Several studies have searched for such a signature, as summarized in Buratti et al.; however, none has yet produced sufficient evidence to dissipate doubts. In the radiation environment characteristic of Saturn’s icy moons, the presence of crystalline H2O ice has been used as a marker of a high-temperature region. Because ion bombardment will, over time, drive crystalline ice toward an increasingly amorphous state, the current phase of the H2O ice can be used to gauge the temporal temperature evolution of the surface. We adopt a technique described by Dalle Ore et al. to map the fraction of amorphous to crystalline H2O ice on Dione’s surface, observed by the Cassini Visible and Infrared Mapping Spectrometer, and provide an ice exposure age. We focus on a region observed at high spatial resolution and centered on one of the faults of the Wispy Terrain, which is measured to be fully crystalline. By assuming an amorphous to crystalline ice fraction of 5% , significantly higher than the actual measurement, we obtain an upper limit for the age of the fault of 152 Ma. This implies that the studied fault has been active in the last ~100 Ma, supporting the hypothesis that Dione might still be active or was active a very short time ago, and similarly to Enceladus, might still be harboring a body of liquid water under its crust.We examine the H For example, Enceladus owes its activity to the tidal interplay with Saturn and Dione , being lSeveral studies have searched for signs of current activity on Dione, as summarized by However, bright streaks, dubbed “Wispy Terrain,” that stretch across Dione’s trailing hemisphere, indicate that the moon was at least active in geologically recent times. This system of bright tectonic faults crosscutting the underlying cratered surface  and the 2O ice in its crystalline phase  on board the spacecraft. VIMS consisted of two slit spectrometers, or channels, covering the visual (VIMS-V) and infrared (VIMS-IR) spectral ranges from 0.35 to 1.05 ectively . Each VI2) focused on one of the faults, the Padua Chasmata, in the Wispy Terrain. This is located on Dione’s trailing hemisphere, approximately on the equator, at ~110°W.Two data sets were adopted in this study. The global data set consists of a mosaic of more than 600 cubes prepared as described in the 2O ice phase measurements have historically been based mostly on the presence of the 1.65 μm band, where the absence of the band indicated that the ice was amorphous or crystalline warmer than 150 K , and temperature of the ice in different parts of the surface and covering a full range of fractions of amorphous to crystalline ice. The end member spectral variations across the surface were found by means of a clustering tool focused on the 1.5 μm band region, known to be sensitive to all parameters to a similar degree.The first—Delta Measurement—consisted of measuring the change in shape in a quantitative fashion. We achieved this goal by comparing in turn all the spectra in each grid with a Gaussian fit to the 2.0 μm band, taking the ratio of the spectra to the corresponding fits. We then measured the difference in normalized albedo between the two minima at 1.95 μm and 2.1 μm, which we named “delta.” From these measurements, we obtained the calibration curves linking delta, the asymmetry parameter, to the level of crystallinity for all the regions shown in The second step—Phase Fraction Determination—consisted of measuring delta for all pixels in the mosaic and associating a value of the fraction of amorphous to crystalline ice making use of the appropriate calibration curve determined based on the geographical location of each pixel.The third step—Each step is explained in detail in the 3.3.1.2O ice was measured as described in the The fraction of amorphous to crystalline H2O ice , is predominantly crystalline . This is due to the continuous infalling of E-ring particles, known to be mainly composed of crystalline H2O ice , 2009. I2O ice  and ther2O ice phase distribution is more varied and the story more complicated. Because of the position of Dione’s orbit with respect to Saturn’s magnetosphere, the trailing hemisphere is bombarded by particles that overtake the satellite and have two known effects on the surface: they darken it, due to the composition of the magnetospheric grains hitting the surface , the H surface , and the2O ice to the degree allowed by Dione’s diurnal temperatures and thermal recrystallization. As described in If no other mechanism were at work on Dione, then most of the trailing hemisphere would be covered in amorphous H2O ice phase, some clearly corresponding to tectonic features in the Wispy Terrain region. In this region, the amount of amorphous ice decreases distinctly to just a few percent from the more common 25%–30% levels