically advanced WWTP effectively promoted low-nutrient conditions in the longer term. This was translated into transition of the bacterial community in the riverbed hyporheic sediment from members favoured under nutrient-rich and reducing conditions to those adapted to nutrient-poor and oxic conditions, indicating that microbial communities are good sentinels and integrators of environmental change. The low-organic condition did not have a detrimental impact on VC respiration as one of the important hyporheic functions at CAH-contaminated sites fuelled by organic matter. Overall, the results of this study showed a positive impact of the technologically advanced WWTP on the sediment bacterial community, in line with sustainable water resource management aims.3/s in dry weather conditionscis-dichloroethene and in particular VC as degradation products of reductive dechlorination of PCE and TCE, were reaching to the Zenne riverbed sediment12The study site is a short transect of the Zenne River located in an industrial area near Brussels, Belgium. The test site was previously described in detailSediment cores were collected from the top 80-cm layer of the riverbed sediment in December 2005, May 2010 and September 2011, at post 25 P25,  using a 2. Approximately 37 g (wet) sediment was transferred to 160-ml glass serum bottles containing 70 ml groundwater from the monitoring well SB-1. All microcosms were prepared in duplicate. Two extra microcosms prepared from the core C sample of 2011 were supplemented with 1 g tree bark as a source of solid organic carbon and electron donor as described elsewhereRiverbed sediment microcosms were prepared directly after each sampling campaign with samples taken from the top (0–20 cm depth) sediment layer of the core C samples. The microcosms were prepared in an anoxic glove box  containing an atmosphere of ND. mccartyi and selected protein-encoding genes commonly used as biomarkers for specific functional guilds, i.e. sulphate reducers (dsrB), denitrifiers , methanogens (mcrA), and bacterial/archaeal ammonium oxidizers  were quantified for the 40 selected samples. The qPCR measurements were performed in triplicate in 25-μL reactions in an iQ5 iCycler  using the iQ SYBR Green Supermix kit (Bio-Rad). See Methanosarcina mazei  was used as standard for mcrA quantification.All A and B sediment cores were divided into 1-cm slices until a depth of 60–70 cm. At 5 cm intervals, a total of 80 sediment samples of approximately 2 g were used for DNA extraction as described previouslyBacterial 16S rRNA genes were first amplified from the DNA extracts using the general 27F/1492R primer setPyrosequencing data were processed and analysed using QIIME v1.8.0Concentrations of CAHs, ethene, and ethane were determined via head-space analysis on a Varian GC-FID (CP-3800) as described previouslyUnivariate statistical analysis  were performed within the Matlab environment , unless otherwise stated. For all statistical analysis, Benjamini-Hochberg59Accession codes: The complete dataset is available at the European Bioinformatics Institute (www.ebi.ac.uk) under accession number PRJEB8703.How to cite this article: Atashgahi, S. et al. Impact of a wastewater treatment plant on microbial community composition and function in a hyporheic zone of a eutrophic river. Sci. Rep.5, 17284; doi: 10.1038/srep17284 (2015).


Immune recognition of foreign proteins by T cells hinges on the formation of a ternary complex sandwiching a constituent peptide of the protein between a major histocompatibility complex (MHC) molecule and a T cell receptor (TCR). Viruses have evolved means of "camouflaging" themselves, avoiding immune recognition by reducing the MHC and/or TCR binding of their constituent peptides. Computer-driven T cell epitope mapping tools have been used to evaluate the degree to which particular viruses have used this means of avoiding immune response, but most such analyses focus on MHC-facing 'agretopes'. Here we set out a new means of evaluating the TCR faces of viral peptides in addition to their agretopes, integrating evaluations of both sides of the ternary complex in a single analysis.Janus Immunogenicity Score (JIS), bringing together a well-established method for predicting MHC binding, with a novel assessment of the potential for TCR binding based on similarity with self. Intuitively, both good MHC binding and poor self-similarity are required for high immunogenicity .This paper develops what we call the Focusing on the class II antigen-processing pathway, we show that the JIS of T effector epitopes and null or regulatory epitopes deposited in a large database of epitopes (Immune Epitope Database) are significantly different. We then show that different types of viruses display significantly different patterns of scores over their constituent peptides, with viruses causing chronic infection  strongly shifted to lower scores relative to those causing acute infection (Ebola and Marburg). Similarly we find distinct patterns among influenza proteins in H1N1 (a strain against which human populations rapidly developed immunity) and H5N1 and H7N9 .The Janus Immunogenicity Score, which integrates MHC binding and TCR cross-reactivity, provides a new tool for studying immunogenicity of pathogens and may improve the selection and optimization of antigenic elements for vaccine design. Cellular immune defenses against infectious diseases are dependent on the immune system's ability to identify and control invading pathogens without causing too much collateral damage. To accomplish that, the immune system employs complex networks of interacting cells and molecules. In one key network, Antigen Presenting Cells (APCs) process and then present to circulating T cells peptides that they have derived from pathogenic proteins. An APC loads such a peptide, called a T cell epitope, onto its Class II Human Leukocyte Antigen (HLA), also known as Major Histocompatibility Complex (MHC). Engagement of the T Cell Receptor (TCR) with the T cell epitope:MHC II complex on the surface of the APC triggers a cascade of events leading to activation of the T cell response. Thus the ternary MHC:T-cell epitope:TCR complex connects the APC to the responder T cell ,2 Figur.This antigen-presenting pathway is constantly processing proteins that have been sampled from the extracellular environment. In the case of viruses and bacteria, peptides (derived from their constituent proteins) that bind MHC and TCR are recognized as foreign and trigger an immune response. T helper (MHC class II-restricted) epitopes within viral and bacterial proteins are a necessary component of immune protection against these pathogens since the presentation of a T-cell epitope in the context of MHC Class II molecules is essential for a B-cell to be stimulated and produce high-affinity and high-titer antibodies. Since this interaction is the one that initiates a specific immune response against the pathogen, one means by which pathogens evade immune response is to present proteins with lower immunogenic potential . .Computational T-cell epitope mapping tools have been used to evaluate the degree to which selected pathogens have reduced their epitope content in order to evade immune response. For example, one study of CD8+ T cell epitopes in viruses showed that those infecting humans contained fewer CD8+ T-cell epitopes than non-human-host viruses, with human-host viral proteins expressed earlier in the life cycle having a lower epitope density than those expressed later . SimilarCharacterizing and making predictions for the TCR side of recognition is substantially harder than for the MHC side, due to the essentially unpredictable variability in the complementarity determining regions of the TCR. However, in the context of pathogenic immune escape, there is one critical aspect that is more tractable for analysis: similarity to self. Autologous proteins are also processed and presented by antigen presenting cells, but they do not usually trigger immune responses by T cells due to mechanisms of central and peripheral tolerance. For T cells, initial self/non-self discrimination occurs in the thymus during neonatal development when medullary epithelial cells express tissue-specific self-proteins and present the epitopes therein to immature T cells. T cells recognizing self-antigens with high affinity are deleted; auto-reactive T cells with moderate affinity may escape deletion and be converted to function as 'natural' regulatory T cells (Treg) cells . While ccross-reactive (binding the same TCRs). Cross-reactivity is an intrinsic characteristic of the T cell receptor, in that each TCR can potentially interact with many different T cell epitopes [Assuming then that autologous proteins are less likely to generate T effector responses, an immune escape mechanism for a pathogen is to ensure that MHC-binding peptides within its proteins present human-like TCR faces. This is a more relaxed requirement than full "humanization" ; instead, the peptides only need to be epitopes -22. T ceepitopes ,24.Thus one means of immune escape by viruses may be to present T cell epitopes recognized by nTregs or iTregs. TCR-level cross-reactivity with autologous proteins is of great interest in general as T cells responding to a foreign protein may have diminished or altered type of immune response, and auto-reactive T cells may be triggered by