I Cu active pocket in the laccase through hydrophobic interactions. Cations with shorter alkyl chains (C2~C6) entered the channel inside the pocket, exhibiting a high compatibility with laccase (competitive inhibition constant Kic = 3.36~3.83 mM). Under the same conditions, [Omim]Cl (Kic = 2.15 mM) and [Dmim]Cl (Kic = 0.18 mM) with longer alkyl chains bound with Leu296 or Leu297 near the pocket edge and Leu429 around TI Cu, which resulted in stronger inhibition. Complexation with alkylimidazolium cations shifted the pH optima of laccase to the right by 0.5 unit, and might, thereby, lead to invalidation of the Hofmeister series of anions. EtSO4− showed higher biocompatibility than did Ac− or Cl−, probably due to its binding near the TI Cu and its hindering the entry of alkylimidazolium cations. In addition, all tested ILs accelerated the scavenging of 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, which, however, did not play a determining role in the inhibition of laccase.Water-miscible alkylimidazolium ionic liquids (ILs) are “green” co-solvents for laccase catalysis, but generally inhibit enzyme activity. Here, we present novel insights into inhibition mechanisms by a combination of enzyme kinetics analysis and molecular simulation. Alkylimidazolium cations competitively bound to the T Anio,1,1,1]+ . The abo,1,1,1]+ ,16. To d,1,1,1]+ ,12,13,14,1,1,1]+ . These to) and inhibition constants (Ki), and the docking sites of five alkylimidazolium chlorides  (m) increased with increasing [I] while the maximum velocity (Vmax) remained constant. Competitive inhibition constants (Kic) were thereby calculated from Equation (2) and shown in ic value decreased with the increase of alkyl chain length or n value. In particular, a dramatic inhibition was detected when n > 6. For [Omim]Cl (n = 8) or [Dmim]Cl (n = 10), the Kic was determined as 2.15 and 0.18 mM, respectively. Lower Kic corresponded to stronger inhibition capacity. The reaction velocities under regular conditions  . All ploions See  were meaMth laccase in [Bmim]Cl/buffer solution, followed with lyophilization and thorough washing with absolute ethanol. The solid resultant was totally re-dissolved in water. The UV/Vis spectrum of the complex had both specific absorption bands of [Bmim]Cl (at around 210 nm) and Mth laccase (260~300 nm). There was obvious red shift from the absorption peak of laccase protein at 272 nm to the peak of laccase/[Bmim]Cl complex at 292 nm, which suggests the complexation of laccase with [Bmim]Cl absorbed energy. In addition, the re-dissolved complex still retained 65% of the initial activity (data not shown).According to enzyme kinetics theory, competitive binding means that the inhibitors irreversibly occupy the specific binding sites for substrates and form an enzyme–inhibitor complex . The forI Cu site  in Mth laccase could accommodate substrate molecules such as 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and three other phenolics  features. Instead, acetate and chloride ILs performed more chaotropic (weakly hydrated) as stronger inhibitors. For example, near 50% of laccase activity was lost by 100~130 mM acetate or chloride ILs at pH 3. KCl caused more activity loss than alkylimidazolium chlorides.Effects of salt species at various concentrations on laccase activity were studied to characterize the role of IL anions. The results  demonstr− and EtSO4− favorably bound with the same sites in Mth laccase, forming hydrogen bonds with Thr246 and Arg281 and hydrophobic interactions with His245. The optimal binding energy for Ac− and EtSO4− were −3.42 and −4.06 kcal/mol, respectively. This site was also the most frequent one for Ac− among 200 docking runs  was tested with addition of alkylimidazolium ILs . The addition of alkylimidazolium ILs (50 mM) in the buffer remarkably accelerated the elimination of ABTS+  or residual enzyme activities . However, the mechanisms underlying the kinetics and molecular interactions remained unexplored. Our results indicated that a competitive inhibition mechanism existed in the interactions between Mth laccase and alkylimidazolium chlorides  and also found dual inhibition mechanisms. The competitive constant, Kic, and the uncompetitive constant, Kiu, were 0.35 and 18.1 mM, respectively. Therefore, competitive inhibition was the dominant reason of the laccase inhibition by chloride salts BF4, while not being affected by [Bmim]PF6. Another study showed that the optimal pH of alkaline phosphatase was left shifted by Na+ and K+ salts [Tve laccase could be activated by [Mmim]MeSO4 at elevated pHs, i.e., 7 and 9 EtSO4 exhibited a good compatibility with laccase, probably due to its difficulty in forming the laccase/[Emim]+ complex. Then, the pH optima of laccase were less affected by [Emim]EtSO4 –(3)), respectively, as shown in the following formula.Our results showed that the change in laccase optimal pH may be linked to ILs’ hydrolyzabilities. Acetate ILs induced more shifts than those paired with strong anions. [Emim]Ac dissociated insufficiently into isolated ions in aqueous solution . It acte2 . Our res+ in experiments. The radical scavenging was thereby studied as an inhibition pathway. Our previous work showed that ABTS+ could be transformed into a reductive state by many common organic solvents EtSO4 than [Emim]MeSO4, [Emim]Ac, or [Emim]DMP at pH 4.5, which was in accordance with our results. Eshtaya et al. ) and IL ([I]). The Lineweaver–Burk diagrams were then constructed by plotting 1/vo against 1/[S] to estimate Km and Vmax according to Equation (1). The features of inhibition by different ILs were also deduced from the diagrams. Ki was correspondingly calculated from Equations (2), (3) or (4).A series of vLaccase preparation with initial activity of 20 U was mixed with 50 mM [Bmim]Cl at 25 °C for 1 h. Then, the mixtures were directly freeze-dried to remove water. The obtained solids were washed with absolute ethanol 6 times. Centrifugation was performed to remove loosely bound ILs as well as other contaminants soluble in ethanol. The resultant solids were re-dissolved in 10 mL water. Then UV/Vis spectra were measured using water as blank. The reference sample was obtained following exactly the same process without addition of any IL.+) were obtained from the oxidation of ABTS (10 mmol/L) with the laccase (50 U/L) in 50 μmol/L phosphate–citrate buffer (pH 3) at 40 °C for 2 h. Then, the reaction mixtures were centrifuged in an ultrafiltration tube  at 9000× g for 10 min). Enzymes and polymerized substances were removed. The oxidized products were incubated with buffer  and ionic liquids (50 mM) at 25 °C for 6 min. The concentration of each product was monitored by the spectrophotometer.ABTS cation radicals -Ln(At), versus time (t) gave straight lines (Equation (5)). Half-life (t1/2) was a constant value and could be estimated from the rate constant (K) according to Equation (6).The first-order kinetics model was assumed to describe the time-dependent scavenging of ABTSear axes . The varI Cu active pocket. Small cations with shorter alkyl chains (C2~C6) favourably entered the channel inside the pocket through hydrophobic interactions, exhibiting a good compatibility with laccase. Meanwhile, the species with long alkyl chains (C8~C10) formed stereo hindrance by occupying several key amino acids and finally led to a significant inhibition, i.e., at sites Leu296/Leu297 and Leu429. Complexation with alkylimidazolium cations caused a right shift of the laccase pH optima, thereby resulting in the invalidation of Hofmeister series of anions. In particular, EtSO4− could bind near the TI Cu active pocket, change the surrounding electric distribution and finally prevented the competitive binding of alkylimidazolium cations. In addition, alkylimidazolium ILs could accelerate the scavenging of ABTS+. However, it may not play a dominant role in the inhibitory effects of ILs.This work provided new insights into the Hofmeister effects of alkylimidazolium ionic liquids (ILs) on fungal laccase activity. The alkylimidazolium cations competitively bound to the T


In this paper, we present a benchmark dataset to evaluate the currently available analysis methods and visualizations for epiproteomic data. The benchmark dataset is a subset of a high-throughput time-series study of phosphoevents occurring upon insulin stimulation. Our dataset is provided in multiple formats for use with four currently available tools. We also provide a file containing the kinase assignments for the sites, as well as a simple kappa model on phosphorylation changes in insulin signalling. A detailed description of the tools, their analysis methods, and the visualizations generated using the input files described here, are discussed in detail in the accompanying review titled “Visualization and analysis of epiproteome dynamics" [1]. Such a selection had many advantages: a dataset of 103 profiles is easily managed compared to the total of 37,248. Furthermore, the phosphosites in this dataset are well studied, as they have been the subject of Figure 5 in Humphrey et al., and in subsequent publications Thus, from Humphrey In the originating paper et al. are provided in the file Original.txt. This dataset was converted to various input formats for use with the tools: DiBS, DynaPho, PhosphoPath and PHOXTRACK. Additionally, to demonstrate the use of mathematical modelling to evaluate our understanding of these data, we created a sample kappa model on phosphorylation changes in insulin signalling by surveying the literature, and provide it for use with kappa tools. The input files for these five tools are described in the sections below.The 103 profiles from Humphrey 2.1Input for this tool is provided in the file Dibsvis.csv. It is a simple CSV file