found in the neighboring parts of the hemisphere. To further analyze the nature of the ice in those areas where the ice phase varies from amorphous to crystalline, we created a mosaic of high-spatial-resolution cubes and performed the same analysis outlined in the However, further mechanisms are at work, e.g., the flash heating and cooling during impact cratering leads to the formation of crystalline ice, which is amorphized over time by charged particle bombardment . On Dion3.2.2O ice in and around one of the faults (Padua Chasmata) belonging to the Wispy Terrain region. The map on the right shows the high-resolution subset in comparison to the global map. The ice in the fault (identified by a red arrow in the map) is almost completely crystalline, in contrast with neighboring regions away from the tectonic features, where there is as much as ~30% amorphous ice. 2 could have gone undetected by the Cassini CIRS scans if the temperature of the ice were reported to be in the range ~105–110 K. Our best-fitting models, described in detail in the There is also a number of “super-crystalline” pixels (no amorphous contribution) covering an area of about 2% of the mosaic. Since the change in ice phase is driven by changes in temperature, these pixels are likely marking the position of the warmest areas in the region  in temperature . In the 2O ice into its disordered amorphous phase, following an exponential behavior , as measured in the center of the fissure, to 5% (ΦAmax). The latter is a fraction that is higher than the measurement errors  or if the depth were changed to 7 μm or 9 μm, the age would change to 68, 133, or 171 Ma, respectively. Furthermore, if the penetration depth was decreased, the value of K changed to that of protons instead of He ions, and a 1% variation in the amount of amorphous were adopted at once, the resulting age would be ~71 Ma, exposing a large degree of uncertainty in the measurement tied to the many assumptions made in the derivation—and showing which  carry the most weight in the calculation.Our calculations yielded an age, for a 5% fraction of Hμm, and a K value consistent with He ion bombardment.In 2O ice is very small, have to be quite young to be purely crystalline, and therefore a temperature enhancement either must exist or have existed very recently has existed in order to produce them, thus supporting the idea of an active Dione.We should point out that, while calculating the age of the ice, we are not discussing the fact that the actual phenomenon that produced the gradient in crystallinity observed around the wispy terrain fissure is a gradient in temperature, i.e., that the ice farther out than the center of the fault is or was not warm enough to crystallize completely. Once the temperature lowers to the background one, it is only ion bombardment that can bring the ice phase back to being partially amorphous. The ages shown in 4.2O ice phase, specifically the amorphous ice fraction, as it has been shown that variations in temperature will cause a change in the ice phase from amorphous to crystalline  crystalline. We find a clear correspondence of the pure crystalline regions and the wispy terrain.We apply a technique previously adopted in a similar study of craters on Rhea , this ti2O ice, marking those regions that have had temperature enhancements. Based on previous work by We study in detail one of the faults in the wispy terrain, making use of a high-resolution mosaic of the region. Here, the fault is seen to be associated with areas dominated completely by crystalline H2O ice in one of the faults is zero or close to zero, and subject to the assumptions and caveats described in 2O ice. This amount is significantly higher than the measured fraction in the fault. We obtain a value of <200 Ma for the slightly amorphized ice, implying that the fault has to be even younger. This estimate is lower than the age estimates based on stratigraphic relations (Based on the fact that the fraction of amorphous to crystalline Helations . This im


Long acting injectable (LAI) antipsychotics are commonly used in the treatment of schizophrenia to improve adherence and clinical outcomes. Concerns have been reported in relation to their non-systemic or injection site adverse effect profile. As such, this study aims to review and evaluate all evidence reporting injection site adverse effects with LAI antipsychotics.An electronic search was systematically conducted through four databases  in order to identify studies investigating injection-site reactions associated with LAI antipsychotics. Unpublished studies such as conference proceedings and clinical trial registries were also searched. The search was limited to literature published in English without year limits.Of a total of 189 citations that were identified