Craniofacial structures change dynamically in morphology during development through the coordinated regulation of various cellular molecules. However, it remains unclear how these complex mechanisms are regulated in a spatiotemporal manner. Here we applied natural cubic splines to model gene and microRNA (miRNA) expression from embryonic day (E) 10.5 to E14.5 in the proximal and distal regions of the maxillary processes to identify spatiotemporal patterns of gene and miRNA expression, followed by constructing corresponding regulatory networks. Three major groups of differentially expressed genes (DEGs) were identified, including 3,927 temporal, 314 spatial, and 494 spatiotemporal DEGs. Unsupervised clustering further resolved these spatiotemporal DEGs into 8 clusters with distinct expression patterns. Interestingly, we found 2 clusters of differentially expressed miRNAs: 1 had 80 miRNAs monotonically decreasing and the other had 97 increasing across developmental stages. To evaluate the phenotypic relevance of these DEGs during craniofacial development, we integrated data from the CleftGeneDB database and constructed the regulatory networks of genes related to orofacial clefts. Our analysis revealed 2 hub miRNAs, mmu-miR-325-3p and mmu-miR-384-5p, that repressed cleft-related genes Adamts3, Runx2, Fgfr2, Acvr1, and Edn2, while their expression increased over time. On the contrary, 2 hub miRNAs, mmu-miR-218-5p and mmu-miR-338-5p, repressed cleft-related genes Pbx2, Ermp1, Snai1, Tbx2, and Bmi1, while their expression decreased over time. Our experiments indicated that these miRNA mimics significantly inhibited cell proliferation in mouse embryonic palatal mesenchymal (MEPM) cells and O9-1 cells through the regulation of genes associated with cleft palate and validated the role of our regulatory networks in orofacial clefts. To facilitate interactive exploration of these data, we developed a user-friendly web tool to visualize the gene and miRNA expression patterns across developmental stages, as well as the regulatory networks (https://fyan.shinyapps.io/facebase_shiny/). Taken together, our results provide a valuable resource that serves as a reference map for future research in craniofacial development.

Interferon gamma has critical antiviral properties by inducing type-1 immunity. In addition important immunoregulatory functions have been described that may go beyond antiviral immunity. We used single cell transcriptomics to comparatively analyze wild-type (n=30) and interferon-gamma receptor knockout (IFNγR−/−, n=30) mice infected with murine gamma herpesvirus 68 (MHV-68). Lytic virus was cleared from the lungs within 14 days but with incompetent and delayed response in IFNγR−/− mice. The IFNγR−/− mice showed a type-2 bias of immune response and developed irreversible lung fibrosis with many features of IPF within 100 days after virus infection. We harvested the lungs for scRNA-seq at days 3/6/15/28/45 and 100 post infection, and sequenced a total of 70.000 single cells. Single cell analysis provided an unprecedented resolution of MHV68 and host transcriptomes at single cell level with the identification of the infected cell types. We analyzed cell type specific responses to the virus and resolved gene expression kinetics for >30 individual cell types, revealing prominent type-1 and type-2 immune polarization patterns in WT and IFNγR−/− mice, respectively. Cell-cell communication analysis revealed distinct cellular circuits associated with the evolution of immunopathology in IFNγR−/− mice. For instance, the discovery of an immune recruiting state of the alveolar type-2 pneumocyte overexpressing Ccl3 and Ccl9 chemokines and the evolution of pro-fibrotic macrophage states. Overall, we present time-resolved single cell data on virus-host interactions in the context of type-2 interferon immune modulation that reveals important functions of IFNγ beyond antiviral immunity.

Type-2 immunity is essential for tissue repair processes and is a common contributing factor in the development of fibrosis. We used single cell transcriptomics to comparatively analyze how type-1 and type-2 driven immunity unfolds over a time-course of 100 days in mouse lungs after infection with the murine gamma herpesvirus strain MHV68. In wild type (WT) mice, the virus is cleared within 28 days in an interferon-gamma (IFNγ) dependent manner. In contrast, the IFNγ knock out (IFNγR-/-) mice with MHV68 leads to an incomplete virus clearance, a type-2 bias of the immune response, and the development of irreversible pulmonary fibrosis within 100 days after virus infection. We infected WT (n=30) and IFNγR-/- (n=30) mice with the MHV-68 virus, harvested the lungs for scRNAseq at days 3/6/15/28/45 and 100 post infection, and sequenced a total of 70.000 single cells. Single cell analysis enabled us to derive an unprecedented resolution of the MHV-68 virus and its host transcriptomes at the same single cell level. This facilitates a deep insight into the differential response of infected versus uninfected cells and provides strict monitoring of the general course of the disease. We analyzed cell type specific responses to the virus and resolved gene expression kinetics for more than 30 individual cell types, revealing prominent type-1 and type-2 immune polarization patterns in WT and IFNγR-/- mice, respectively.