this cross-reactivity . We are Computational analyses of sequence similarity between predicted epitopes in pathogens and in the human genome have been used to study TCR-level cross-reactivity. Previous studies  showed fSo as to enable broad computational studies of cross-reactivity, we recently developed a new immunoinformatic tool, JanusMatrix . This toBuilding on this observation, this paper presents a new framework for quantitatively characterizing immunogenicity risk, unifying the MHC and TCR sides of immune recognition. The Janus Immunogenicity Score (JIS) provides a probabilistic assessment of the potential of a peptide to induce a T effector response, combining evaluation of MHC binding potential with identification of cross-reactivity against the human genome. We show that JIS is predictive of Teff data deposited in the immune epitope database (IEDB). We then show that it reveals patterns of camouflage that differ between hit-and-stay viruses that cause chronic infection  and hit-and-run ones that cause acute infection . We also consider a wide range of other human-host viruses and see that most appear quite similar in terms of their relative camouflage, while some are striking outliers. We finally zoom in and apply a similar analysis to specific influenza proteins from different strains, characterizing their relative visibility to the human immune system.J is defined for a peptide p with respect to a set A of MHC alleles. Throughout this manuscript, we use for A eight common representative alleles  that "cover" the genetic backgrounds of most humans worldwide [p into a set of overlapping 9mers, and assessing each such 9mer e for both MHC binding to each allele a ϵA, FMHC, and also conditional TCR recognition of the epitope: MHC complex, FTCR|MHC if e is an MHC binder. Then J combines information across alleles and epitopes.The Janus immunogenicity score orldwide . The anaEpiMatrix is a pattern-matching algorithm used for identification and prediction of T cell epitopes . EpiMatre that is predicted to bind allele a , FMHC is defined as the cumulative value in the Normal distribution for the Z-score  is undefined. Except for one illustrative discussion point, the 5% threshold is used throughout the results.For an epitope e Figure , left - JanusMatrix  examinesFTCR|MHC is then the tail probability in this empirical distribution . Note tn Figure , right -E be the set of 9mer epitopes within p that are predicted to bind one or more alleles. Each such epitope e is evaluated against each allele a in A. A joint probability of MHC binding and conditional TCR binding ensures that both sides of the epitope sandwich are satisfied - the combination of good MHC and good TCR binding implies a higher probability of being immunogenic. A joint probability over the alleles A encodes the notion that a promiscuous epitope is more likely to be immunogenic [J is computed as:Let unogenic . AllelesT cell data  for five relevant cytokines  MHC-typed to one of the eight representative alleles listed above was downloaded from the IEDB . A peptiTo study the two extremes of viral camouflage, four viruses from the UniProt reviewed set were selected: Epstein-Barr virus  and cytomegalovirus  were selected to serves as representatives of long-lived infection viruses, and Ebola (strain Mayinga-76) and Marburg (strain Musoke-80) were selected to serve as representatives of short-lived/zoonotic viruses. The JIS for each overlapping 15mer "fragment" was computed for each virus; 15mer fragments with no predicted epitopes were separately noted. As a control for the zoonotic extreme, a set of 73 plant-host viruses from  was likeFor a more diverse set of human-host viruses, the all-fragment analysis was performed on all 51 human-host viruses . Each viTo study differences among related strains of the same virus, JIS was computed for each 15mer fragment in three representative influenza A strains: H1N1 , the most recent pandemic strain; H5N1 (A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd), a representative H5 avian influenza strain that has been responsible for many human deaths; and H7N9 (A/Shanghai/2/2013), also a recently emerged avian strain that caused more than 100 infections and 44 deaths in 2013. The JIS distribution was evaluated separately for the fragments in each of the two surface proteins hemagglutinin (HA) and neuraminidase (NA), as well other common proteins: nucleoprotein (NP), matrix proteins (M1 and M2), and non-structural proteins (NS1 and NEP).A total of 588 peptides with Teff-pos peptide responses and 76 with Teff-neg/null responses were identified from the IEDB. To assess whether JIS is characteristically different for Teff-pos and Teff-neg/null, the distributions of scores for each set were separately characterized. The epitope predictor threshold separating MHC binders vs. non-binders is typically set to control the false positive rate (by taking only the most confident predictions), but it also naturally impacts the false negative rate. In this dataset, the standard 5% threshold eliminated 11% of the Teff-pos peptides and 20% of the Teff-neg/null peptides as having no predicted epitopes, while a more relaxed 10% threshold eliminated 4% and 5%, respectively. While these data points could be considered false negatives for immunoinformatics analysis, they might also be false positives in the T cell assay. To be consistent with standard practice and keep false positive rate low, the 5% threshold was employed for the remainder of the analysis.Figure While JIS is computed over the eight standard alleles (capturing increased likelihood of immunogenicity for promiscuous binders), most peptides in the IEDB were tested for only one allele. The dataset includes 646 allele-specific characterizations for the 588 Teff-pos peptides and 79 for the 76 Teff-neg/null ones. Computing JIS for just the tested allele  results in the distributions illustrated in Figure -6 for Ebola vs. plant-host viruses, and 6*10-4 for Marburg vs. plant-host viruses. Most of these relationships hold even among just the binders, indicating a significant shift in the quantitative scores, though the EBV vs. CMV p-value increases to .03 and EBV vs. Marburg to .01; CMV vs. Marburg crosses the usual line up to .06. Thus we conclude that the immunogenicity profiles of commensal hit-and-stay viruses are significantly different from hit-and-run viruses that cause acute disease and high mortality, in two ways. Commensal viruses appear to avoid immune response by (1) reducing their epitope content and (2) adopting more human-like TCR faces among the remaining epitopes, when compared to acute viruses and plant-virus controls.As Figure y coordinates to avoid overlap). Most of those on the low JIS side  do indeed chronically infect humans and thus face evolutionary pressure to camouflage themselves reducing immune response. Figure Figure FTCR|MHC as well as values for human genome. As is well-known (and encapsulated in epitope predictors), T cell epitopes are enriched in hydrophobic residues ; the plot shows that in this dataset, the enrichment comes particularly at the expense of negatively charged residues  as well as a few others . The acute Marburg has similar trends, while the chronic EBV is less consistent and even reverses some of them. Human also reverses some of the trends. Rubella goes against the trends to an extreme degree, often in the same direction as human and away from binders, but much further. Quantitatively, the Euclidean distance from the background frequency distribution to the Rubella one is .117, while the distance from background to EBV is .057, to Marburg .058, to binders .067, and to human .037. Rubella is even further away from the set of binders, at a distance of .153; thus, Rubella has strikingly different amino acid prevalence. While the implication of this finding is uncertain, it may relate to an as-yet undescribed means of immune escape. In future studies we plan to identify pathogens that have similar amino acid distributions and examine their relationship with human hosts.The striking outlier in this analysis is rubella, for which a substantial fraction 50.8%) of fragments contain no predicted binders and thus which cannot be shown in the median-JIS plot. In order to better understand why this virus exhibits this pattern, and how that relates to some of the other representative viruses, the overall amino acid content within the different genomes was characterized. Figure % of fragFor comparisons within virus species, JIS distributions can be separately characterized for different proteins. Using influenza as a case study, Figure In contrasting the different strains, H7N9 is generally more deimmunized than H5N1 and H1N1, and is for both HA and NA in particular. HA5 is also more deimmunized than HA1. The low immunogenicity scores of H7N9 has been previously reported by our group. The significance of this finding is unknown, since neither virus would have normally interacted with the human immune system; this finding may be relevant to immune responses that are directed against the virus by its other hosts (birds and swine).While cross-reactivity is now understood as an intrinsic characteristic of the T cell receptor, critical to training and protection, this paper presents the first attempt to quantitatively evaluate it, and to use such a characterization in a genomic-scale study