from the electronic database search, 12 were selected for inclusion in this review. Various injection site reactions were reported in these studies, including pain, bleeding, and swelling. Overall, the studies reported a low incidence of these injection site reactions. Only a minority of the included articles compared injection site reactions between different LAI antipsychotics.Injection site pain was the most commonly reported injection site adverse effect across all articles reviewed. The low incidence of injection site adverse effects associated with LAI antipsychotics indicates that these formulations appear to be well tolerated by patients. More head-to-head trials comparing second generation LAI antipsychotics are needed. Long acting injectable (LAI) antipsychotics  are concentrated formulations which, following intramuscular injection, release the antipsychotic drug slowly over time. This allows an effective maintenance dose of the antipsychotic to be delivered with injections at intervals that range from 2 weeks to up to several months.10First-generation LAI antipsychotics consist of the antipsychotic esterified to a decanoate, which is dissolved in an oily vehicle.15Considering the relatively limited attention that has been paid to non-systemic side effects associated with LAI antipsychotics, and which are rarely reported in clinical trials, this study aims to systematically review all the available evidence reporting injection-site adverse effects associated with the administration of LAI antipsychotics. This review of local adverse events of single LAI antipsychotics will provide evidence-based recommendations for their safe use in practice and may help clinicians in tailoring their choice of treatment.Appendix 1), which outlined the different steps of this systematic review of the literature, an electronic database search was conducted from database inception using PubMed, SCOPUS, Embase and Cochrane databases in order to identify studies investigating injection-site reactions associated with LAI antipsychotics. Search terms included “LAIAPs”, “long acting depot”, “antipsychotics”, “neuroleptics”, “local reactions” and “injection site adverse effects”. To avoid publication bias, unpublished studies such as conference proceedings and clinical trial registries were also searched. In addition, a quick update of the literature was undertaken to identify articles published between the time of the initial search and the time of publication.Based on a pre-determined protocol . Studies were selected for full review based on the following inclusion criteria: articles in English that investigated injection-site reactions associated with LAI antipsychotics or studies reporting any form of adverse effects relating to injection-site administration of antipsychotics. By injection site adverse effects, only local reactions of the skin and underlying tissue were considered. As such, non-localized adverse effects resulting from the intramuscular administration of the antipsychotic (such olanzapine-induced post-injection delirium/sedation syndrome) were excluded. Articles were also excluded if injection-site related adverse effects were not included in the study outcomes. The full text of all potentially relevant articles were retrieved and distributed among three reviewers  to confirm eligibility. The quality of the articles was assessed using the Critical Appraisal Skills Programme (CASP) Systematic Review checklist.Of the 189 citations identified in the initial electronic database search, 25 full-text articles were selected to undergo a more comprehensive review based on the inclusion/exclusion criteria. However, following the full-text review, 13 articles were excluded because these articles did not provide information on injection-site adverse effects. Therefore, a total of 12 articles were selected for full review and data extraction using a standardized data collection tool.ore*1Hay 9517CrossThe quality assessment scores reported in In this study, a systematic review was conducted on all the available evidence reporting injection-site adverse effects associated with the administration of LAI antipsychotics aiming to provide evidence-based recommendations for their safe use in practice. The most commonly reported injection site reaction across the 12 articles included in this study was pain at the injection site. In an indirect comparative study of injection-site pain associated with first-generation LAI antipsychotics versus paliperidone palmitate (PP), researchers suggested that PP may be associated with lower mean pain severity than first-generation products.Most studies investigated in the current systematic review were of relatively high quality . All theIn conclusion, the majority of the articles discussed in this review