containing the columns ‘Gene name’, ‘Phosphorylated amino acid’, ‘IPI position’  and the abundance ratios at 8 time points.2.2Input for this tool is provided in the file Dynapho.txt. It is a tab-separated file containing the human UniProt identifier, site (phosphosite position), residue (amino acid), 13-amino acid peptide sequence, and the abundance ratios at 8 time points. All the columns were directly extracted from the original dataset, except for UniProt identifier and site. This is because DynaPho only accepts human data, therefore, the mouse proteins and the phosphosites were converted to their human equivalents by performing a BLAST search 12.3Two input files are required for creating a visualization using this tool. Thus, two files are provided, namely, PhosphoPath_network.txt and PhosphoPath_timeseries.txt.The file PhosphoPath_network.txt provides a network structure for representing a protein and its phosphosites. Since PhosphoPath is a plugin for Cytoscape, where a network is represented in the form ‘source node’ and ‘target node’, the same structure is used here: the protein node as a source node and the site node as a target node, in columns 1 and 2, respectively. Columns 3 and 4, contain the site node display name and the peptide number, respectively.The file PhosphoPath_timeseries.txt contains time series values, which are used to build the heatmap in the visualization . These data are in two columns, where the first column is in the format “UniProt Id-Residue Site-Peptide Id-Time point number”, and the second column contains the quantified abundance ratio value at that particular time point for the phosphosite. We additionally also provide a script (getTpPhosData.py) and an example input file (phosphopath_input.tab) which can be used to generate this file. Details needed to run this script are found in the README.md file.2.4Data for this tool are provided in the file Phoxtrack.txt. It is a tab-separated file containing the 13-amino acid peptide sequence, followed by the mass spectrometry abundance ratios at 8 time points.2.5We provide a model of phosphorylation changes in response to insulin stimulation in the Kappa language format, in the file InsulinSignallingModel.ka. This model was compiled from the literature. It does not aim to provide an accurate representation of the underlying biology, but was built only for demonstration purposes .The model contains 13 proteins, 2 additional molecules (GTP and GDP), and a total of 33 reactions. The initial concentration of all molecules was set to 10, and the concentration of GTP was set to 1000, GDP was set to 10 (bound to ras protein). We observed the output of Insr Y1175 and Erk1 T203. A list of modelled reactions are shown in 2.6In this paper, a benchmark dataset is presented, in various formats, consisting of profiles of 103 phosphosites on 58 proteins, and their associated kinases. Although the sites in this dataset are well studied, a number of limitations are present.et al.The original study by Humphrey Secondly, of the 37,000 sites quantified, the benchmark dataset is a very small subset with only 103 sites. Furthermore, even though these 103 sites have been well studied and depict both phosphorylation and dephosphorylation changes, greater emphasis has been laid on studying the phosphorylation changes, and thus the phosphatases potentially causing the dephosphorylation changes are not included in our benchmark. Instead, dephosphorylation was considered only indirectly, as a result of deactivation of the kinases Finally, in spite of the focus on phosphorylation events, even for the well-studied 103 sites, not all causative kinases are known. These are presented in the kinase assignments file (Kinase_assignments.xlsx) as “?”. Furthermore, while the kinases included in the benchmark are well known and the assignments are based on good evidence, there is still the possibility that some of these assignments are not entirely accurate since, for example, the evidence may be generated under different experimental conditions or in different cell lines.


Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment. Integrin-mediated adhesion is a central regulatory process in cell biology, including development, immune system regulation and cancer. In immune cells, integrins are involved in almost every aspect of the immune response . IntegriB-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in western countries characterized by accumulation of neoplastic B-lymphocytes in the bone marrow (BM), secondary lymphoid organs and blood . CompareBruton's protein tyrosine kinase (BTK) was identified as a crucial protein kinase in B-CLL biology, regulating cell response to B-cell receptor (BCR) engagement , 28. SinTo address BTK role in chemokine signaling in B-CLL, we performed a comparative analysis of BTK signaling in healthy versus CLL B-lymphocytes. We applied an articulated approach consisting of adhesion assays and biochemical studies, to define the involvement of BTK in chemokine-triggered integrin activation. We found that CXCL12 activates BTK in normal as well as in CLL B-lymphocytes, with kinetics consistent with rapid integrin triggering. BTK inhibition by Ibrutinib prevents CXCL12-induced LFA-1 and VLA-4 mediated adhesion. Moreover, we show that BTK governs CXCL12-induced LFA-1 conformational changes leading to increased heterodimer affinity. Biochemical analysis shows that BTK is an upstream regulator of RhoA, thus explaining its role in integrin affinity triggering. Very importantly, we provide evidence showing that JAK2 is a crucial regulator of BTK activation by mediating BTK phosphorylation on critical tyrosine residues upon CXCL12 stimulation. This signaling mechanism is also fully conserved in normal versus CLL B-lymphocytes. Finally, we show that the JAK2-BTK interplay also regulates BCR signal transduction. Taken together, our data identify BTK as a key event in the signaling cascade generated by chemokines and BCR and leading to integrin-mediated cell adhesion. Importantly, JAK2 emerges as an upstream regulator of BTK activation in both CXCR4- and BCR-mediated signaling, thus suggesting a conserved hierarchical regulation of JAK2 versus BTK. This arises the possibility of JAK2-BTK inhibition as a possible synergistic combinatorial approach to B-CLL therapy.To characterize the role of BTK in the intracellular signaling leading to integrin activation in healthy primary B-lymphocytes, we first evaluated, in tyr-phosphorylation assays, BTK activation state upon CXCL12 stimulation. We found that BTK was rapidly phosphorylated on Y223 by CXCL12, with kinetics consistent with rapid adhesion triggering Figure  and 1C. To further characterize the role of BTK in integrin activation by chemokines, we analyzed the effect of BTK inhibition on chemokine-triggered integrin conformation changes, focusing on LFA-1 as prototypic and best characterized example of leukocyte integrin undergoing structural conformational changes corresponding to progressive affinity increase. We found that Ibrutinib pretreatment almost completely prevented LFA-1 transition to extended conformations, specifically evidenced by KIM127 Figure  and 2B aWe have previously demonstrated that JAK2 has a main role in the inside-out signaling mediating LFA-1 affinity upregulation by chemoattractants , 25, andWe, then, proceeded to investigate whether BTK is involved in chemokine-triggered inside-out signaling also in B-lymphocytes isolated from patients with a diagnosis of B-CLL, by studying 36 patients at the diagnosis and, thus, without any previous treatment Table . We founTo further confirm the involvement of BTK in integrin activation in CLL B-lymphocytes, we focused, as above, on the analysis of LFA-1 affinity triggering. Pretreatment with Ibrutinib resulted in a marked inhibition of CXCL12-induced LFA-1 conformational changes corresponding to transition to low-intermediate Figure  and 5B aWe have previously shown the main role of JAK2 in chemokine-induced integrin activation in CLL B-lymphocytes . ConsideUpon BCR engagement, B-lymphocytes develop adhesive interactions with other cells and matrix proteins , 49–54. Notably, in the context of BCR signaling to integrin activation, we observed some peculiarities. Indeed, and in sharp contrast with chemokine triggering, we could not detect any upregulation of LFA-1 affinity, as measured by conformation epitope antibodies (data not shown). Furthermore, we could not detect an increase of JAK Y1007/Y1008 phosphorylation upon BCR engagement (data not shown), and this contrasts with the observed effect of JAK2 inhibitors on both BTK activation and adhesion by BCR.Overall, these data demonstrate that the interplay between JAK2 and BTK is a central mechanism of integrin activation, although in a context of diversity of chemokine versus BCR signaling, and that these mechanisms are conserved both in normal as well as CLL B-lymphocytes.In previous studies we investigated the role of the rho module of integrin affinity triggering and of JAK protein tyrosine kinases (PTKs) in integrin activation by CXCL12 in B-CLL . We founBTK activation relies on phosphorylation on tyrosines 223 and 551, both involved in upregulation of enzymatic activity , 40. NotInterestingly, recent data show that, in mouse neutrophils, BTK is involved in classical chemoattractant, such a fMLF, but not chemokine, such as the CXCR2 ligand CXCL1 (IL-8), pro-adhesive signaling . These fNotably, we showed that in primary normal and CLL B-cells, RhoA activation by CXCL12 is mediated by BTK, thus explaining the inhibitory effect of Ibrutinib of integrin affinity triggering. In contrast, previous data obtained in a chicken cell line show that BTK is not involved in Rac1 and Rap1 activation by CXCL12 . This coOverall, our data demonstrate a novel function for BTK in CXCL12-induced rapid integrin activation and dependent adhesion in normal and CLL B-lymphocytes. Moreover, we identify the regulatory role of JAK2 on BTK activation by CXCR4, evidencing diversities with respect to BCR signaling. BTK seems a point of intersection between chemokine- and BCR-triggered signal transduction, providing novel insights in the molecular events occurring in leukemic cells after exposure to stromal cues. This could help envisioning new protocols of combined treatments in B-CLL.2 anti-human IgM was from Southern Biotech ; Tyrphostin and rabbit polyclonal anti-actin antibody were from Sigma; Ibrutinib was from Selleck Chemicals ; PE mouse anti-BTK (pY223) antibody was from BD Biosciences ; rabbit monoclonal anti-JAK2 (D2E12) was from Cell Signaling Technology ; siRNAs (ON-TARGETplus SMARTpool) were from Thermo Fisher Scientific .Human E-selectin/Fc, human ICAM-1/Fc, human VCAM-1/Fc and human CXCL12 were from R&D Systems ; goat anti-human IgM was from SouthernBiotech ; fluorescein isothiocyanate (FITC) goat secondary antibody to mouse was from Sigma ; KIM127 mouse monoclonal antibody was from American Type Culture Collection ; 327A mouse monoclonal antibody was kindly provided by Dr. Kristine Kikly ; goat F(ab’)6/ml in RPMI + Glutamine 2 mM + FBS 10% for 2 h before treatment with inhibitors or Trojan peptides.Normal and CLL B-lymphocytes were isolated from PBMCs after blood separation on Ficoll Paque Plus  and purification by negative selection . Purity of B-lymphocyte preparations was checked by flow cytometry with anti-CD19 mAb . PBMCs were recovered by buffy coats from the venous blood of normal healthy volunteers, from the Blood Transfusion Center at the Verona University Hospital, after informed consent. The study involved a total of 36 B-CLL patients. To avoid any bias regarding age and/or gender, all available patients were studied . Patients were, however, selected for complete absence of any previous treatment. The diagnosis of B-CLL was made upon clinical and laboratory parameters, including the complete blood cell count and immunophenotype of the circulating lymphoid cells, according to the current guidelines and fulfilling diagnostic and immunophenotypic criteria for common B-CLL  at the S6/ml in standard adhesion buffer . Adhesion assays were performed on 18-well glass slides coated with human ICAM-1 or VCAM-1, 1 μM in PBS. 20 μl of cell suspension were added to the well and stimulated at 37°C with 5 μl of CXCL12, 0.5 μM final concentration, for 120 sec, or with anti-IgM, 10 μg/ml final concentration, for 5 min. After washing, adherent cells were fixed in glutaraldehyde 1.5% in ice-cold PBS and counted by computer-assisted enumeration.B-lymphocytes were suspended at 5 × 106/ml in standard adhesion buffer. Cell behavior in under-flow conditions was studied with the BioFlux 200 system . 48-well plate microfluidics were first co-coated overnight at RT with 2.5 μg/ml human E-selectin together with 10 μg/ml human ICAM-1 or with VCAM-1 alone in PBS. Immediately before use, microfluidic channels were washed with PBS and then coated with 4 μM CXCL12 in PBS for 3 h at RT and the assay was done at wall shear stress of 1 dyne/cm2. After extensive washing of microfluidics with adhesion buffer, the behavior of interacting lymphocytes was recorded on digital drive with a fast CCD videocamera  and analyzed subframe by subframe. Single areas of 0.2 mm2 were recorded for at least 60 sec. Interactions of 20 msec or longer were considered significant and scored. Lymphocytes that remained firmly adherent for at least 10 sec, thus including also events of adhesion stabilization, were considered fully arrested and scored. Rolling interacting and arrested cell behaviors were automatically detected and quantified with BeQuanti .B-lymphocytes were suspended at 1.5 × 106/ml, were briefly pre-incubated with 10 μg/ml of KIM127 or 327A mAbs and then stimulated for 120 sec with 0.5 μM CXCL12  at 37°C. After rapid washing, cells were stained by FITC secondary polyclonal antibody and analyzed by cytofluorimetric quantification.B-lymphocytes, suspended in standard adhesion buffer at 2 × 106 cells were treated and stimulated as indicated and, after a rapid wash, fixed in formaldehyde 4% for 30 minutes at 4°C. Then cells were briefly washed and suspended in 50 μl of permeabilization buffer (PBS + FBS 5% + saponin 0.5%) containing PE-conjugated anti-pY223-BTK antibody, for 30 minutes at 4°C. After rapid wash, cells were suspended in ice-cold PBS and analyzed by cytofluorimetric quantification.BTK activation was measured by means of tyrosine 223 phosphorylation. Briefly, 0.5 × 106 cells were treated and stimulated as indicated and then lysed at 4°C. Cell lysates were tested for pYpY1007/1008 JAK2 and colorimetric signals were detected by a plate reader .JAK2 activation was measured by ELISA kit , following manufacturer's instructions. Briefly, 5 × 106 cells were treated and stimulated as indicated and then lysed at 4°C. Cell lysates were tested for GTP loaded RhoA and colorimetric signals were detected by a plate reader .RhoA activation was measured by using commercial kit , following manufacturer's instructions. Briefly, 5 × 10Silencing was performed in normal B-lymphocytes by AMAXA nucleofector (program U-15), following manufacturer's instructions, and the efficacy of gene silencing was assessed after 48 h by immunoblotting).Cells were lysed in ice-cold 1% NP-40 lysis buffer containing complete protease inhibitor cocktail (Roche). After quantification by Bradford assay (Bio-Rad), equal amounts of proteins were subjected to 10% SDS-PAGE. After incubation with JAK2 primary antibody and HRP-coupled secondary antibody , immunoreactive bands were visualized by ECL detection (EMD Millipore), acquired by using ImageQuant Las4000  and quantified by densitometric analysis .t-test or one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test at the 95% confidence level. Significance threshold was set at p < 0.05. All statistical analyses were performed using the GraphPad Prism version 6 software .Results are expressed as mean ± standard deviation (SD). Statistical significance was assessed by two-tailed Student's


Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria. Aerosol and local soil depositions on the glacier surface resulted in distinct microbial communities developing in glacier ice and cryoconite holes. Based on a source tracking algorithm, we found that cryoconite microbial assemblages were a potential source of organisms, explaining up to 36% of benthic microbial mat communities in the lake. However, the major biotic sources for the lake ecosystem are still unknown, illustrating the possible importance of englacial and subglacial zones. The Anuchin Glacier may be considered as a vector in a biological sense for the bacterial colonization of the perennially ice-covered Lake Untersee. However, despite a thick perennial ice cover, observed “lift-off” microbial mats escaping the lake make a bidirectional transfer of biota plausible. Hence, there is an exchange of biota between Lake Untersee and connective habitats possible despite the apparent sealing by a perennial ice cover and the absence of moat areas during austral summer.Ultra-oligotrophic Lake Untersee is among the largest and deepest surface lakes of Central Queen Maud Land in East Antarctica. It is dammed at its north end by the Anuchin Glacier and the ice-cover dynamics are controlled by sublimation — not melt — as the dominating ablation process and therefore surface melt during austral summer does not provide significant amounts of water for recharge compared to subsurface melt of the Anuchin Glacier. Several studies have already described the structure and function of the microbial communities within the water column and benthic environments of Lake Untersee, however, thus far there have been no studies that examine the linkages between the lake ecosystem with that of the surrounding soils or the Anuchin Glacier. The glacier may also play an important role as a major contributor of nutrients and biota into the lake ecosystem. Based on microbial 16S rRNA amplicon sequencing, we showed that the dominant bacterial signatures in Lake Untersee, the Anuchin Glacier and its surrounding soils were affiliated with  The largest ice sheet on Earth covers more than 99.8% of continental Antarctica . Here, tThe most prominent habitat on glacier surfaces is cryoconite holes that cover 1 to 10% of glacial ablation zones . They foGlaciers and ice sheets are hydrologically connected ecosystems that constantly receive abiotic and biotic matter and subsequently export material to adjacent downstream environments . TherefoIn many cases, Antarctic glaciers are the only obvious hydrological connection to adjacent perennially ice-covered lakes and hence the only source of nutrients and biota . BesidesHere, the perennially ice-covered Lake Untersee is dammed by the Anuchin Glacier that gradually drains into the lake. Among others, studies at the Untersee Oasis included biogeochemical analyses of the water column , microbiAs shown in the case of Lake Untersee, the majority of scientific investigations on specific polar environments have mostly been dealt with on a solitary basis, but not in a connective sense. Examples thereof are scarce e.g., . HoweverHere, we hypothesized that in the supraglacial zone (a) cryoconite hole microbial communities were dominated by microbial assemblages found in aerosols and adjacent soils and therefore differed from communities originating from bare glacier ice. Despite the presumably small volumetric fraction of cryoconite to the annual glacial melt water input, (b) cryoconite microbial