of how pathogens may use this means of escaping an immune response. Our model unifies into the novel Janus Immunogenicity Score both sides of recognition: MHC-based epitope prediction and extent of TCR-level cross-reactivity against self peptides (conditioned on MHC binding). The predictive power of the JIS is demonstrated by comparing epitopes in the IEDB that are associated with T effector cytokines ("Teff-pos") vs. those that are associated with regulatory T cell cytokine profiles or lack of immune response ("Teff-neg/null"), and finding a statistically significant difference in scores.With this immunogenicity score in hand, we studied profiles of viruses associated with chronic infection (hit-and-stay) vs. those that cause acute disease and higher mortality (hit-and-run), and found them to be significantly different. Commensal viruses appear to avoid immune response by (1) reducing their epitope content and (2) adopting more human-like TCR faces among the remaining epitopes, when compared to acute viruses and plant-virus controls. When expanding the analysis to a broader set of viruses, several of these also appear to have evolved sequences that contained reduced T cell epitope content and increased human cross-reactivity at the TCR facing residues of T cell epitopes (stronger low-JIS tail in the distribution). Rubella is a striking outlier, which may relate to an as-yet undescribed means of immune escape. In future studies we plan to identify pathogens that have similar amino acid distributions and examine their relationship with human hosts.And finally, among influenza viruses, H7N9 is generally more deimmunized than H5N1 and H1N1, and is for both HA and NA in particular. HA5 is also more deimmunized than HA1. The low immunogenicity scores of H7N9 has been previously reported by our group. The significance of this finding is unknown, since neither virus would have normally interacted with the human immune system; this finding may be relevant to immune responses that are directed against the virus by other hosts (birds and swine).APC: antigen presenting cell; CMV: cytomegalovirus; EBV: Epstein-Barr virus; IEDB: immune epitope database; JIS: Janus immunogenicity score; MHC: major histocompatibility complex; TCR: T cell receptorCoauthors ADG, WM and LM are employees of EpiVax, a vaccine and therapeutic design company, and two  are majority stockholders. These authors recognize the presence of potential conflicts of interest and affirm that the information represented in this paper is original and unbiased observations. In addition to his role as a faculty member at Dartmouth, CBK is co-founder and CTO of Stealth Biologics, LLC, a therapeutic protein design company. Dartmouth has worked with him to manage all potential conflicts of interest arising from his commercial affiliation, and he likewise affirms that this paper presents work free of any bias. LH and ANG declare no competing interests.LH, ADG, WM, and CBK conceived of the idea; LH and CBK designed the methods with feedback from the others; LH implemented the methods and collected the results; LH and CBK analyzed the results with feedback from the others; LH, ADG, and CBK wrote the paper. All authors read and approved the final manuscript.


Results from geochemical analyses, microscopy , 16S rRNA gene amplicon-sequencing and metatranscriptomics were compared to results from previous analyses of biofilms growing on black smoker chimneys at LCVF. Based on our results, we constructed a conceptual model involving the geochemistry and microbiology in the LCVF. The model suggests that CH4 and H2S are important electron donors for microorganisms in both high-temperature and low-temperature areas, whereas the utilization of H2 seems restricted to high-temperature areas. This further implies that sub-seafloor processes can affect energy-landscapes, elemental cycling, and the metabolic activity of primary producers on the seafloor. In the cotton-like microbial mats on top of the active barite chimneys, a unique network of single cells of Epsilonproteobacteria interconnected by threads of extracellular polymeric substances (EPS) was seen, differing significantly from the long filamentous Sulfurovum filaments observed in biofilms on the black smokers. This network also induced nucleation of barite crystals and is suggested to play an essential role in the formation of the microbial mats and the chimneys. Furthermore, it illustrates variations in how different genera of Epsilonproteobacteria colonize and position cells in different vent fluid mixing zones within a vent field. This may be related to niche-specific physical characteristics. Altogether, the model provides a reference for future studies and illustrates the importance of systematic comparative studies of spatially closely connected niches in order to fully understand the geomicrobiology of hydrothermal systems.In order to fully understand the cycling of elements in hydrothermal systems it is critical to understand intra-field variations in geochemical and microbiological processes in both focused, high-temperature and diffuse, low-temperature areas. To reveal important causes and effects of this variation, we performed an extensive chemical and microbiological characterization of a low-temperature venting area in the Loki's Castle Vent Field (LCVF). This area, located at the flank of the large sulfide mound, is characterized by numerous chimney-like barite (BaSO These disequilibria support primary production by diverse chemolithoautotrophic microorganisms , located at the Arctic Mid-Ocean Ridge (AMOR) in the Norwegian-Greenland Sea, is a sediment-influenced, basalt-hosted hydrothermal field with emanating fluids with high concentrations of H4)   equipped with video facilities was used to collect samples during research cruises to the LCVF in 2009 and 2010 with the research vessel G.O. Sars. From the barite field we collected three microbial mats using a 1 L hydraulic sampling cylinder (biosyringe), and two barite chimneys and one sediment sample using an aluminium scuffle box  containing two distinct central flow channels with gray to black colored walls , air-dried, mounted on an aluminium specimen stub and coated with iridium in a Gatan 682 coater. The sample was studied by a Zeiss Supra 55VP field emission scanning electron microscope , equipped with a Thermo Noran System SIX energy dispersive spectrometer (EDS) system .Sulfurimonas phylotype and labeled with Atto RHO 101. NONEUB338 probes were labeled with Atto RHO 101 and used as control for non-specific staining  .DNA was extracted from the microbial mats using the MasterPure™ Complete DNA and RNA Purification Kit . The FastDNA spin kit for soil and the FastPrep® -24 Instrument  was used for DNA extractions of chimney and sediment sections. The amounts of nucleic acids were determined by AThe 16S rRNA gene (the V5–V8 region) was amplified with the primers Un787f (5′-ATTAGATACCCNGGTAG)  with added random hexamer primers . The RNA used was extracted simultaneous with DNA of Mat1 using the MasterPure™ Complete DNA and RNA Purification Kit . The MinElute PCR purification kit  was used for sample clean-up and concentration. The cDNA protocol was implemented using triplicate samples that were later pooled and concentrated using Eppendorf concentrator 5301 . In total, 673 ng of double stranded cDNA, as measured by SYBR-Green quantification , using BLASTN , and denoted as putative mRNA reads.cDNA reads were filtered in MOTHUR .In addition, mRNA transcripts were analyzed in MG-RAST  using scripts distributed with AMPLICONNOISE  and the active barite chimneys , the most abundant taxa were the proteobacterial classes Epsilonproteobacteria and Gammaproteobacteria, and the archaeal phylum Euryarchaeota Figure . In contSulfurimonas was by far the most abundant genus  and BaCh2 (22.5–37.0%), whereas in the sediment Sulfurimonas comprised a minor fraction of the community. The genus Arcobacter was also present in the microbial mats and Sulfurovum was identified in the barite chimney channels and the black sediment. Epsilonproteobacteria are widespread and account for a significant fraction of deep-sea vent chemoautotrophs  where s Figure . SulfuriIn the white interiors of the barite chimneys , Methanomicrobia were highly abundant comprising 50.3 and 37.1%, of the total reads, respectively. The Methanomicrobia were of uncultured, anaerobic, methanotrophic archaea (ANME-1) and GOM Arc-1, except in the rusty sediment where they were of the AAA-subcluster Figure . ANME-1 Thiomicrospira in the exterior section of chimney 1 (BaCh1O). Thiomicrospira spp. isolated from hydrothermal vents utilizes sulfur compounds , a high proportion of Gammaproteobacteria was observed Figure . Most noNitrosopumilus, which includes ammonia-oxidizers  dominated the black sediment  had a high abundance of Thaumarchaeota, of which MGI was dominating Figure . In addit Figure . These tt Figure . The Sh7t Figure , comprist Figure . Unique Sulfurimonas , H2 (4.7–5.5 mmol kg−1), −1) and CO2 (22.3–26.0 mmol kg−1) concentrations . Finally, hydrogenase transcripts were not detected in the Sulfurimonas dominated mat  and denitrification . As previously noted for Epsilonproteobacteria, carbon-fixation by the Sulfurimonas population is mediated by use of the rTCA cycle and transcript of RubisCO known to operate in sulfur-oxidizing Gammaproteobacteria