Human lungs enable efficient gas exchange, and form an interface with the environment which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in health. We report location-dependent airway epithelial cell states, and a novel subset of tissue-resident memory T cells. In lower airways of asthma patients, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report presence of pathogenic effector Th2 cells in asthma, and find evidence for type-2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identify a shift from airway structural cell communication in health to a Th2-dominated interactome in asthma.

Background With the advent of the age of big data in bioinformatics, large volumes of data and high performance computing power enable researchers to perform re-analyses of publicly available datasets at an unprecedented scale. Ever more studies imply the microbiome in both normal human physiology and a wide range of diseases. RNA sequencing technology (RNA-seq) is commonly used to infer global eukaryotic gene expression patterns under defined conditions, including human disease-related contexts, but its generic nature also enables the detection of microbial and viral transcripts. Findings We developed a bioinformatic pipeline to screen existing human RNA-seq datasets for the presence of microbial and viral reads by re-inspecting the non-human-mapping read fraction. We validated this approach by recapitulating outcomes from 6 independent controlled infection experiments of cell line models and comparison with an alternative metatranscriptomic mapping strategy. We then applied the pipeline to close to 150 terabytes of publicly available raw RNA-seq data from >17,000 samples from >400 studies relevant to human disease using state-of-the-art high performance computing systems. The resulting data of this large-scale re-analysis are made available in the presented MetaMap resource. Conclusions Our results demonstrate that common human RNA-seq data, including those archived in public repositories, might contain valuable information to correlate microbial and viral detection patterns with diverse diseases. The presented MetaMap database thus provides a rich resource for hypothesis generation towards the role of the microbiome in human disease.

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

Motivation The MetaMap resource contains metatranscriptomic expression data from screening >17,000 RNA-seq samples from >400 archived human disease-related studies for viral and microbial reads, so-called “metafeatures”. However, navigating this set of large and heterogeneous data is challenging, especially for researchers without bioinformatic expertise. Therefore, a user-friendly interface is needed that allows users to visualize and statistically analyse the data. Results We developed an interactive frontend to facilitate the exploration of the MetaMap resource. The webtool allows users to query the resource by searching study abstracts for keywords or browsing expression patterns for specific metafeatures. Moreover, users can manually define sample groupings or use the existing annotation for downstream analysis. The web tool provides a large variety of analyses and visualizations including dimension reduction, differential abundance analysis and Krona visualizations. The MetaMap webtool represents a valuable resource for hypothesis generation regarding the impact of the microbiome in human disease. Availability The presented web tool can be accessed at

17.0%±4.1,p=0.01.There was no significant GLS or RLS improvement in C Group patients. GLS in STEMI patientsConclusions: After STEMI and ventricular dysfunction, only patients treated with G-CSF showed a significant improvement of EF at 6 months.Moreover, only in these patients, 2D GLS improved at 6 months.This effect was observed in remote areas, too, maybe showing long lasting and diffuse effects of the cytokine.

The formation of amyloid-like fibrils was studied by using the well-known serine protease trypsin as a model protein in the presence of ethanol as organic solvent. Trypsin forms amyloid-like fibrils in aqueous ethanol at pH = 7.0. The dye Congo red (CR) was used to detect the presence of amyloid-like fibrils in the samples. The binding of CR to fibrils led to an increase in absorption intensity and a red shift in the absorption band of CR. Thioflavin T (ThT) and 8-anilino-1- naphthalenesulfonic acid (ANS) binding assays were employed to characterize amyloid-like fibril formation. The ThT binding assay revealed that the protein exhibited maximum aggregation in 60% (v/v) ethanol after incubation for 24 h at 24 (o)C. The ANS binding results indicated that the hydrophobic residues were more exposed to the solvent in the aggregated form of the protein. The effects of polyethylene glycol (PEG) on the formation of amyloid-like fibrils was studied in vitro. The aggregation of trypsin was followed via the kinetics of aggregation, the far-UV circular dichroism (CD) and transmission electron microscopy (TEM) in the presence and absence of PEG. The CD measurements indicated that the protein aggregates have a cross-beta structure in 60% ethanol. TEM revealed that trypsin forms fibrils with a thread-like structure. The inhibitory effect of PEG on the aggregation of trypsin increased with rising PEG concentration. PEG therefore inhibits the formation of amyloid-like fibrils of trypsin in aqueous ethanol.