communities can be mirrored in Lake Untersee environments. Biotic fluxes from the Anuchin Glacier to Lake Untersee were put in context by estimating the annual glacial melt volume and the volumetric fraction of cryoconite holes thereof.2 large ice wall  build up due to methanogenesis in surface sediments and the water column  and drains into the Lake Untersee oxic basin. The surface velocity of the glacier is about 9 m a-1  and -35.2°C (July 2012). The number of days above freezing ranged from 7 to 49 days. The prevailing wind direction was south with an average daily maximum of 15 m s.7 m s-1 .During the 2015 summer field season, an additional custom-built weather station recorded wind speed, direction and temperature with a high temporal resolution . The station was set up in the Aurkjosen Cirque, at the north-western shore of Lake Untersee from November 25, 2015 until December 14, 2015 .Two aerial images  and a satellite image from 2015 provided by Digital Globe were imported as layers into Adobe Photoshop and subsequently transformed to account for different angles during image acquisition. Then, the scene was cropped to unify all image-excerpts and the glacier-lake boundaries from each image were mapped into evenly spaced data points with defined x and y pixel coordinates using the software Fiji . The y cThe melt rate of the glacier into the lake was estimated by combining data from a bathymetric map  and the n = 10), north of the glacier/lake ice ridge (n = 6) and white ice patch within the lake ice (n = 3). Batch modifications of all histograms with the software Fiji (4 × Area / (π + Major Axis2).Images of the glacier surface were recorded with a drone  during two flights between 17:00 and 18:00 UTC. In total, 19 images with a resolution of 12 megapixel were analyzed from the medial moraine  ethanol and cleaned with an extra ice core that was drilled next to the cryoconite hole to be discarded. Sediments less than 5 cm deep were collected with a sterile spatula after the ice lid was removed with a sterilized ice axe. Cryoconite hole samples were distinguished into two regions, namely “medial moraine” at the upper part of the glacier medial moraine and “glacier terminus” in proximity of the glacier-lake boundary.We sampled glacial ice along the N-S orientated medial moraine (AT_1) and collected additional transects in clean ice 150 m west (AT_2) and east (AT_3) from AT_1. Each transect consisted of six transect points. The four sampling points closest to the lake were in a distance of 500 m to each other and the upper two sites were kept each at 1000 m to the next point.At each sampling site, three ice cores from the upper 20 cm of ice were collected with a Kovacs ice corer powered by a 2-cycle Tecumseh motor (2014) or a Bosch Hammer GBH 36V-LI PLUS electrical drill with 4 Ah batteries (2015). A custom-made drill guidance system enabled a standardized spatial sampling of triplicate ice cores due to a rotational axis that kept the distance between the ice samples at fixed levels .Additionally, we sampled two one-meter long ice cores from the Lake Untersee ice cover and the so-called “White Ice Patch” which are further referred to as “US” and “WIP,” respectively. WIP appears to be glacial ice but is part of the lake ice-cover, south of an ice ridge that separated the glacier from the lake at the surface . Each saFor all drilled samples, post-sampling decontamination procedures  could noSamples of lake water at the anoxic basin were collected in 2008 at 8, 30, 40, 50, and 70 m with a Niskin bottle. Water samples were kept frozen throughout storage in the field and subsequent transport to the home university with dry ice. Lake sediments from the oxic (169 m) and anoxic (100 m) basins were collected with an Ekman dredge connected to a parachute cord. The samples were transported to the campsite and immediately transferred into sterile bags using a baked spatula. Other benthic samples, namely flat mats and pinnacle mats were collected by a scuba diver at a depth of ∼30 m in the oxic basin using methods as described by Soil samples were collected with a baked spatula at the south-western shore of the lake. They were associated with a microbial mat, buried 3 cm deep in gravel and a gray crust on a rock.-min ranging from 3,000 to 12,000 liters per sample, yielding to 45,000 L total volume. The captured airborne particles had to be pooled into one sample to gain sufficient DNA content.Samples of near-ground air were collected in 2015, at the windward side of the campsite to avoid contamination from the camp itself. Airborne particles were accumulated in a sterile sodium chloride solution (0.9%) with a Bertin Coriolis-μ air sampler. To prevent the liquid from freezing during the sampling procedure, the sampling container was placed in a Nalgene bottle with hot water. After sample collection, the sampling containers were stored with previously collected ice cores that served as a cooling agent. In total, seven samples have been collected over a period of 3 days with a flow rate of 300 L®DNA Isolation Kit. PowerWater®DNA Isolation Kit was used for the bioaerosols, ice and water samples following their respective manual’s instructions. The DNA content was measured with a QubitTM dsDNA HS Assay Kit before 10 ng DNA of each sample were applied as template in subsequent PCR reactions.All samples were thawed thoroughly in the dark at 4°C. DNA from benthic microbial mats, terrestrial samples and a cryoconite holes was extracted using the PowerSoilThe 16S rRNA gene amplicons for sequencing were obtained using Illumina-tagged universal primers F515 and R806 as proposed by the Human Microbiome Project and the Earth Microbiome Project  and archRaw sequences were pre-processed and filtered using the R package dada2 (version 1.4.0) according to the proposed processing pipeline . Brieflypost hoc test (Bonferroni corrected). Analysis of beta diversity was performed to examine differences between microbial community structures in Lake Untersee environments. Principal Coordinates Analysis (PCoA) was performed directly on the (to relative abundance transformed) RSV table. Significances were tested using adonis (seed 1) and further confirmed by dispersion tests (permutations: 999). Source tracking was performed using the sourcetracker algorithm as proposed by The RSV table  was used to calculate alpha and beta diversity using the R packages Phyloseq  version SRP145579.Every step of the analysis included negative controls consisting of blank extractions for each group of samples and negative controls during PCR amplifications. RSVs that overlapped negative controls and samples were removed from the data sets. Sequence data have been deposited in the NCBI database under the accession number -1. During the first 56 years (1939–1995) the glacier retreated on average by 1.16 m a-1 and increased up to 5.76 m a-1 during the last 20 years (1995–2015). Combining these calculations with data from a bathymetric map of Lake Untersee . Further, the length of the surface glacier-lake boundary smoothened and therefore decreased by 2.85 km. About 82% of this reduction happened between 1995 and 2015. The position of the Anuchin Glacier for the years 1939, 1995, and 2015 is depicted in The average retreat within 76 years (1939–2015) was 2.37 m aUntersee , the melThe cryoconite holes coverage on the Anuchin Glacier was 3.46 ± 2.34% on average. The highest density was observed near the glacier-lake interface at the SW part of the glacier (6.36 ± 0.98%) and the lowest coverage was observed along the medial moraine (1.73 ± 0.36%).-1 and from 8.96 to 32.94 m a-1 in recent years (1995–2015), respectively. The maximum volumetric portion of cryoconite to the annual melt was 1.32 × 10-3% on average within 76 years, representing 1.56 × 10-6% of the present estimated lake volume.Assuming an average cryoconite layer thickness of 1 cm, we estimated that the long-term (1939–2015) average contribution to the lake ranged between 3.69 and 13.56 m a-1) and maximum wind speed (24.6 m s-1) were associated with the prevailing wind directions. Average hourly wind speeds above 7 m s-1 occurred only between 02:00 and 10:00 and the average temperature was -0.08°C .About 77% of the time local winds from 90 to 160° were recorded during a 20-day period . The higFor bacterial community profiles, the prevailing information was grouped into seven different habitats: “Air” (air sample), “Terrestrial” (buried mat and crust from a rock), “Pelagic” (water column of Lake Untersee), “Benthic” , “Lake ICE” (lake ice from Lake Untersee), “Glacier ICE” (surface ice of Anuchin Glacier) and “Cryoconite” (cryoconite holes from Anuchin Glacier). Amplicon sequencing resulted in a total of 1,414,290 read counts, including 4,772 taxonomic observations in 52 samples. The amplicon read counts for all samples are depicted in Proteobacteria , followed by Actinobacteria (17.9%), Cyanobacteria (13%) and Firmicutes (7.6%). The highest percentage of reads was assigned to the bacterial genera Methylobacterium , Reyranella (1.7%), Sphingomonas (1.8%), Polaromonas (1.8%) and Acinetobacter (2.4%). Tychonema (6.7%), Chamaesiphon (2.5%), and Leptolyngbya (2.7%) comprised most affiliated reads for the phylum Cyanobacteria. An overview of the most abundant phyla across the sampling sites is depicted in Overall, signatures of 42 bacterial phyla were detected, whereas most of the RSVs were affiliated with p < 0.05) and differed significantly . This was also true for the normally distributed dataset observed for the Shannon index .The bacterial alpha diversity was calculated using the indices Observed, Shannon and InvSimpson and is depicted in a boxplot . Data fop = 3.261232 × 10-5 and 4.102203 × 10-2, respectively; Dunn’s Test, the