were not detected  of SEEP-SRB1 was identified in the barite chimney, indicating that the ANME-1 population was free-living as previously observed in Nyegga pockmarks  (Knittel and Boetius, Candidatus Scalinuda, which may grow by anaerobic ammonium oxidation, putative aerobically ammonium-oxidizing Nitrosopumilus as well as nitrite-oxidizing Nitrospina in the rusty surface sediment in the barite field. Furthermore, a high proportion of MGI was seen in the black sediment horizon. These results indicate that ammonium oxidation is in fact a biologically relevant energy source in LCVF and point to a possible hotspot for biological nitrogen cycling, in the sediments. However, it should be noted that a high fraction of MG1 was also seen in the distinct barite-silica chimneys. Furthermore, planctomycetes performing anammox are found to carry out additional metabolic properties (Strous et al., Candidatus Scalindua in the nitrogen cycling at LCVF will ultimately require in situ rate measurements of nitrification and anammox.Neither the low-temperature venting barite chimneys Figure  nor the Sulfurimonas, which where interconnected and polarly attached to ultrathin EPS threads (Figure Sulfurimonas mat structure appeared to represent a specific adaptation to life in this environment as it seems to position the cells optimally for an efficient acquisition of energy on top of the venting barite chimney. This point is made even more clearly when we compare the Sulfurimonas mats from this study with the mat comprising Epsilonproteobacteria of the Sulfurovum genus observed on a black smoker chimney wall of LCVF (Stokke et al., Sulfurovum-dominated mat was made up of long filaments surrounded by thermotolerant sheaths, presumably composed of a polymer resembling chitin or cellulose. A similar structure was also observed for Sulfurovum epibionts growing on the anomuran crab, Shinkaia crosnieri (Watsuji et al., Arcobacter, forming sulfur-filaments (Sievert et al., Whereas energy availability may be key factors controlling the distribution of functional groups of organisms in the diffusely venting site in LCVF, our data also indicate that the distribution of specific ecotypes may be controlled by specific physical factors constraining the structuring of the microbial mats. The microbial mats situated on top of the barite chimneys consisted almost exclusively of members of s Figure . To our IS designed the experiments, conducted the research and wrote the paper. HD bioinformatic data analysis and writing. RS bioinformatic data analysis. IR lab work and writing. FD lab work and writing. HR environmental sampling and writing. RP environmental sampling and writing. IT lab work and writing of paper.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.


The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion. E.coli (Eco-MscL) is the best-known protein channel.[Mechanosensitive channels are integral proteins located on the bacterial cytoplasmic membrane, whose function is that of a natural pressure valve preventing cell lysis in case of hypoosmotic stress.,2 Among  channel.,4 In res channel.,6 in whiet al. [Mutagenesis experiments in Eco-MscL identified residues L19-V23 to be critical for channel gating; the corret al. : in thiset al. ,17 it haIn light of such remarkable applications, several aspects of the charge-induced gating mechanism deserve further investigation, among which i) the scale of protein conformational changes upon charge introduction, ii) the extent of the channel structural expansion and iii) the selectivity and geometric limitations of molecular transport across the channel. Such details demand an atomistic level understanding of the interplay between the induced electrostatic effects and the protein structural rearrangements. In this regard, molecular dynamics (MD) simulations can be profitably used to provide structural characteristics and dynamical features of protein channels, while retaining explicitly the environmental effects mainly due to the lipid bilayer and the solvent. Previous computational studies have focused on the gating conformation transitions of the wild-type MscL protein –21, the sn-glycero-3-phosphocholine) spanning ~100 Ang along the lateral dimensions  using the CHARMM-GUI server,[+ and Cl- were added to the system, setting the ion concentration to about 1 M. The engineered model with five ligands (hereafter referred to as 5L) was then minimized to remove steric clashes, slowly heated up from 100 K to 300 K in about 1 ns simulation and then equilibrated for 10 ns in a NPT ensemble. The production run was performed in a NVT ensemble, applying a homogeneous external electric field (Ez) along the z-axis (Lz) perpendicular to the membrane proportional to a defined voltage . Different engineered models were then generated by a sequential replacement (every 10 ns) of each ligand by the charged group   and the α atoms in adjacent subunits. Water and ions around a permeating K+ were estimated from the average number of corresponding species within a predefined cut-off radius, as a function of the z-coordinate. Channel conductance was estimated from the ratio of the average current and applied voltage.[α atom distances. Trajectory visualization was performed using the Caffeine software.[α geometric centroid of residue 21.Root mean square deviations (RMSD) were computed performing a least-square fitting to a reference structure, either the starting one or the X-ray crystallographic structure. The pore radius profile was obtained using the HOLE program. The ion  voltage.,38 Intersoftware. Figures software.,41 Unles+ translocation through the channel using the adaptive biasing force methodology (ABF) implemented in the NAMD package.[+ ion in the pore. The ions in the bulk solution were restrained to obtain the single-ion PMF profile.A computational approach similar to that reported in previous studies ,43 was e package.,45 In sh15DLAVAVVIGTAFTALVTKFTDSIIT40). First, we have analyzed and compared the structures of different engineered MscL channels issuing from the sequential replacement of the attached  photo-sensitive ligands, i.e. 6-nitroveratryl alcohol, by negatively charged acetate groups. It should be noted that in the original experimental work[The Tb-MscL protein is a relatively small homopentamer channel of 151 residues (17KDa) . In partntal work, even ifThe stability of the simulated MscL models was assessed by evaluating the structural deviations from starting configurations. RMSD of the backbone atoms, depicted as function of simulation time, have shown a plateau after about 10 ns ., followα atoms  at the same residue along a stretch of aminoacids flanking the functionalized site. Results depicted in The extent of channel structural expansion in each engineered MscL model was examined by evaluating the average pore radius along the channel axis, considering only the backbone atoms for the sake of comparison (i.e. the steric effects of side chains and ligands were neglected). In Moreover, it is worth nothing that the WT simulation was initiated from an equilibrated configuration of the NL model after replacing each charged group at the functionalized sites with Valine. Such a hydrophobic substitution led to a fast channel closure (a few nanoseconds) as indicated by the radius profile . A simil+ ions were able to permeate through the engineered MscL channels from the periplasmic side. As reported in + ions has shown the occurrence of ion flux only through 1L and NL models. In 1L model, the bulky ligand in the pore largely attenuated the permeation events (~11%), albeit a structural expansion similar to the NL model is observed  similarly exposed to the pore with respect to V21, and located on either side of V21. We have investigated the effect of charge incorporation at these alternative sites , performrbations . Howeverrbations . The intRecent studies have demonstrated that introducing only one charge in a single subunit of the MscL hydrophobic pore (at position G22 in Eco-MscL) is sufficient to trigger channel gating in the absence of membrane tension.,17 Note In order to better investigate, at molecular level, the present gating mechanism, we conceived an additional model (WT_1e) in which the same negatively charged group was directly attached to the constricted site (V21) in a single subunit (subunit 1) of the WT protein, therefore mimicking the light-actuation of a single subunit of MscL. Inspection of the average pore radius along the channel axis revealed a clear expansion of the WT_1e model with respect to the WT protein ., albeitFurthermore, the pore hydration upon single subunit modification was examined by monitoring the water molecules located within a defined radial distance from the channel central axis and spanning 10 Ang along the z-direction on either side of position 21. The 2D distribution of water molecules depicted as a function of axis/radial coordinates inside the pore showed a marked difference in the hydration pattern between WT and WT_1e models . In the We also monitored the changes in inter-subunit contacts with time. In MscL, each TM1 helix does form contacts with other two TM1 helices from neighboring subunits. For example, residues of subunit 1 are in contact with residues belonging to subunit 2 and 5. We monitored the total contacts between the TM1 helix of subunit 1 (modified subunit) and TM1 helices of subunit 2 and 5 relative to the starting configuration . The plot of variations in inter-helix contacts as a function of time is shown in off”state with all subunits containing the neutral bulky photo-active ligands and an “on” state with all subunits converted to the corresponding charged moieties upon light irradiation. Here, taking inspiration from this pioneering study, we investigated in more detail through atomistic MD simulation the effect of charge activation. Starting from the “off” state (5L model), the photo-active ligands were sequentially replaced by charged groups and the corresponding functionalized MscL proteins thoroughly studied. Simulation results have shown a gradual expansion of the channel proportional to the increasing number of charges in the pore lumen (off (5L model) to the on state (NL model). In particular, the NL model displayed a conductive pore, in agreement with experiments. It is worth noting that after each chemical (electrostatic) change in the channel interior, the following structural relaxation occurred in the nanosecond timescale, thus supporting a relatively fast dynamical response of the protein channel. This result is in stark contrast to the millisecond-timescale opening observed in the native protein via the tension-induced mechanism.