The formation of amyloid-like fibrils of α-chymotrypsin was studied in aqueous ethanol, methanol, tertbutanol, dimethylformamide and acetonitrile. Thioflavin T (ThT), Congo red (CR) and 1-anilino-8-naphthalenesulfonic acid (ANS) binding, turbidity, intrinsic fluorescence and far-UV circular dichroism measurements were employed to characterize the amyloid fibril formation. The greatest extent of fibril formation after incubation for 24 h at pH 7.0 and at 24 °C was in ethanol at 55%, in methanol and dimethylformamide (DMF) at 60-70% and in tert-butanol at 60-80%. The ANS binding and intrinsic fluorescence results showed that the hydrophobic residues are more solvent-exposed in the aggregated form of α-chymotrypsin. The ThT, CR binding and far-UV CD measurements indicated that the formation of the cross-β structure of α-chymotrypsin depends on the polarity of the organic solvent. To determine the role of surface charges in the aggregation, chemically modified forms of α-chymotrypsin were prepared. The citraconylated and succinylated enzymes exhibited a higher and the enzyme forms modified with aliphatic aldehydes a lower propensity for aggregation. These results suggest the important role of surface charges in the aggregation of α-chymotrypsin.

Salicylic acid (SA) applied at 10(-3) m in hydroponic culture decreased stomatal conductance (g(s)), maximal CO(2) fixation rate (A(max) ) and initial slopes of the CO(2) (A/C(i)) and light response (A/PPFD) curves, carboxylation efficiency of Rubisco (CE) and photosynthetic quantum efficiency (Q), resulting in the death of tomato plants. However, plants could acclimate to lower concentrations of SA (10(-7) -10(-4) m) and, after 3 weeks, returned to control levels of g(s), photosynthetic performance and soluble sugar content. In response to high salinity (100 mm NaCl), the pre-treated plants exhibited higher A(max) as a function of internal CO(2) concentration (C(i) ) or photosynthetic photon flux density (PPFD), and higher CE and Q values than salt-treated controls, suggesting more effective photosynthesis after SA treatment. Growth in 10(-7) or 10(-4) m SA-containing solution led to accumulation of soluble sugars in both leaf and root tissues, which remained higher in both plant parts during salt stress at 10(-4) m SA. The activity of hexokinase (HXK) with glucose, but not fructose, as substrate was reduced by SA treatment in leaf and root samples, leading to accumulation of glucose and fructose in leaf tissues. HXK activity decreased further under high salinity in both plant organs. The accumulation of soluble sugars and sucrose in roots of plants growing in the presence of 10(-4) m SA contributed to osmotic adjustment and improved tolerance to subsequent salt stress. Apart from its putative role in delaying senescence, decreased HXK activity may divert hexoses from catabolic reactions to osmotic adaptation.

Increased aldose reductase (ALR) activities were detected in the leaf tissues of tomato plants grown for 3 weeks in culture medium containing 10 -7 or 10 -4 M salicylic acid (SA), and in the roots after the 10 -4 M SA pretreatment.The ALR activity changed in parallel with the sorbitol content in the leaves of the SA-treated plants.Salt stress elicited by 100 mM NaCl enhanced the accumulation of sorbitol in the leaves of control plants and as compared with the untreated control the sorbitol content in the SA-pretreated leaves remained elevated under salt stress.DEAE cellulose anionexchange column purification of the protein precipitated with 80 % (NH 4 ) 2 SO 4 revealed two enzyme fractions with ALR activity in both the leaf and the root tissues.The fraction of the leaf extract that was not bound to the column reacted with glucose and glucose-6-P as substrates, whereas glucose was not a substrate for the bound fraction or for root isoenzymes.The root enzyme was less sensitive to salt treatment: 50 mM NaCl caused 30 % inhibition in the leaf extract, whereas the enzyme activity of the root extract was not affected.It is suggested that increased ALR activity and sorbitol synthesis in the leaves of SA-treated tomato plants may result in an improved salt stress tolerance.

Pre-treatment with 10−4 M salicylic acid (SA) in hydroponic culture medium provided protection against salinity stress in tomato plants (Solanum lycopersicum L. cv. Rio Fuego). The effect of 10−7 or 10−4 M SA on the water status of plants was examined in relation to the biosynthesis and accumulation of abscisic acid (ABA) in order to reveal the role of SA in the subsequent response to salt stress. Both pre-treatments inhibited the K+(86Rb+) uptake of plants, reduced the K+ content of leaves, and caused a decrease in leaf water potential (ψw). Due to the changes in the cellular water status, SA triggered the accumulation of ABA. Since the decrease in ψw proved to be transient, the effect of SA on ABA synthesis may also develop via other mechanisms. In spite of osmotic adaptation, the application of 10−4 M, but not 10−7 M SA, led to prolonged ABA accumulation and to enhanced activity of aldehyde oxidase (AO1, EC.1.2.3.1.), an enzyme responsible for the conversion of ABA-aldehyde to ABA, both in root and leaf tissues. AO2–AO4 isoforms from the root extracts also exhibited increased activities. The fact that the activities of AO are significantly enhanced both in the leaves and roots of plants exposed to 10−4 M SA, may indicate a positive feedback regulation of ABA synthesis by ABA in this system. Moreover, during a 100 mM NaCl treatment, higher levels of free putrescine or spermine were found in these leaves or roots, respectively, than in the salt-stressed controls, suggesting that polyamines may be implicated in the protection response of the cells. As a result, Na+ could be transported to the leaf mesophyll cells without known symptoms of salt toxicity.