p-values were adjusted using the Bonferroni method). In total, 4 groups were significantly different when observing the Shannon Index . The pooled air sample had to be excluded from statistical analysis.There were significant differences between the groups “GLACIER ICE” / “GLACIER CRYOCONITE” and “GLACIER ICE” / “LAKE BENTHIC” when using the Observed index (p < 0.001). Most of cryoconite holes from the medial moraine overlapped with lake samples while cryoconite holes from the glacier terminus were rather isolated from other lake samples. Except for the lake ice cover, lake samples also clustered with terrestrial samples. The glacier ice formed three distinct clusters; two of them were associated with white ice patch samples and one with the air sample, respectively.Regarding the beta diversity, the samples clustered according to their grouping and were significantly different from each other  with glacier ice communities and 79 (55.6%) with cryoconite holes, respectively. The supraglacial environment contained in total 713 genera, 112 of them (10.55%) were present in both, glacial ice and cryoconite holes while 38 genera (3.58%) were exclusive to cryoconite holes and 563 genera (42.47%) were only found in glacial ice. From 108 OTU’s that were identified in terrestrial sources 36 (33.3%) of them were also discovered in cryoconite holes, glacial ice and the lake. The lake harbored 284 different genera. Lake sediments and the water column shared 62 (21.8%) of them. In the lake and on the glacier, 177 genera (21.5% of genera affiliated with the lake) were identical with glacier affiliated genera (We observed 12 out of 1247 genera (0.96%) that occurred in all habitats. They were affiliated with d genera .p < 0.05). The majority were abundant in the glacier ice but not abundant in cryoconite holes. Most of the RSVs could not be assigned to any known taxon.We focused on the supraglacial habitats cryoconite holes and glacial ice as they were different from each other in richness and diversity. Taxa with significantly different abundances were determined using the Mann–Whitney test . In total, 92 RSVs were significantly different abundant . Here, the highest amount of genus-assigned RSV counts was affiliated to Chamaesiphon in the group “GLACIER CRYOCONITE”  and Pseudanabaena in the group “LAKE PELAGIC” (2.5%).Three genera were significantly different abundant: Euryarchaeota and the highest amount of all archaeal RSV counts was classified as Methanomicrobia (51.8%), followed by Halobacteria (43.2%) and low numbers of Thermoplasmata (>5%), Methanobacteria and Thaumarchaeota (both > 1%).In total, 13 samples did not comprise any RSV affiliated to archaea (amongst them the “AIR” sample and most of the “LAKE ICE” samples). From all other samples, 185,004 archaeal RSV counts were obtained. Exclusively, all RSVs were affiliated to Methanobacteriales-SMS-sludge 7 . Methanosaeta  and Methanocellaceae  comprised also a high RSV count, while low abundant RSVs were affiliated to Methanobrevibacter and Methanosarcina (<1%) (Methanogens were exclusively detected in the groups “LAKE ICE” and “LAKE BENTHIC” as well as a high majority of unassigned na (<1%) .The archaeal alpha diversity was calculated as described in the section “bacterial alpha diversity.” The highest diversity and richness were detected in the group “LAKE BENTHIC,” followed by “GLACIER ICE” and “GLACIER CRYOCONITE.” The groups “TERRESTRIAL” and “LAKE ICE” comprised a very low diversity and almost all samples within these groups did not contain any Archaea at all . Statistx-axis (51.4%) to other assemblages, namely “GLACIER CRYOCONITE” and lake sediments from the group “LAKE BENTHIC.” The remaining “ANUCHIN CRYOCONITE,” “TERRESTRIAL,” and “LAKE BENTHIC” samples formed a third cluster  lined up in the same area and showed the largest distance on the  cluster . An analTo estimate source environments for the microbial distribution, we applied a source tracking algorithm . GeograpA diverse range of habitat types at the Lake Untersee Oasis host microbial life including glacier surfaces, ice-free areas, and a perennially ice-covered lake with its pelagic and benthic zones. These refugia are geographically connected as aerial and local depositions inoculate cryoconite holes on the glacier surface and subsequently colonize Lake Untersee. Generally, extremely short periods in the year provide subsurface melt water within cryoconite holes that ensure biotic exchange and the surrounding glacial ice. However, sub-surface glacial melt into Lake Untersee exists throughout the year with sub-annual varying rates .-3%, the proportion of cryoconite hole communities as source environment appeared to be substantial.We used a Bayesian source tracking algorithm  to estimMost biotic sources that inoculated Lake Untersee benthic and pelagic zones were not identified in this study and the proportion of known source environments differed across the sink samples. We expected biota entering the lake being present in the water column and benthic zones but pelagic microbial communities did not appear as an important sink environment. Considering that the lake was not permanently ice covered  until atFirmicutes, Proteobacteria and Actinobacteria. Cyanobacteria, which are known to be deposited as bioaerosols , whereas Gammaproteobacteria (AT2) and Alphaproteobacteria (AT3) were more abundant in the clean-ice parallel transects.Surface ice samples from the Anuchin Glacier were divided into three transects, namely the medial moraine (AT1) that was flanked by two parallel transects  with clear ice. We found Cyanobacteria that were present in the lake ice and the white ice patch were also discovered in at least one of the glacier transect points. Within Cyanobacteria, Chroococcidiopsis was found in AT1 and AT2 while the genera Geitlerinema, Microcoleus, Nostoc, Phormidium, and Pseudanabaena were mainly present along the medial moraine (AT1), indicating that different genera of Cyanobacteria prefer specific local conditions or that biotic sources differ among transects.Genera belonging to Cryoconite holes are considered as microbial hotspots in supraglacial Arctic environments due to a high biodiversity . ContradActinobacteria in cryoconite holes and Thermoleophilia in glacier ice, less than 50% of the genera within the significantly different classes  could not be assigned either. Microbial assemblages in cryoconite holes and the glacier ice were significantly different . The mosPelagic microbial communities from Lake Untersee were dominated by Proteobacteria, Bacteroidetes, Actinobacteria, Planctomycetes, and Cyanobacteria. In comparison, within three interconnected lakes located in the nearby Schirmacher Oasis, Chloroflexi was among the most abundant phyla and another series of connected lakes from the same area was rather dominated by Firmicutes than by Bacteroidetes  were no exception from previous observations and harbored Cyanobacteria, Proteobacteria, Bacteroidetes, Planctomycetes, Verrucomicrobia, and Actinobacteria. In contrast, the main phyla in the oxic sediment belonged to Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, and Planctomycetes. In the anoxic sediments we found Proteobacteria, Chlorobi, Actinobacteria, Planctomycetes, and Armatimonadetes as dominating phyla. It is noteworthy that differences in sampling strategies between the benthic microbial mat samples (carefully collected by a scuba diver) and deep lake sediments (Ekman dredge) hampered this comparison. Based on in situ images in the oxic basin . Together with Bacteroidetes and Actinobacteria, they comprised 99.2% of the bacteriome in Lake Untersee sediments. Cyanobacteria in three distinct laminae of a benthic microbial mat from Lake Untersee decreased with depth ranging from about 90% (top) to 20% (bottom). In our study, only bulk samples were processed and hence, results are not directly comparable. However, the relative abundance of Cyanobacteria within benthic microbial mats in both studies was in the same range.Further, Cyanobacteria are the most important phototrophs in freshwater and terrestrial ecosystems  and anoxic sediments (33.11%), mainly belonging to the class Methanomicrobia. In contrast, the first discovery of archaea in Antarctic cryoconite holes was mainly linked with Thaumarchaeota  than with aerosols and soils combined (53%) and therefore we reject our initial hypothesis that cryoconite hole microbial communities were dominated by microbial assemblages found in aerosols and adjacent soils. Further, from 150 genera in cryoconite holes, 69.3% of them were also present within Lake Untersee (including the lake ice cover). Considering this finding in combination with the source tracking analysis, we accept our hypothesis (b) that cryoconite hole microbial communities are mirrored in Lake Untersee environments.The combination of wind driven temperature changes and global rising temperatures makes the Lake Untersee Oasis prone to rapid changes compared to other Antarctic regions. Rising temperatures will increase the availability of liquid water and hence induce glacio-morphological changes, reshaping habitats and will possibly alter the microbiome of the oasis. These changes require thorough monitoring to fully understand potential shifts in the microbiome. For this purpose, the present study has provided a reference database for future studies in context with climate change at Lake Untersee Oasis.KW designed the study and collected samples  during the Antarctic expedition 2015 that was led by DA. KW, and AP extracted DNA and did all subsequent genome analyses. KW and BS wrote most of the manuscript that was reviewed by all authors. Statistical analyses were done by AP.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.