[In the experimental context of ref. , where tre lumen ., in goiechanism. To cope echanism.,11. In aechanism.,55On the other hand, inspired by recent experiments that demonstrated channel activation upon chemical modification of a single subunit of Eco-MscL,,17 an adOur investigation has also unraveled further molecular details concerning the asymmetric motion of the only charged subunit with respect to the others. Such a structural transition, not triggered by a strong repulsive interactions, is possibly another confirm of the highly flexible character of the MscL channel. This is perhaps not surprising if we consider that the natural activation mechanism of the mechanosensitive channels does require a considerable structural change driven solely by membrane tension, whereas other protein channels activated by different stimuli  are significantly more rigid and insensitive to membrane stress. Furthermore, the MscL transition from the closed to the open state has been described as an iris-like symmetric motion of all subunits.–59 HowevWhile the present computational investigation has provided an overall consistent picture with respect to known experiments, especially for the WT_1e and NL models representing activated MscL protein channels, some important details contrast with experimental findings. In particular, the extent of pore expansion achieved in our simulations is significantly smaller than experimental predictions. The introduction of a single charge on a pore residue in one subunit of Eco-MscL has been estimated to lead to a pore diameter of up to ~10 Ang. In our wE.coli spheroplasts (in-vivo) or reconstituted in azolectin liposomes (in-vitro), a feature that was observed when the crystals of Tb-MscL was resolubilized and functionally reconstituted.[In our case, we envisage that the expanded pore likely represents a sub-conducting state of the channel. MscL has been shown to visit sequential intermediate states during the transition from the closed to the open state. The charge-induced activation apparently populates the same initial substates achieved by the natural trigger, i.e. membrane tension. Accordinstituted. Hence, dstituted. structurThe bacterial mechanosensitive channel of large conductance (MscL) has been conceived as an ideal activated nanovalve for controlled drug delivery with several advantages that include: i) large conductance, ii) non-selective pore, iii) easy isolation from cells, iv) in-vitro translation, v) reconstitution in synthetic lipids and vi) charge-induced activation of channel gating. In particular, incorporation of photo-active groups into the hydrophobic channel core through site-specific mutations and chemical synthesis has enabled a light-activated gating mechanism, which offers a high spatio-temporal control without affecting the environment. In addition to these relevant features, the present computational study has unraveled a few interesting details concerning the MscL charge-induced gating: (1) a fast pore-opening dynamics  leading to initial conducting substates, (2) a high sensitivity of the channel structural expansion to several factors  and (3) an asymmetric subunit movement upon single-charge incorporation into the pore. We note that the observed sensitivity of charge perturbations towards selected channel interior sites may support the need for a preliminary molecular modeling of the detailed triggering events leading to pore gating. As well, we note that independent subunit movements upon charge activation not only support the proposed asymmetric motion in MscL channel opening but also do suggest the possibility of a subunit-specific triggering to achieve pores of desired sizes.S1 FigBackbone RMSD, calculated with respect to the starting configuration of respective simulations. The right panel represents the distribution of the RMSD values over the last 20 ns.(TIF)Click here for additional data file.S2 FigBackbone RMSF mapped onto the average structure. The protein is represented as ribbon whose color (green to red) and thickness is proportional to the RMSF values.(TIF)Click here for additional data file.S3 FigPore area estimated for a stretch of residues lining the TM1 helix for WT (gray), 5L (black) and NL (magenta). The vertical dashed line corresponds to the X-ray structure.(TIF)Click here for additional data file.S4 FigAverage pore radius along the axial positions, obtained considering the backbone atoms.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Accumulation of carboxylated polyethylene glycol (PEG) CdSe/ZnSquantum dots (QDs) has been monitored in living fibroblasts using confocal microscopy for fluorescence intensity and fluorescence-lifetime imaging (FLIM). The wide range of mean photoluminescence (PL) lifetime values was observed for the intracellular QDs in different intracellular microenvironment, which revealed structural heterogeneity of endosomes and enabled the distinguishing among endosomes of different maturity. It is highly sensitive to the changes at the molecular level and ensures high spatial resolution of the collected data. However, the analysis of the fluorescence results gained from a biological object is often complicated due to such limiting factors as light scattering and absorption. Fluorescence-lifetime imaging (FLIM) has emerged as one of key techniques allowing visualizing the interaction of nanoparticles and other fluorophores with surrounding environment in living cells , i.e., DSPE-PEG2000  peak at 625 nm have a CdSe core passivated with a ZnS shell and are capped with a micelle-like coating of amphiphilic 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-c radius . The culc radius . Fluorescence-lifetime images were acquired using Lifetime and FCS Upgrade for Nikon C1si . The system consisted of a 405-nm pulsed diode laser with a pulse width of 39 ps and a repetition rate of 10 MHz, which allowed for the use of a measurement time interval of 100 ns. Detected photons were counted by a time correlated single-photon counter PicoHarp 300 (PicoQuant GmbH). Initialization, scanning, and data acquisition were controlled by the same Nikon C1si microscope system. PL lifetime signal of QDs in living cells and in water suspensions of the different pH values was detected with a single channel unit of single photon-counting avalanche photodiodes (SPADs) at a spectral range of 578 ± 52.5 nm. Each lifetime image was obtained by collecting 1000 counts at the peak value, and the image resolution was fixed at 512 × 512 pixels. FLIM images were reconstructed using a three-exponential fitting model , in which an average lifetime of QDs is displayed for every given pixel. Histograms for all average lifetimes  of QDs were plotted using the SymPhoTime v.5.2 (PicoQuant GmbH) software.The fluorescence spectra as well as confocal fluorescence microscopy images of intracellular distribution of carboxylated CdSe/ZnS-PEG QDs were found to be very similar despite the fact that during observation period QDs were translocated to different organelles. It was demonstrated for the first time that FLIM enabled the distinguishing of QDs accumulated among different endosomes. At the first phase of accumulation, when QDs adhered to the plasma membrane, the PL lifetimes were the longest. When QDs are internalized into endocytic vesicles, the shorter lifetimes appeared. The vesicles bearing more complex internal structures with several mean PL lifetimes appeared at the last phase of accumulation.Two of the possible reasons for the shortening of PL lifetimes of intravesicular QDs are the acidic medium and the altered interactions of QDs with surrounding biomolecules in the heterogeneous microenvironment.The observed spatial heterogeneity of organelles implies the possibility of applying QDs in combination with FLIM technique for the imaging of the sub-structure of endosomes and the accurate distinction between early and late endosomes on the basis of morphology, when appropriate time gates are chosen, but much more effort is needed to relate the PL lifetimes of intracellular QDs with particular biomolecular structures.