When the primary amino groups of α-chymotrypsin were modified with acetic, propionic, succinic, citraconic and phthalic acid anhydrides, the modifications enhanced the stability of the enzyme in 60% aqueous solutions of 1,4-dioxane, ethanol and acetonitrile. The acetylation of the amino groups resulted in the lowest stabilization, but the citraconylated, propionylated and succinylated forms of α-chymotrypsin exhibited increased stabilities in all the aqueous organic solvents studied. The modification of α-chymotrypsin with phthalic anhydride was accompanied by very extensive activation. The near-UV circular dichroism spectroscopic measurements did not indicate significant structural changes in the acylated forms of α-chymotrypsin, with the exception of the phthalic anhydride-modified enzyme. The spectral changes in the phthalic anhydride-modified enzyme suggest enhanced interactions between the aromatic chromophores and alterations in the disulfide contribution. The improvement in stability may be related to the modifications caused by the reorientation and the increased interactions of the aromatic side-chains, particularly in the case of the phthalic anhydride derivative.

Papain was modified with the anhydrides of various monocarboxylic (acetic or propionic) and dicarboxylic (citraconic, maleic or succinic) acids. 7–10 of the 11 primary amino groups of the enzyme were modified. The organic solvent tolerances of the modified enzyme forms were increased (especially in the concentration range of 10–60%) in comparison with the unmodified enzyme. Acylation enhanced the catalytic activity and stability of papain both in buffer and in aqueous organic solvents (ethanol and acetonitrile). Decrease of the positive charges on the surface of papain resulted in a higher enzyme stability than when they were replaced by negative charges. The kinetic parameters revealed that in aqueous ethanol the maximum rates (Vmax) and Michaelis constants (KM) of the modified papain forms were increased, and higher catalytic efficiencies (kcat/KM) were detected as compared with the native enzyme. The results of near-UV circular dichroism and tryptophan fluorescence spectroscopic studies suggested that the modifications caused only local changes around the aromatic residues. The modified enzyme forms led to higher N-acetyl-l-tyrosine ethyl ester synthesis conversions in aqueous ethanol; acetyl and propionyl papain furnishing the highest productivity.

BACKGROUND: The use of enzymes in organic solvents has extended the scale of their practical applications. Papain has been widely used in chemical syntheses because of its broad substrate specificity. The aim of the present study was to improve the stability of papain in aqueous tetrahydrofuran (THF) by using different saccharides. The effects of these carbohydrates on the structure of papain were followed by means of circular dichroism (CD) and fluorescence spectroscopic measurements.RESULTS: In contrast with most organic solvents, 60% (i.e. 600 mL L-1) THF practically inactivated the enzyme within 30 min. Sugars protected papain from THF-induced inactivation in the sequence D-ribose > D-fructose > D-glucose > D-saccharose > D-raffinose. Ribose at 1.6 mol L-1 proved the most effective stabiliser: in 60% THF in the presence of ribose, papain preserved about 55% of its initial activity after 2 h. Fluorescence and near-UV CD spectroscopic measurements revealed local changes in the papain conformation. With decrease in the free amino group content of the enzyme, protein-carbohydrate interactions (Schiff base formation) were detected.CONCLUSION: These results demonstrate that the catalytic activity and stability of papain may be increased in aqueous THF by using different carbohydrates, when a more compact structure of the enzyme is formed. (C) 2008 Society of Chemical Industry

The effects of aqueous solutions of ethanol, acetonitrile and 1,4-dioxane in the concentration range 10-90% (v/v) on the activity of porcine pepsin were studied. The enzyme retained its activity in aqueous ethanol and aqueous acetonitrile with increasing organic solvent concentration up to 60%, and in aqueous 1,4-dioxane up to 30%, but thereafter a considerable decrease in activity was observed. The changes caused in the catalytic activity by the water-miscible organic solvents may be related to structural changes, which were followed by means of intrinsic fluorescence and circular dichroism spectroscopy measurements. (C) 2007 Elsevier Ltd. All rights reserved.