Ribosomes are among the largest and most dynamic molecular motors. The structure and dynamics of translation initiation and elongation are reviewed. Three ribosome motions have been identified for initiation and translocation. A swivel motion between the head/beak and the body of the 30S subunit was observed. A tilting dynamic of the head/beak versus the body of the 30S subunit was detected using simulations. A reversible ratcheting motion was seen between the 30S and the 50S subunits that slide relative to one another. The 30S–50S intersubunit contacts regulate translocation. IF2, EF-Tu, and EF-G are homologous G-protein GTPases that cycle on and off the same site on the ribosome. The ribosome, aminoacyl-tRNA synthetase (aaRS) enzymes, transfer ribonucleic acid (tRNA), and messenger ribonucleic acid (mRNA) form the core of information processing in cells and are coevolved. Surprisingly, class I and class II aaRS enzymes, with distinct and incompatible folds, are homologs. Divergence of class I and class II aaRS enzymes and coevolution of the genetic code are described by analysis of ancient archaeal species.  Thermus thermophilus ribosome as an example, and numbering throughout is for T. thermophilus. Discussion of transfer ribonucleic acid (tRNA), genetic code, and aminoacyl-tRNA synthetase (aaRS) evolution relies on ancient archaeal species because these functions are the most primitive in archaea. Although some factors have been substituted in evolution, translation mechanisms are similar in archaeal, bacterial, and eukaryotic systems. In our discussion, we stress the homologous binding and functions of IF2, EF-Tu, and EF-G G-protein GTPases in translation initiation and elongation. As part of an ancient pre-ribosome, the 30S subunit and the decoding center appear to be older than an associated 50S subunit, and the 50S subunit may have been recruited from a captured, formerly mobile peptidyl transferase center (PTC). The messenger ribonucleic acid (mRNA) and tRNAs lie across the neck of the 30S subunit between the body and the head/beak. The head/beak reversibly swivels and/or tilts relative to the body to function as a translocation ratchet. The 30S and 50S subunits reversibly rotate relative to one another to form an additional ratchet for elongation and initiation. Interactions between the 30S and 50S subunits and events at the A-site latch regulate translocation. A channel forms between the 30S and the 50S subunits, allowing tRNAs to align, advance, and pass through without major obstructions. We describe translation initiation and elongation using images taken from recent structures. Characteristics of tRNAs that make a relatively stiff and efficient translation adapter are discussed. Translation systems appear to have evolved around tRNA. The referencing in this review is not exhaustive, and we apologize to anyone whose original work we may have overlooked. Excellent and more detailed reviews are available for initiation .,17.fMet itiation . PresumaConsidered as a molecular motor, the ribosome appears to be driven by a complex thermal ratchet translocation mechanism with an overall step length of ~14 angstroms [1 codon (3 nt) on an extended mRNA] . BecauseT. thermophilus. In −1, and accommodation appears to be the rate-limiting step   and rotaEF-Tu·GTP binds aa-tRNA to form the aa-tRNA·EF-Tu·GTP ternary complex, and this is the form in which aa-tRNA enters the ribosome . EF-Tu·GThe accuracy of translation requires the tight closing of the A-site codon-anticodon latch to confirm base pairing . An overIle (UAU), which is very rarely used in prokaryotes versus tRNAMet (CAU)—only pyrimidine-purine discrimination is possible at the wobble position. By contrast, a near-cognate tRNA generally fails to maintain a closed conformation of the latch and the 30S subunit, releasing and replacing the aa-tRNA·EF-Tu·GTP/GDP ternary complex before inaccurate aa-tRNA CCA accommodation and aa misincorporation can occur. A near-cognate tRNA would be one with a G~U wobble pair in the 1st or 2nd codon position, which correspond to the 2nd or 3rd anticodon position (reading 5′→3′). The codon-anticodon latch, therefore, is tight enough to accurately select Watson-Crick pairs from wobble pairs in the 1st and 2nd codon positions (the 2nd and 3rd anticodon positions). From x-ray crystallography studies, it appears that near cognate G~U pairs within a tightened latch are forced into Watson-Crick geometry, which requires keto → enol tautomerization of either G or U and is not energetically favorable nor kinetically stable, which leads to near-cognate aa-tRNA release . By contThe mechanism of EF-Tu GTPase activation by the 50S ribosomal subunit is shown in Accommodation requires a large rotation of the A-site aa-tRNA from the A/T-site to the A/A-site (~100 angstrom transition of the incoming amino acid) . Figure Accommodation is described in two major steps: (1) elbow accommodation; and (2) CCA accommodation ,30,33. IOnce EF-Tu dissociates, the free 3′-CCA-aa end of the A-site tRNA is single-stranded and flexible and must penetrate the PTC ,35,36,37The codon-anticodon A-site latch remains closed through peptidyl bond formation, but opens to enable translocation. CCA accommodation results in a kink in the mRNA between the P-site and A-site tRNAs in order to position the tRNA 3′-ends for peptidyl transfer . After bBringing and finally locking the 3′-CCA-aa end of the A/A-site tRNA close to the P-site tRNA peptide in a dehydrated environment with appropriate molecular positioning and crowding is sufficient to support peptide bond synthesis. The peptide chain is transferred from the P-site tRNA to the A-site tRNA, where the peptide chain mostly remains stationed. In the presence of EF-G·GTP/GDP, reversible excursions between A-site and P-site contacts are observed for peptidyl-tRNA, described as A/A, ap/A, and ap/ap hybrid states ,48. In tThe PTC is considered to be a molecular crowding and dehydration chamber generally lacking any particular ribozyme activity . AlthougPhe and P-site fMet-tRNAfMet). A high kinetic solvent isotope effect (hydrogen/deuterium exchange) for protein synthesis indicates proton transfers from water, supporting the proton shuttle pathway. Potentially, some amino acid substrates may prefer the alternate reaction pathway, which is general base catalyzed. The pH dependence of the protein synthesis mechanism so far observed, however, is not generally consistent with heavy use of the base-catalyzed path. In contrast to peptide synthesis, translation termination mechanisms utilize the base-catalyzed path, leading to P-site peptidyl-tRNA hydrolysis and peptide chain release mediated by a protein translation release factor . Th. Th21]. 2+. The somewhat loose binding allows restrained sliding of the subunits relative to one another. Ratcheting of the 30S subunit relative to the 50S subunit by ~7° helps to drive forward translocation [The 30S and the 50S subunits of the ribosome are somewhat loosely attached. Subunit association is stabilized by mRNA binding and Mglocation . Before location ,48. EF-Glocation . From siEF-G·GTP/GDP undergoes multiple conformational distortions during binding, peptidyl transfer, GTPase, and translocation ,29,58,59Because the 30S and the 50S subunits rotate relative to one another in translocation, intersubunit contacts regulate dynamics ,62. In tWe posit that A-site, P-site, and E-site codon-anticodon (mRNA-ASL), 3′-CCA tRNA attachments, and the exiting peptide chain form the primary pawls that help maintain the ribosome translocation phase. The E-site does not bind acylated tRNA, so tRNA cannot slip from the P-site to the E-site without the transfer of the peptide chain to the A-site. As might have been expected, mutation of the E-site near the 3′-CCA contact causes frameshifting errors . This isThe A-site finger  that contacts the 30S S13 protein might be considered a micro-pawl to maintain the register for translocation. Transitioning from the A/A-site to the ap/ap-site, tRNAs contact the A-site finger . Type I In concert with the larger ratcheting, tilting, and swiveling motions of the ribosome are smaller movements. The ribosome is described as a flexible, semi-stable, and dynamic molecular motor . The 50STranslation systems evolved around tRNA. Internal tRNA homologies and tRNA evolution are described in Type I tRNA evolved by the ligation of three 31 nt minihelices followed by two symmetrical 9 nt deletions within ligated 3′- and 5′-acceptor stems ,64,65. TAs explained above, tRNA evolved from repeats  and inverted repeats  ,64. In F8 proteins. By contrast to class I aaRS, class II aaRS enzymes have an active site of antiparallel β-sheets. Rodin and Ohno posited that, despite their incompatible folds, class I and class II aaRS enzymes were somehow related, possibly by antisense transcription-translation [Methanobacterium bryantii) and GlyRS-IIA  enzymes with e-values as low as 5 × 10−11, strongly indicating homology.Aminoacyl-tRNA synthetases  charge tRNAs with amino acids . These enslation ,76,77,78nslation . Our labPyrococcus furiosis, an ancient species that is very similar to LUCA . Because of the proximity of P. furiosis to LUCA, this comparison gives an accessible and qualitative view of early aaRS evolution and radiation. In P. furiosis, which is similar to LUCA. The smaller the e-value, the more closely related are the enzymes. From column 1 of the genetic code , ValRS-IrRNA is posited to have evolved after and around tRNA ,80. The Ile (UAU) is rarely used. Furthermore, single base discrimination of A, G, C, and U at the anticodon wobble position is difficult. Effectively, only purine versus pyrimidine discrimination is achieved at the wobble position for archaeal tRNAs. At the base of code evolution, therefore, the genetic code capacity in tRNA shrinks to 32 distinct anticodons at most [Ile (UAU) is rarely used, so tRNAIle (UAU) and tRNAMet (CAU) should not be considered 1-codon sectors at the base of code evolution. We prefer the classic structure and representation of the genetic code to circular code diagrams because code structure is most apparent using a codon-anticodon table  code, as previously described ,46. EvolThe ribosome is a large and dynamic thermal ratchet molecular motor at the heart of information processing in cells. The mRNA and bound tRNAs traverse the 16S rRNA neck. Translocation is supported by a reversible swiveling motion of the 16S rRNA head/beak versus the body, by an apparent tilting of the head/beak relative to the body, and by a reversible ratcheting motion between the 50S and the 30S ribosomal subunits. Directionality of translation appears to be due to GTPase elongation factors EF-Tu and EF-G that bind in turn to the same site on the ribosome. EF-Tu is a primary fidelity factor for translation. EF-G mostly functions in peptidyl transfer and translocation. Attachments of tRNAs to mRNA and to the 50S subunit appear to act as pawls to enforce the direction and the mRNA register of translation. The 30S-50S subunit contacts regulate translocation and appear to communicate events at the A-site ASL latch to translocation ratchets. The coevolution of ribosomes, the genetic code, mRNA, and aaRS enzymes appears to have been around the tRNAs that evolved from ordered repeat sequences and stem-loop-stems that include a U-turn within a 7 nt loop (Ac and T loop).