reported that patients tolerate LAI antipsychotics formulations relatively well. The most commonly reported injection site reaction across all the studies was pain at the injection site, especially with oil-based formulations of LAI antipsychotics. Several strategies have been recommended to minimize injection site pain, such as selecting appropriate injection administration techniques, using suitable needle specifications, increasing the interval between injections and administering low volume of highly concentrated formulations of LAI antipsychotics. Noticeably, such strategies should be further investigated in order to assess their influence on improving injection-site reactions. Similarly, the selection of the muscle site for injection administration is an important consideration that is worth exploring further. Although the gluteal region is the preferred site for administering depot antipsychotics, the deltoid area is less intrusive for patients; hence, this may influence patient acceptability and compliance.32–34Although this review generated important findings, some limitations in the literature review process need to be highlighted. First, the article by Kern Sliwa et al


Schizosaccharomyces pombe phosphoproteome in relation to changes observed in global cellular protein synthesis upon TOR inhibition. We find that after TOR inhibition, the rate of protein synthesis is rapidly reduced and that notable phosphorylation changes are observed in proteins involved in a range of cellular processes. We show that this reduction in protein synthesis rates upon TOR inhibition is not dependent on S6K activity, but is partially dependent on the S. pombe homologue of eIF4G, Tif471. Our study demonstrates the impact of TOR‐dependent phospho‐regulation on the rate of protein synthesis and establishes a foundational resource for further investigation of additional TOR‐regulated targets both in fission yeast and other eukaryotes.Cell growth is orchestrated by a number of interlinking cellular processes. Components of the TOR pathway have been proposed as potential regulators of cell growth, but little is known about their immediate effects on protein synthesis in response to TOR‐dependent growth inhibition. Here, we present a resource providing an in‐depth characterisation of  Schizosaccharomyces pombe reveals a TOR‐dependent network of phosphoproteins involved in the regulation of protein synthesis rates.Phosphoproteomic analysis of  TOR is a serine/threonine protein kinase that is a member of the phosphatidylinositol kinase‐related kinase (PIKK) family. It was discovered in budding yeast through characterising mutants resistant to the growth inhibitor rapamycin  and eIF4F complex, are among the most widely discussed proteins as potential downstream targets of TOR in having an impact on protein synthesis upon inhibition   began to reduce by 80–90 min, with the rate of mass doubling reducing to a third compared to cells treated with the DMSO solvent alone  and sampled at intervals from 5 to 85 min. The samples were labelled with isobaric tandem mass tag labels (TMT10plex) for relative quantification of both the protein and individual phospho‐peptide levels .T = 0 min), in either wild type or Torin1‐resistant cells, after 40 min of Torin1 treatment , a well‐established biochemical read‐out of TORC1 and TORC2 activity , and an illustrated example of the workflow is provided in Fig This phosphorylation study identified a total of 20,072 phosphosites on 2,271 phosphoproteins, over 3 times the number of phosphosites identified from previous TOR phosphoproteomics studies in both budding yeast and mammalian cells. We controlled for non‐specific and off‐target phosphorylation changes caused by DMSO or Torin1 treatment by taking into account the phosphorylation values of the Torin1‐resistant strain at each time point  Biological Process annotations  account for the largest group of proteins (37/160) that exhibit rapid phosphorylation changes upon TOR inhibition. In particular, enrichment analysis identified a 5.6‐fold enrichment for those involved in the negative regulation of intracellular signal transduction  is not involved in the rapid inhibition of protein synthesis . This second time course largely replicates the critical part of the first time course, and examination of the same phosphosites from the two experiments indicates that the data are reproducible.Given that the esis Fig , we focutor1∆ cells across all time points (Table tor1∆). The corresponding levels of individual proteins in tor1∆ cells were compared to the levels measured in wild type cells before background subtraction and before Torin1 addition. 26 proteins showed over a 1.5‐fold difference in expression in tor1∆ compared to wild type cells before Torin1 treatment  were also in common with the 85‐min time course Table . 