Next-generation sequencing has led to the discovery of a number of disease-associated genes in patients with uncharacterized autoinflammatory diseases and has pointed to new pathways regulating immune function.To investigate the pathogenesis of disease in patients with the deficiency of the CCA-adding enzyme tRNA nucleotidyltransferase 1 (TRNT1). The TRNT1 enzyme catalyzes an essential step for tRNA maturation and protein synthesis, however it is largely unknown how abnormalities in this pathway lead to inflammation and immunodeficiency.Patients and Methods: Whole exome sequencing (WES) was performed in a consanguineous Saudi family with 2 affected siblings and in a parent-child trio; candidate gene screening was subsequently performed in 3 sporadic Caucasian patients. Cytokine profiling, tissue immunohistochemistry, and deep RNA and tRNA sequencing were performed in patients’ primary cells. Protein function was studied in zebrafish embryos.TRNT1 gene. Three patients died due to multiorgan failure. We identified 6 disease-associated missense mutations that affect evolutionarily conserved amino acid residues. These variants are either novel or found at an allele frequency less than 0.0001 in public databases, consistent with recessive disease inheritance. Two apparently unrelated patients shared the same genotype. Recently, loss-of-function mutations in the same gene were reported in patients with a syndrome termed SIFD . Four mutations from our study have not been reported in patients with SIFD. Knockdown of the zebrafish TRNT1 homologue caused abnormalities resembling the human phenotype. Preliminary results of next-generation tRNA sequencing showed a significant down-regulation of mature tRNAs in patient's fibroblasts compared to healthy control. RNA-sequencing of patients’ whole blood showed an up-regulation in the expression of neutrophil-related genes. Consistent with these data, analysis of lesional biopsies from one patient's colon showed cryptitis with neutrophilic infiltration. Cytokine profiling of stimulated patients’ leukocytes and serum samples suggest that the inflammatory phenotype is likely driven by IL-6 and interferon. Treatment with TNF inhibitors has shown promising results in attenuating the systemic inflammation and stabilizing the anemia in 3 patients who experienced recurrent fevers and required transfusions.We have identified 7 patients with biallelic homozygous or compound heterozygous mutations in the TRNT1 are associated with a new autoinflammatory disease manifesting a variable phenotype of fevers, congenital sideroblastic anemia, immunodeficiency, and developmental delay. Study of the underlying disease mechanisms might lead to the discovery of a new pathway regulating immune function and inflammation.Hypomorphic mutations in


Over the past decade, the focus of molecular biology has shifted from being predominately DNA and protein-centric to having a greater appreciation of RNA. It is now accepted that the genome is pervasively transcribed in tissue- and cell-specific manner, to produce not only protein-coding RNAs, but also an array of noncoding RNAs (ncRNAs). Many of these ncRNAs have been found to interact with DNA, protein and other RNA molecules where they exert regulatory functions. Long ncRNAs (lncRNAs) are a subclass of ncRNAs that are particularly interesting due to their cell-specific and species-specific expression patterns and unique conservation patterns. Currently, individual lncRNAs have been classified functionally; however, for the vast majority the functional relevance is unknown. To better categorize lncRNAs, an understanding of their specific expression patterns and evolutionary constraints are needed. Drosophila melanogaster, these species exhibit dramatically differing levels of phenotypic complexity. To account for this disparity, there must exist a multi-level regulatory mechanism enabling such drastic diversity from a similar number of protein-coding genes.Recent developments in RNA sequencing (RNA-Seq) technology have given scientists an in-depth view of the human transcriptome . It is aThere is a direct correlation between the proportion of ncRNAs in an organism’s genome and its developmental complexity . The larThe dysregulation of lncRNAs expression has been implicated in a number of diseases across different tissue types. Merely 7% of disease-associated single nucleotide polymorphism (SNPs) is located within protein-coding regions compared to the 93% of SNPs that are found in noncoding regions . DespitelncRNAs have been shown to be expressed in a distinct pattern across a number of tissue types. A number of lncRNAs have also been shown to be expressed in discrete cell types and within distinct subcellular structures . These fAnother interesting property of lncRNAs is their rapid evolution across species. Previously, the conservation of sequence was thought to be evidence of functionality but lncRNAs have proved that this is not always the case. The tissue-specific expression patterns of lncRNAs, coupled with their distinctive conservation patterns, make lncRNAs a unique transcriptional element that warrants further investigation.LncRNAs exhibit notably higher degree of tissue specificity when compared to protein-coding genes . The appThe brain is the most complex tissue in the human body. Beyond its billions of neurons, the brain comprises a number of other cell types, such as oligodendrocytes, astrocytes and microglia with heterogeneous distribution across anatomical subregions. Due to this complexity in terms of both structure and function, the brain requires a similarly complex regulatory system and as a result is the richest source of lncRNAs in the body . LncRNAsOLMALINC) [OLMALINC is a primate-specific transcript that has been shown to play an essential role in the regulation of genes responsible for human oligodendrocyte maturation [OLMALINC is highly expressed in the white matter of the human frontal cortex with expression levels of 71.5 fragments per kilobase of exon per million fragments mapped (fpkm) as determined by RNA-Seq. Such high level of expression indicates a strong regulatory role in oligodendrocytes, which comprise the majority of white matter. The differential expression of OLMALINC in gray and white matter  demonstrates the dynamic nature of the brain transcriptome and the tissue specificity of lincRNAs.There is a growing amount of data on the highly tissue-specific lncRNA transcripts located between protein-coding gene loci, known as long intervening ncRNAs (lincRNAs), and their role in regulation of fundamental cellular processes . The linLMALINC) . OLMALINturation . OLMALINA recent profiling of the transcriptome patterns of gray and white matter highlighted the tissue-specific nature of lincRNAs in a healthy brain . The expThere is a growing need for the development of more comprehensive expression profiles of lncRNAs for all regions of the human brain . Recent cis-regulatory elements, and adjacent protein-coding genes has also been observed in the human brain [The purported functional relationship between lncRNAs, as an brain . It has an brain . These lan brain .Despite an incomplete annotation of the long noncoding transcriptome and a general lack of functional information, the dysregulation of tissue-specific lncRNAs has been strongly associated with a number of diseases . The difTestis is a rich source of many unique lncRNA transcripts; however, very little is known about lncRNAs expressed solely in this organ. In-depth analyses of the testis transcriptome using RNA-Seq data have shown a widespread and diverse transcription of both protein-coding and ncRNAs . The tesA comparative study investigating the five most common cell types involved in spermatogenesis found that in addition to expressing a greater palette of lncRNAs transcripts than cells of the brain or liver, the expression of unique lncRNAs differed significantly between the cells of the testis producing highly specific expression patterns . This waCurrently there are limited studies into human testis-specific lncRNAs expression and as a result we must rely on animal models. A recent study produced lncRNA expression profiles for the testis of a neo-natal and adult mouse . This stlncLSTR) was found to regulate the clearance of triglyceride and help maintain systemic lipid homeostasis through a novel lncRNA signaling pathway. Its apparently key role in this crucial metabolic process highlights the potential physiological importance of lncRNAs in the liver.The role and function of lncRNAs in the liver is largely unknown but the dysregulation of specific transcripts has been associated with liver diseases such as hepatocellular carcinoma  and nonaFendrr) is a lateral mesoderm-specific lncRNA that is essential for the development of the heart wall in mouse and was shown to have an orthologous transcript in humans [Fendrr was found to modulate chromatin signatures that define gene activity by binding directly to the histone-modifying complexes Polycomb repressive complex 2 (PRC2) and histone–lysine N-methyltransferase 2A (KMT2A), which play a central role in the activation of genes responsible for cell differentiation and lineage commitment. PRC2 and KMT2A act as a repressor and activator of cellular proliferation, respectively, in the heart during embryonic development. The knockdown of Fendrr in mice was shown