To overcome these limitations, we prepared genetically modified resveratrol-enriched rice (RR). We previously reported the strong antiaging potential of RR against ultraviolet B/reactive oxygen species-induced toxicity in normal human dermal fibroblasts (NHDF). As aging is characterized by neuroinflammation and neurodegeneration, we further evaluated the role of RR against LPS-induced neuroinflammation. RR inhibited nitric oxide production and the expression of inflammatory proteins such as iNOS and COX-2. RR significantly modulated mitogen-activated protein kinase signaling, activator protein AP-1 signaling, and nuclear factor kappa B (NF-κB) mediated transcription of inflammatory proteins via inhibition of NF-κB translocation, IkB phosphorylation, and proinflammatory cytokine productions such as interleukin IL-6, IL-1β, tumor necrosis factor alpha (TNF-α), and prostaglandin E2 (PGE2). These findings show that the strong antineuroinflammatory effects of RR can be beneficial for aging-mediated neurodegenerative conditions as well as disorders of the central nervous system caused by neuroinflammation.Resveratrol, a natural stilbenoid, is produced by several plants, especially grape vines. Its strong potency against obesity, metabolic disorders, vascular disease, inflammation, and various cancers has already been reported. Large amounts of wine or grapes need to be consumed to obtain the amount of resveratrol required for biological activity. Pure resveratrol at concentrations as low as 10  This mip < 0.05 was considered statistically significant.The results were evaluated using the Statistical Analysis System . The results are presented as mean ± standard error of the mean (SEM), and all results are the mean of at least three independent experiments. A statistical comparison of different treatment groups was performed by one-way analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison test. A value of μg/mL). However, NR and RR showed better potency than resveratrol at concentrations of 1 and 10 μg/mL. Although RR extract has lesser amount of pure resveratrol than resveratrol, it has higher potency with lower IC50 value, suggesting that RR does not cause cellular toxicity. The effect of RR (100 μg/mL) on nitrite production is statistically significant to that of NR alone suggesting that RR has better potency than that of the NR. NR and RR treatment protected against LPS-induced toxicity, especially up to a concentration 100 μg/mL, by increasing the number of viable cells. In case of resveratrol treatment, only 10 μg/mL protected against LPS toxicity by increasing the viable cells, but not 100 μg/mL. NR and RR treatment at concentrations of 1 μg/mL and 10 μg/mL demonstrated higher potency in inhibiting nitrite production than L-NMMA, suggesting that NR and RR are better alternatives than L-NMMA for nitrite production inhibition as shown in . LPS-activated microglia showed significantly increased phosphorylation of JNK, ERK, and P38, which might be responsible for the induction of transcription of inflammatory mediators. Treatment of LPS-activated BV2 cells with NR and RR significantly reduced expression of phosphorylated JNK and P38. In case of ERK phosphorylation, only RR showed high efficacy, while NR did not demonstrate significant inhibition. The effect of RR is statistically significant in both the treated concentration to inhibit the ERK phosphorylation in comparison to the NR alone. This data suggested that additive effect of NR and resveratrol in RR is more significant than in NR and R alone. Resveratrol showed slightly different pattern in the MAPK modulation where it showed increase of JNK phosphorylation although it is not significant. Also, it did not show any changes in ERK phosphorylation whereas it significantly inhibited the p38 phosphorylation. NR and RR exhibited high potency to inhibit P38 phosphorylation than that of the ERK and JNK as shown in . Among aκB. NR and RR treatment at a concentration of 100 μg/mL inhibited the expression of p-C-Fos where NR and RR significantly inhibited the expression of p-C-Jun at both the treated concentrations (10 and 100 μg/mL). The effect of RR is statistically significant on both the treated concentration in comparison to the NR alone suggesting that additive effect of NR and resveratrol in RR is more significant than the NR and R alone. P38, JNK, and ERK activation is required for C-Jun activation. RR treatment downregulated p-C-Jun expression, indicating its strong anti-inflammatory effects against activated microglia as shown in Next, we assessed the expression of AP-1 proteins. Toxicant-induced activation of MAPK is responsible for the activation of AP-1 (C-Fos and C-Jun) in inflammatory response and other conditions . In thisκB and AP-1 play the key roles as transcription factors under inflammatory conditions. In this study, we evaluated the role of NR, RR, and resveratrol against activated microglia-induced NF-κB translocation and I-kB phosphorylation. LPS treatment to BV2 cells significantly increased the translocation of NF-κB from the cytosol to the nucleus, as well as the phosphorylation of I-kB. These events together can increase transcription. However, NR, RR, and resveratrol treatment reversed these events. LPS-activated BV2 cells treated with RR showed the highest expression of cytosolic NF-κB and the lowest expression of nucleolar NF-κB. RR treatment significantly increased the cytosolic NF-κB and decreased the nuclear one which is statistically significant to that of NR-treated group as shown in κB translocation and NF-κB/I-kB-mediated transcription of inflammatory proteins.Transcription factors play an important role in the increased production of inflammatory proteins and proinflammatory cytokines. NF-We evaluated the role of NR, RR, and resveratrol in altering the activation/phosphorylation of MAPK proteins  under chronic (24 h) LPS activation condition in BV2 cells. Inhibition of pJNK by NR and RR was more significant in chronic activation than in acute activation. None of the samples inhibited ERK phosphorylation in chronic activation condition, rather NR treatment significantly increased pERK expression that might suggest for its cell survival capacity against chronic activation of microglia. RR treatment also showed a slight increase in pERK expression, but it was not significant when compared with LPS-only-treated group. In case of P38 phosphorylation, although all the treated samples inhibited pP38 expression, only RR treatment showed significant inhibition when compared with LPS-only-treated group as shown in .κB translocation and NF-κB/I-kB transcription revealed the reason for the marked inhibition of nitrite levels and iNOS/COX-2 expression. This was further confirmed through the measurement of proinflammatory cytokine levels in LPS-activated BV2 microglial cells. NR, RR, and resveratrol demonstrated almost similar potency to inhibit PGE2 secretion, while resveratrol showed the highest potency for inhibition of TNF-α and IL-6 production as shown in  in comparison to NR  and resveratrol alone. NR, RR, and R treatment showed the different potencies in different concentrations for inhibition of inflammatory parameters. The highest concentration of resveratrol, i.e., 100 μg/mL, showed the highest potency but it also possessed the cellular toxicity. We found the potential effect of RR that seems to act in synergistic effect of NR and resveratrol. NR also showed good potency to inhibit the inflammatory cascades. Therefore, we hypothesized that the presence of phytochemicals α-tocopherol and γ-tocopherol in NR can boost the activity of RR either they can help for the protection of cells against resveratrol-mediated cell death or morphological changes. We evaluated the potential antineuroinflammatory effect of α-tocopherol and γ-tocopherol in rice and found that treatment of α-tocopherol and γ-tocopherol inhibited not only the nitrite production but also the expression of iNOS and COX-2 without cellular toxicity up to the concentration of 1 mg/mL. This data supports that active phytochemicals including α-tocopherol and γ-tocopherol in NR are responsible for the anti-inflammatory potential in LPS-treated BV2 cells. Also, we found that the RR-treated group significantly showed the higher potency of RR than resveratrol-treated group alone in experimental group. Hence, the most notable achievement of this study was the improved efficacy of resveratrol following preparation of pharmaceutical crops (rice) containing resveratrol, which showed negligible toxicity on microglial cells.NO production by overactivated microglia is a key biomarker for neuroinflammation in CNS disorders. In a preliminary study, the treatment of NR, RR, and resveratrol showed significant inhibition of nitrite production in LPS-activated BV2 microglial cells, without cellular toxicity. Therefore, we decided to perform a mechanistic study. The inhibition of nitrite production and the ICproteins , 39. Simtic acid . NR contredients , 41 whicredients . In specal cells . This fi of pain , 44, theytokines . Treatmeytokines , 46. RR ytokines , 47, 48.ytokines . The incytokines . Resveraα-tocopherol and γ-tocopherol in rice. The anti-inflammatory effect by RR treatment seems to be mediated through inhibition of nitrite production, MAPK phosphorylation, NF-κB mediated production of proinflammatory cytokines, and expressions of inflammatory proteins which are shown in in vitro and in vivo studies.In this study, we not only discovered the safe and effective role of RR against aging and neuroinflammation but also identified the antineuroinflammatory potential of NR itself is because of the presence of active phytochemicals such as