372 photor1∆ within 40 min  family, which are among the most widely discussed targets, hence we decided to further investigate and understand their potential role in TOR regulation of protein synthesis. There are 3 functional homologues of the S6Ks in fission yeast, Psk1, Sck1 and Sck2 (Nakashima 2012). After 40 min of Torin1 treatment, we found that the relative protein expression of all 3 proteins remained unchanged in both wild type and ells Fig . Sck1 waells Fig , and oneells Fig . Furtherells Fig . Previouells Fig . The chaells Fig ).et al, To investigate this possibility, we undertook a genetic analysis of the S6K homologues. Previous genetic analyses have suggested that deletion of any one or combinations of the three S6K homologues does not affect cell viability in fission yeast (Nakashima Translation Initiation Factors (TIFs), and together with Lcp5, an essential ribonucleoprotein involved in 18S rRNA maturation, these 5 proteins play crucial roles in recruiting and engaging the ribosome for initiating translation. Additionally, one other translation‐related protein Erf1 was identified in our analysis, which is shown to have functions at the end of the translational process, and is responsible for coordinating the release of newly synthesised peptides from the active ribosome.Another set of putative targets of TOR‐dependent regulation on protein synthesis are those related to cytoplasmic translation. These represent a notable proportion of the 27 proteins that are part of the conserved TORC1‐dependent core interactome network, as identified in the cytoplasmic translation cluster Fig . In parti. One Tif212 phosphorylation site was identified in the TORC1‐dependency analysis, showing rapid dephosphorylation in both wild type and tor1∆ cells within 12 min of Torin1 addition   Fig , as welltif471_18A was reduced to 17% of its starting level, over 3 times higher than the control (5%)  Fig . This deWe have investigated how the TOR‐signalling pathway regulates eukaryotic cell growth, with a focus on understanding the control of global cellular protein synthesis. Using fission yeast, we describe an in‐depth analysis of the proteomic and phosphoproteomic changes that occur after inhibition of both TOR protein kinases with Torin1 in two time course studies at fine temporal resolution. Our results establish that TORC2, which contains the non‐essential Tor1 kinase, is dispensable for regulating global cellular protein synthesis, and so we have characterised changes that are primarily dependent on the essential Tor2 kinase containing TORC1. A total of 20,894 phosphosites located on 2,306 different phosphoproteins were identified in the two studies. Of these, 908 phosphosites on 343 proteins exhibited a more than 2‐fold change in phosphorylation levels after 40 min of Torin1 treatment, by which time protein synthesis rates were reduced over 90%. Two‐thirds of these proteins (213/316) shared homologues in both budding yeast and human cells, and a total of 88% (279/316) of these had homologues in at least one of these two organisms.et al, Our database of phosphoproteomic changes after TOR kinase inhibition provides an in‐depth resource for investigating the TOR pathways' involvement in regulating cell growth, with many of the identified phosphoproteins conserved among eukaryotes. We investigated two possible candidates, the S6 protein kinases and the translation initiation factor, eIF4G (Tif471), examining their potential roles in cell growth control with respect to protein synthesis. The S6 kinases have widely been suggested as downstream regulators of the TOR kinase pathways and implicated in growth control via modulation of translation initiation activity (see reviews in (Wullschleger tif471_18A), we find that in response to TOR inhibition, the residual rate of protein synthesis is reduced to 17% of the starting level, more than three times higher than in wild type. This, to our knowledge, is the first demonstration of a component regulated by phosphorylation as a consequence of TOR inhibition that has an immediate impact on reducing the global cellular rates of protein synthesis. The partial inhibition observed suggests that there are multiple points of control distributed within the TOR downstream network, and complete inhibition will require the coordination of various components.One of the proteins most heavily regulated by phosphorylation identified in our study was the translation initiation factor Tif471, the fission yeast homologue of eIF4G. In a phosphomutant of Tif471  analyses of the TOR‐dependent phosphoproteome identified a range of molecular processes with four broad categories that were particularly enriched: signalling‐related processes, cell polarity and actin cytoskeletal