to be lethal to the embryos due to heart wall deficits and significantly impaired heart function demonstrating its importance for normal heart function.Little is known about the role of lncRNAs in the heart; however, a heart-specific lncRNAs has been found to be involved in cardiac development. FOXF1 adjacent noncoding developmental regulatory RNA (n humans . Fendrr Linc-MD1) has been identified to have a significant role in myogenesis through its control of muscle differentiation [Linc-MD1 expression is temporally dynamic in order to control the progression through the stages of muscle differentiation where it functions as a competing endogenous RNA for the binding of the microRNAs (miRNAs) miR-133 and miR-135. The two miRNAs regulate the binding of the transcription factors that promote muscle differentiation. Hence, Linc-MD1 plays a crucial role in the regulation of muscle terminal differentiation through its action as part of a network of regulatory interactions.Long intergenic ncRNA, muscle differentiation 1 (ntiation . Linc-MDSix3OS) is promoter-associated lncRNA found to play a role in the regulation of retinal cell differentiation through knockdown and overexpression studies [Six3OS was also shown to modulate the expression of associated protein-coding genes through the recruitment of histone modification enzymes. Six3OS acts as a molecular scaffold that leads to the recruitment of histone modification enzymes. A retina-specific lncRNA ventral anterior homeobox 2, opposite strand (Vax2os) was also shown to regulate the cell cycle during mammalian retina development [Vax2os is so far the only example of a cell type-specific lncRNA regulating the cell cycle during mammalian development.Several retina-specific lncRNAs in mice have been identified and determined to be of functional importance in retinal cell development and differentiation. Six3 opposite strand transcript . One of the well-characterized lncRNAs, HOX transcript antisense RNA (HOTAIR), is believed to have conserved structures but divergent sequences across species [Malat1) [The sequence of RNAs can differ whilst their secondary structure can be conserved . Many ln species . RNAs ca species , GU base species , adenosi species . These m[Malat1) . The maj[Malat1) . This th[Malat1) .Xist), which is involved in X-chromosome inactivation. The function of Xist is conserved across mammals, even though the sequence is evolving at a high rate [The functional role of the lncRNA may also be conserved. One established lncRNA is X-inactive-specific transcript . It has been suggested that HAR1 has been evolutionarily selected for increased stability [HAR1 [FOXP2) and abnormal spindle-like microcephaly associated protein (ASPM), which are involved in speech production and brain size respectively, have undergone the same kind of evolutionary change [One example of a noncoding gene that is thought to have evolved a unique function in humans is the human accelerated region 1  [BACE1-AS) [More recently, slight modifications of the template preparation stage of RNA-Seq have allowed for the strand of origin from which an RNA molecule is transcribed from to be tracked, thus allowing for the identification of antisense transcription. These techniques are known as strand-specific RNA-Seq . While aTSIX)  and the ACE1-AS) . It is eACE1-AS) . FurtherACE1-AS) . AnotherACE1-AS) . This isACE1-AS) . As riboRaw RNA-Seq data needs to be processed and analyzed to answer all sorts of bioinformatics enquires, including investigation of gene/transcript expression levels, detection of alternative splicing events and identification of unannotated genes/transcripts. In brief to analyze RNA-Seq data, first the reads must be mapped to the reference genome, next transcripts are assembled followed by a differential expression analysis. A common workflow currently used by researchers is known as the Tuxedo suite, which utilizes the software packages Tophat, Cufflinks and Cuffdiff . This wohttp://aws.amazon.com/). AWS allows researchers to dynamically adjust the computing power and storage requirements based on current requirements and has potential computing power much larger than any HPC cluster.A typical RNA-Seq experiment will produce vast amounts of data. Generally it is not feasible to analyze data on a personal computer due to limitations in storage size and raw processing power. These problems can be overcome through the use of high-performance computing (HPC) clusters. A HPC cluster consists of multiple nodes, with each node containing one or more central processing units (CPUs), each with numerous cores. HPC clusters are normally a resource shared across a major institute such as a university or hospital. Another alternative is to take advantage of cloud computing services such as Amazon Web Services (AWS) (PTENP1), through retroviral vectors, revealed its role in the regulation of the phosphatase and tensin homolog (PTEN) [OLMALINC in human oligodendrocytes [Only recently has technology been able to identify lncRNAs using high-throughput methods such as RNA-seq. Questions still remain as to how many of the proposed lncRNAs are functional, what that function is and the role that they have played in evolution. More knockdown and overexpression studies are necessary to explore the diverse roles that lncRNAs possess. For example, the overexpression of the 3’UTR region of the phosphatase and tensin homolog pseudogene 1 (g (PTEN) . RNAi ofdrocytes  revealedIn order to better determine lncRNA role as part of a regulatory network it is essential to produce comprehensive, functional annotations for lncRNAs similar to those that exist for protein-coding genes. This is especially relevant for those novel lncRNAs associated with human diseases. As a result of advances into ncRNA research, there are several public databases of annotated lncRNAs; however complete functional characterization of all lncRNAs is needed beyond merely basic sequence and transcript information . A numbehttp://www.lncrnadb.org/) provides a summary of known eukaryotic lncRNAs. lncRNAdb differs from many other databases as entries must be supported by literature and they do not pull their data from unconfirmed sources to ensure validity [in vitro and/or in vivo experiments.The lncRNAdb  is a web-based lncRNA catalog containing annotations of male germ-cell specific lncRNAs [GermlncRNA ( lncRNAs . This ca lncRNAs . A tissuhttp://www.lncipedia.org/) is a comprehensive database for annotated human lncRNAs generated through the incorporation of data obtained from a number of different sources. This allowed for a rapid increase of the gene entries from 21488 annotated lncRNAs in LNCipedia v.1.0 to 111685 annotated lncRNAs in LNCipedia v.3.1. [LNCipedia (a v.3.1. . A stratThese databases were created through the integration of pre-existing public resources. While this allows for large amounts of information to be shared and combined, it also led to lncRNA predictions that greatly vary between individual repositories. This is due to differences in methodology, classification and assembly algorithms, which result in many lncRNAs to be missed or improperly categorized . Constan


Our findings strongly suggest that the methionine salvage pathway is a major player in homeostatic regulation of metabolite pools in prostate cancer due to their high level of flux through the polyamine biosynthetic pathway. Therefore, this pathway, and specifically the MTAP enzyme, is an attractive therapeutic target for prostate cancer.Prostatic epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen. This distinctive characteristic places added strain on the connected pathways, which are forced to increase metabolite production to maintain pools. The methionine salvage pathway recycles the one-carbon unit lost to polyamine biosynthesis back to the methionine cycle, allowing for replenishment of SAM pools providing a mechanism to help mitigate metabolic stress associated with high flux through these pathways. The rate-limiting enzyme involved in this process is methylthioadenosine phosphorylase (MTAP), which, although commonly deleted in many cancers, is protected in prostate cancer. We report near universal retention of MTAP expression in a panel of human prostate cancer cell lines as well as patient samples. Upon metabolic perturbation, prostate cancer cell lines upregulate MTAP and this correlates with recovery of SAM levels. Furthermore, in a mouse model of prostate cancer we find that both normal prostate and diseased prostate maintain higher SAM levels than other tissues, even under increased metabolic stress. Finally, we show that knockdown of MTAP, both genetically and pharmacologically, blocks androgen sensitive prostate cancer growth  Prostate is a secretory gland that produces and secretes massive amounts of polyamines-4, ubiquvia the generation of SAM.SAM pools are consumed in order to synthesize polyamines by decarboxylation through the action of AMD1. This removes SAM and the one-carbon unit from the methionine cycle. The decarboxylated SAM donates the propylamine group to putrescine and spermidine through the actions of spermidine synthase (SRM) and spermine synthase (SMS), respectively. This results in the formation of spermidine and spermine, as well as the byproduct methylthioadenosine (MTA), which carries the one-carbon unit taken from the methionine cycle. Ultimately, this one-carbon unit is derived from one-carbon metabolism and requires folate derivatives to shuttle it to the methionine cycle for the synthesis of methionine and SAM. Therefore polyamine biosynthesis relies on folate from the diet min/+ mice -3-hydroxy-4-(methylthiomethyl)-pyrrolidine, or MT-DADMe-Immucillin-A (MTDIA) was synthesized following the synthesis scheme shown in 6 wild type LNCaP cells were subcutaneously injected onto the flank of pelleted nude mice. At time of injection animals were randomly assigned to one of the following three groups; control, 9 or 21 mg/kg MTDIA in the drinking water with 20 mice in each group. Tumor measurements were assessed and recorded twice a week. Animals were sacrificed once tumors reached 2 cm3 or at the end point of the experiment (91 days).1 × 10