To examine the antiallergic effect of Sesamum indicum Linn. extract (SIE), we treated antigen/immunoglobulin E- (IgE-) sensitized mast cells with extracts of various concentrations. We examined the degranulation release and concentrations of inflammatory mediators. Additionally, the expressions of genes involved in the FcεRI and arachidonate signaling pathways were examined. SIE inhibited the degranulation and secretion of inflammatory mediators in antigen/IgE-sensitized mast cells. SIE reduced the expressions of FcεRI signaling-related genes, such as Syk, Lyn, and Fyn, and the phosphorylation of extracellular signal-regulated kinase in antigen/IgE-sensitized mast cells. Additionally, in late allergic responses, SIE reduced PGD2 release and COX-2 and cPLA2 phosphorylation expression in FcεRI-mediated mast cell activation. Lastly, 250–500 mg/kg SIE significantly attenuated the Ag/IgE-induced passive cutaneous anaphylaxis (PCA) reaction in mice. The potent effect of SIE on RBL-2H3 mast cell activation indicates that the extract could potentially be used as a novel inhibitor against allergic reactions.This study is aimed at determining whether Sesamum indicum Linn. beneficially influences Fc These creleased , which aeceptors , 6. SimiSesamum indicum Linn. (SI) is one of the most important seed crops and traditional health food in Asian countries. Most pharmacological studies on S. indicum seeds have reported hypoglycemic effects in genetic diabetes, antitumor effects, antiestrogenic activity, positive effects on Parkinson's disease patients, antihypertensive effects, and increased vitamin E levels without the use of vitamin E supplements. In addition, aqueous defatted seed extracts from S. indicum have already been demonstrated to exhibit hypoglycemic and hypolipidemic activities. Thus, SI has been shown to have fat-lowering effects in hypercholesterolemic rats; it reduces weight gain caused by cholesterol intake and reinforces the antioxidant defense system in the body [the body . In addithe body . SI contthe body , 10. HowSesamum indicum Linn. extract (SIE) against FcεRI-mediated allergic reactions in mast cells and the mechanism by which SIE suppresses these allergic reactions. The results of this study would be important in the advancement of plant medicines against allergic diseases.The present study investigated the antiallergic activities of the Sesamum indicum Linn. (SI) were obtained as dried herbs from Yeongcheon Oriental Herbal Market  and were authenticated by the Korean Medicine Application Center, Korea Institute of Oriental Medicine. SI (50 g) was extracted using 70% ethanol at 40°C for 24 hr in a shaking incubator. Subsequently, the extract was filtered using a 150 μm filter paper and concentrated in a rotary vacuum evaporator . Samples were freeze-dried and stored at -20°C before use. Sample acquisition was 5.4 g, and the yield was 18.5%. The lyophilized powder was resuspended in distilled water, centrifuged at 10,000×g for 15 min, and filtered through a 0.2 μm sterile filter to prepare the SIE. The HPLC analysis method and results are given in Supplementary Materials (The samples of α (MEM-α) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics  at 37°C in humidified 5% CO2 conditions. The cells were seeded onto a 6-well plate (3 × 105 cells/well) or 96-well plate (2 × 104 cells/well) for 24 hr. At day 2 postconfluence, the medium was changed to MEM-α (10% FBS and 1% antibiotics) containing dinitrophenyl (DNP)-IgE (0.1 μg/mL) for 16 hr. The medium was replaced with serum-free MEM-α (1% FBS and 1% antibiotics). The cells were pretreated with SIE  or 100 nM dexamethasone before DNP-human serum albumin (0.1 μg/mL) treatment for 10 min or 4 hr.Rat RBL-2H3 mast cells were cultured in Minimum Essential Medium-The RBL-2H3 mast cells were treated with various concentrations of SIE for 1 hr. To determine cell cytotoxicity, the cells were incubated with 0.5 mg/mL of MTT reagent for 40 min at 37°C. The medium was discarded, and dimethyl sulfoxide was added to the cells for 5 min. Absorbance readings at 570 nm were obtained using a microplate reader.μL of 4-methyl-umbellyferyl-N-acetyl-β-d-glucosaminidase (10 mM) was added to the supernatant of the sensitized cells with IgE for 1 hour at 37°C. To stop the reaction, the solution was mixed with sodium carbonate buffer , and the absorbance was measured at 405 nm on a microplate reader.RBL-2H3 mast cells were seeded into a 96-well plate and were pretreated with drug. Subsequently, 25 μg/mL) or 100 nM dexamethasone before DNP-human serum albumin (0.1 μg/mL) treatment for 4 hr. The concentrations of TNF-α , IL-4 , IL-6 , histamine , and PGD2  in the cell culture media were measured according to the manufacturer's instructions.The cells were pretreated with Sesamum indicum Linn. extract  containing a protease and phosphatase inhibitor cocktail . Proteins were quantified using the bicinchoninic acid assay and were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto an activated polyvinylidene difluoride membrane for 100 min. The blots were blocked with 5% BSA and incubated with primary antibodies (1 : 1000) at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. Protein expressions were detected using a western blot detection kit  and ChemiDoc™ Touch Imaging System .n = 5), Ag/IgE group , Ag/IgE treated with 10 mg/kg dexamethasone group , Ag/IgE treated with 250 mg/kg SIE group , and Ag/IgE treated with 500 mg/kg SIE group . SIE was prepared in saline, and CTL and Ag/IgE groups received equivalent volumes of saline. All experiments were approved by the Committee on Animal Experimentation and Ethics of KIOM.Male ICR mice, 5 weeks of age, were randomly assigned to five groups after 1 week adaptation period: control group  antibody was subcutaneously injected into the ears of mice. At day 2, IgE-sensitized mice were administered oral SIE (250 or 500 mg/kg) or dexamethasone (10 mg/kg). One hour later, DNP-HSA (300 μg/mL) containing 1% Evans blue was injected into the tail veins. One hour later, the mice were sacrificed using CO2 and tissues from the treated ears were obtained. The Evans blue dye was removed by the ear tissue, which were then incubated with 0.4 mL formamide at 63°C for 16 hr. Absorbance at 620 nm wavelength was measured using a microplate reader.At day 1, anti-DNP-IgE , mounted on positively charged glass slides, and dried in an oven at 60°C for 30 min. The sections were deparaffinized in xylene and then rehydrated in graded ethanol and water. Endogenous peroxidase was blocked by incubation in 3% (v/v) hydrogen peroxide for 10 min. Nonspecific endogenous protein binding was blocked using 1% bovine serum albumin (BSA). Sections were counterstained with hematoxylin-eosin (H&E) staining or toluidine blue. The specimens were mounted with Permount , and images were captured using a Nikon fluorescence microscope equipped with NIS-Elements BR 4.50 software . Histopathological changes of eosinophils in H&E-stained tissues and mast cells in toluidine blue-stained tissues were counted in three different parts by blind observed under microscope with 100x and 400x magnification.For histological analysis, ear tissues were fixed in 100 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). 5 ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0005 were considered statistically significant.Data were expressed as mean ± S.E. from three independent experiments. One-way analysis of variance and Bonferroni's post hoc test were used to determine the statistical significance of differences between each treated group and the negative control (IgE group) using the GraphPad Prism software . μg/mL ; however, they were not effective. Consequently, we conducted the study using relatively high concentrations (100–500 μg/mL), which are the concentrations that have been applied in previous studies [β-hexosaminidase release of mast cells at all doses ranging from 100 to 500 μg/mL. SIE treatment decreased the TNF-α and IL-4 concentrations in the IgE-sensitized RBL-2H3 cells in a concentration-dependent manner , which is involved in the degranulation process of mast cells . Concentrations of Evans blue were significantly lower in the SIE 250 group , the SIE 500 group , and the Dex group   via Lyn kinase, which is bound to the receptor [γ and protein kinase C [Fcignaling . In addireceptor . The phoreceptor . In the kinase C .εRI-mediated allergic reactions in mast cells, we tested the effects of SIE on MAPK activation. Treatment with SIE reduces the phosphorylation of MAPK pathways, including ERK, JNK, and MEK1/2 genes in Ag/IgE-mediated RBL-2H3 mast cells. Additionally, it was found that SIE inhibited the phosphorylation of Akt and IkBα by Lyn/Syk activation [Lyn/Syk activation induces stimulation of MAPKs, which are involved in the production of proinflammatory mediators . MAPK sitivation . These oεRI-mediated allergic reactions through the suppression of cPLA2 activation and inhibition of COX-2 activity.Next, we found that SIE inhibited COX-2 and cPLA2 expressions and reduced the levels of the PGD2 products, which are enhanced in the activated immune cells including mast cells. COX-2 and cPLA2 are pivotal enzymes for the production of proinflammatory lipid mediators and in the metabolism of arachidonic acid associated with allergic actions . PGD2 isFinally, we examined how SIE suppresses IgE-mediated PCA in mice. PCA is characterized by an immediate skin reaction at a localized IgE-mediated allergic response in vivo, typically with increased vascular leakage in the skin that can be assessed by an intravenous injection of Evans blue . In vivoεRI-mediated mast cell activation through the downregulation of FcεRI and arachidonate expression, which might cause inhibition of degranulation in RBL-2H3 mast cells and antigen-IgE-mediated PCA reactions. Inhibition of the degranulation process results in reduced production of allergen mediators, such as histamine, TNF-α, IL-4, IL-6, and PGD2, caused by Ag/IgE interaction. These results indicate that SIE could be a beneficial treatment for allergy-related diseases.This study showed the antiallergic effects of SIE against Fc