organisation, vesicle‐mediated transport and autophagy, and translation‐related processes.et al, et al, et al, The switching off of growth will require a coordinated response from multiple major cellular processes, so it is unsurprising that several signalling network pathways have been implicated in TOR regulatory control. The MAPK‐signalling pathway is thought to be responsible for maintaining structural integrity of a cell through actin cytoskeletal reorganisation . Other components of the translation machineries were also identified, including Erf1, the translation termination and release factor, and Lcp5, the U3 snoRNP‐associated protein, shown to be involved in 18S rRNA maturation in budding yeast  (subsequently used as the tif471+ control). The tif471 genes were amplified from the extracted by PCR with primers (pTM101 & 102) 1.06 kb either side of the ORF. The amplified gene was subcloned into the pSC‐A‐Amp vector using the StrataClone Blunt PCR Cloning Kit (TMp16). The 18 alanine mutations were introduced through 2 consecutive cloning steps, first replacing amino acid position 706 onwards until the end of the ORF with the synthetic gene fragment (gBlock) synthesised by Integrated DNA Technologies (IDT). The gBlock fragment contained 13 alanine mutations: S912A, S919A, S921A, S1293A, S1300A, S1333A, S1349A, T1351A, S1353A and S1357A. The 3′ end the endogenous gene on TMp16 was replaced by the fragment at the using the NEBuilder HiFi DNA Assembly kit and primers pTM111‐114. The final 5 alanine mutations, S882A, T884A, S886A, S896A and S896A, were added by 2 subsequent reactions using the Agilent QuikChange Multi Site‐Directed Mutagenesis Kit and primers pTM141‐146, resulting in the TMp21 plasmid containing all 18 alanine mutants on Tif471.The The entire locus containing mutated amino acid residues was then amplified by PCR with pTM101 and 102 and purified using the QIAquick PCR Purification Kit, then transformed into stable wild type diploid cells (TM21) and selected with antibiotic resistance. The resultant heterozygous diploid was subsequently sporulated into haploids, to check for a 50:50 segregation of wild type to mutant haploids through random spore analysis, and mutant haploids were isolated. Alanine mutations were checked with specifically designed primers on Primer3Plus website  by DNA sequencing from extracted gDNA.595 = 0.1 approximated to 1.26 × 106 cells/ml. All cell cultures were maintained in exponential growth for 48 h in the culturing conditions defined above before any experiments were conducted. Growth rate calculations were made based on the OD measurements, where the logarithm (base 10) of the OD was taken (Log(OD)) and plotted against time. Using the linear regression line fitting function on Prism9, the gradient of the fitted line was used to calculate the generation time (gt) using the formula gt=log10(2)m, where m is represented by the gradient of the regression line. Error bars indicate standard deviations (SD) of three independent experiments unless stated otherwise.Cells were inoculated in static liquid media overnight at corresponding temperature and subsequently diluted to within exponential growth cell density (OD ˜ 0.1 ‐ 0.8). Optical density (OD) was measured at wavelength (λ) = 595 nm using the Ultrospec 2100 pro UV/visible spectrophotometer (Amersham Biosciences). ODCells were grown in EMMG at 25°C and maintained in exponential growth (OD 0.1–0.8) for 48 h in liquid culture; cells were diluted to OD 0.4–0.5 for the start of the assay. The main culture was then split into individual cultures for each time point and allowed to recover for at least 1 h before beginning the assay. The individual cultures were treated with Torin1  for different lengths of time accordingly. The methionine analogue L‐homopropargylglycine (HPG) was added  to cultures 15 min before the first sampling of each time point. Two sample were taken at 10 min apart for the individual time points, and the rate of protein synthesis was calculated from the difference between the two values and then divided through by 10 min.et al, 420 µl of samples were collected at the indicated time points and fixed in 980 µl of 100% ice‐cold ethanol to reach final concentration of 70% ethanol and then kept on ice for 20–30 min. Sample processing and labelling procedures were based on (Knutsen 2SO4) (100 mM), 0.94 µl 488 Alexa Fluor® azide and 37.5 µl 1× Click‐iT HPG buffer additive. Reaction mix was removed, and samples were washed with 500 µl of sodium citrate (50 mM). Cells were subsequently washed and resuspended in 500 µl of sodium citrate and treated with RNase (0.1 mg/ml) for 3 h to overnight at 37°C, protected from light. Following RNase digestion, cells were pelleted and resuspended in 500 µl of sodium citrate with propidium