The model developed predicted samples in the validation set with r2  = 0.80 and SEP  = 1.108, analytical results showed that near infrared spectra had significant potential for the adsorption of ink capacity on the recording coating. The influence of factors such as recording coating thickness, mass ratio silica: binder-polyvinyl alcohol and the solution concentration on the adsorptive ink capacity were studied. With the help of the near infrared spectra, the adsorptive ink capacity of a recording coating material can be rapidly controlled.Near infrared spectra combined with partial least squares were proposed as a means of non-contact analysis of the adsorptive ink capacity of recording coating materials in ink jet printing. First, the recording coating materials were prepared based on nano silica pigments. 80 samples of the recording coating materials were selected to develop the calibration of adsorptive ink capacity against ink adsorption (g/m With the development computers and digital techniques, ink-jet printing has been widely used in many applications, such as organic chemistry Nano-silica is widely applied as pigment particle in recording coating. Its size, distribution and the specific surface area influences ink adsorption. Big specific surface area causes the strong adsorption capacity; the dispersibility of the recording coating is more uniform in terms of the more adsorptive ink. Polyvinyl alcohol is generally used as binders on recording coating. Its molecular structure influences the absorption effect too. The high molecular weight of polyvinyl alcohol leads to a close molecular structure, this structure causes fastness on adsorptive ink drop and excellent water-resistance; small molecular weight causes loose structures, which lead to easy ink impregnation and clear colors. These results influence the clarity and permanent properties of the image. In order to enhance the printing quality, the analysis of the adsorptive ink capacity of RC-IJP is very important. Further, rapid analysis of the adsorptive ink capacity has never been reported.Near infrared (NIR) spectroscopy is a very rapid, accurate and non-destructive method for simultaneous measurements on different constituents in various products 2/g was obtained from Degussa A200, Germany. In addition, the binder-polyvinyl alcohol (PVA), acroart and accessory ingredient is provided from Shanghai Chemical Co., Ltd, China.The inorganic pigments-nano scale silica with the average nanoparticle size of 12 nm, and the specific surface area of 188 m2∶PVA = 3∶1), then, the mixed solution is placed in the oven at 40°C for 120 min. Consequently, the recording coating is prepared. The blade coater method is used in the preparation of recording coating materials, this process is shown in The silicane dispersing agent (gamma-aminopropyltriethoxysilane) is added into distilled water with magnetic stirring for 10 min. Nitric acid is subsequently added until solution pH = 4. Silica is added in the above solution under homogenizer  for 30 min. Solution concentration is 10%, mass ratio between silicane dispersing agent and silica is 0.02∶1. Above preparation product is added in 8% PVA solution by the homogenizer for 10 min (mass ratio SiOThe surface morphologies and roughness of RC-IJP wereobserved using a high-resolution scanning electron microscopy (SEM), energy dispersive spectrum (EDS)  and atomic force microscope . NIR spectra are collected by a diffuse reflection FT-NIR spectrometer . Spectra are collected using contactless way, the light from the sources is focused on to RC-IJP (The distance is 17 cm between light source and samples), and then the diffuse reflectance spectra from the RC-IJP are recorded by the spectrometer.Equipment of adsorptive ink weight of RC-IJP is shown in The micro-structure of RC-IJP is analyzed by SEM. It can be observed from 2, PVA and dispersing agent, these components contain the hydrogen-containing groups. So, NIR spectra can analyze the RC-IJP informationAs the NIR-spectra showed information of the presence of hydrogen-containing groups, the recording coating mainly contained SiO2 and PVA component contents, molecular structure and particle distribution influences the Near-infrared diffuse reflection light absorption degree. Above influence factors jC  provide following equation:The mode of action NIR diffuses reflectance light on RC-IJP is shown in jC : Influence factors;0b: Constant in equation;ijb: Coefficient in equation;iA: Linear function value in i wavelength location.The quantitative relation is found between influence factors and the NIR spectra feature of the samples by statistical methods.The adsorptive ink capacity is confirmed by the recording coating. Nano-silica particle size, distribution and the specific surface area influences ink adsorption. Big specific surface area causes the strong adsorption capacity; the dispersibility of the recording coating is more uniform in terms of the more adsorptive ink. Binder-polyvinyl alcohol molecular structure influences the absorption effect also. High molecular weight lead to the molecular structure of the close, this structure causes fastness on adsorptive ink drop and excellent water-resistance; small molecular weight is loose structure, which leads to ink impregnation easy and colors clear.−1. Each spectrum is obtained by an average of 8 scans with a resolution of 16 cm−1). FT-NIR information of the spectrum is given: the methyl groups and methene groups produced combination bands at 4323 cm−1 and 4250 cm−1, the methyl groups gives an overtone band at 5778 cm−1, the methene groups gives second overtone bands at 8230 cm−1, respectively. In addition, ethenyl demonstrates combination bands at 4772 cm−1. On the other hand, hydroxy gives stretching combination and overtone bands at 5185 cm−1and 6800 cm−1. The above analysis adequately represents the required information of the RC-IJP structure in NIR spectra.Above analysis showed that NIR spectroscopy enables the analysis of the adsorptive ink capacity of RC-IJP. RC-IJP spectra are collected by a diffuse reflection FT-NIR spectrometer in 2R), root mean square error of calibration (RMSEC) and root mean square error of prediction (RMSEP) were calculated to evaluate the model to find the best calibration model. The linearity correlation coefficient is a measure of the consistency between the NIR prediction values and actual values for the calibration sets. In developing model, 97 samples , RMSEC (0.896) and RMSEP (1.108), respectively. The spectral ranges obtained the best wavenumber of 4000–5800 and 6700–9000 cm−1, 5 PLS factors are used. The plots of the actual values versus the predicted values by NIR for calibration set of the adsorptive ink weight were shown in 2∶PVA, solution concentration and disperse method. On the other hand, the distance of the blade coater or press roller, the temperature of the dry tower, the moving velocity can also control the adsorptive ink capacity in preparation of RC-IJP. It is worth taking into consideration that co-adjustments of all the parameters can be complicated. we have only focused on the main parameters in this study although other factors such as the quality of the acroart, environmental humidity, temperature etc., can also affect the RC-IJP quality. Above process is shown in If the processing parameters and conditions are changed, the calibration models would be updated. New samples are selected from the production line and added to the calibration model again. After updating and optimization, the calibration models are developed again. The developing models are implanted to the control system, the processing parameters are adjusted rapidly by NIR spectroscopy. With the help of NIR spectroscopy, RC-IJP adsorptive ink capacity is controlled timely. The preparation processing parameters of the recording coating are adjusted based on recording coating thickness, mass ratio SiOFrom the above analyses, the adsorptive ink weight of the recording coating was successfully performed based on NIR spectra. With the help of NIR spectroscopy, RC-IJP quality is controlled timely. In addition, from the analysis of the adsorptive ink weight, it was observed that when per unit area weight is invariant; the dispersibility of the recording coating is more uniform in terms of the more adsorptive ink.Materials S1The blade coater method is used in the preparation of recording coating materials, this process can be found in the Supporting Information, and NIR spectra of 97 samples  of RC-IJP are shown in Materials S1.(DOC)Click here for additional data file.