iodide (PI) added to a final concentration of 2 µg/ml. Samples were loaded onto 96‐well U bottom plates and ran on BD‐LSR‐Fortessa flow cytometers using the High Throughput Sampler (HTS), and 100,000 events were recorded per sample. Sample flow rate was set to 1 µl/s, with a mixing volume of 100 µl for 2 mixes at 180 µl/s mixing speed, and a wash volume of 400 µl.Composition of reaction mix was modified from the Click‐iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit (Invitrogen). Each sample was stained with 375 µl of reaction mix per reaction, containing 321.56 µl of 1× Click‐iT HPG reaction buffer, 15 µl Copper (II) sulphate (CuVoltage for corresponding parameters was set based on defined criteria. Side scatter (SSC) adjusted to have ˜90% of the cells between 7500 and 15,000 (roughly one doubling). Voltage for the 530/30 blue laser channel (for detection of 488) was set to have the maximum range of intensity whilst keeping 95% of the events below the maximum 250,000 (AU). For the 610/20 yellow laser channel (PI), voltage was set to have the 2C DNA content peak centred around 50 (AU). Cells were manually gated on FlowJo (version.10.3.0) for doublet discrimination, and only cells with 2C DNA content or below were included for further analysis. For the initial experiment in establishing HPG incorporation as a measure for protein synthesis at steady state, median values were calculated for whole populations after data clean‐up and values were exported for statistical analysis on R. Linear regression lines were fitted using the linear model of the geom_smooth function. Error bars indicate standard deviations (SD) of three independent experiments unless stated otherwise.T = 0 min) taken just before Torin1 or DMSO addition. A DMSO sample treated for the duration of the respective time courses was used as a control to account for non‐specific phosphorylation changes. Time points for the 85‐min time course were taken at 0, 5, 10, 20, 25, 30, 40, 60 and 85 (+DMSO 85) min, and for the 40‐min time course, they were at 0, 1, 3, 5, 8, 12, 18, 24 and 40 (+DMSO 40) min.Two time course studies were conducted to investigate the proteomic and phosphoproteomic changes upon Torin1 (5 µM) addition, with the first time point  was reduced with 5 mM dithiothreitol (DTT) for 25 min at 56°C, alkylated with 10 mM iodoacetamide for 30 min at room temperature in the dark and then quenched with 7.5 M DTT. Samples were digested using SP3 on‐bead methodology  being added as a variable modification, MaxQuant output files were imported into Perseus (version 1.4.0.2).MaxQuant (version 1.5.0.13) was used for all data processing. The data were searched against a PomBase  addition for the two respective experiments.All reporter intensities were logT = 0 min in wild type cells for the respective experiments before Torin1 or DMSO addition . For the 40‐min time course study, proteins that showed more than a 1.5‐fold difference (± 0.585) between the 40‐min Torin1‐treated sample and the DMSO control in either wild type (“WT 40 min ‐ DMSO control”) or tor1∆ (“Tor1D 40min ‐ DMSO control”) cells were excluded from subsequent analysis in order to account for DMSO non‐specific changes.  relative to the start (T = 0 min) in the Torin1‐resistant strain (“tr.85.min relative control”) were excluded. Phosphorylation levels were subsequently corrected for non‐specific Torin1‐related changes in the 85‐min time course by subtracting the phosphorylation values of the Torin1‐resistant strain from the values measured in wild type cells in the corresponding time points for the individual phosphosites. Values are then background subtracted to have the relative phosphorylation levels before Torin1 addition (T = 0 min) starting at zero for direct comparison among sites and subsequent fold change analyses (labelled “sub.x.min” in Tables Phosphosites were first filtered for more than 2‐fold change in phosphorylation in wild type cells (“wt.85.min” and “WT 40 min”) at the end of the respective 85‐min and 40‐min time course experiments Tables  and EV3.tor1∆ cells relative to their respective phosphorylation levels before Torin1 addition (T = 0 min). Sites showing more than 2‐fold change by 40 min in wild type cells (“WT 40 min” and “WT 40min ‐ DMSO control”) but less than 1.3‐fold in tor1∆ (“Tor1D norm 40 min” and “Tor1D 40min ‐ DMSO control”) were classified as TORC2‐dependent sites, and TORC1‐dependent sites were defined as those showing more than 2‐fold change in both wild type and tor1∆ cells ” button in the sidebar panel for downloading the faceted plots of the individual phosphosites, or the camera icon as shown in Figure In the “40‐min study” tab, similar navigation and selection fields can be found, but the “TOR Complex dependency” filter is in place of “Conserved in” field in the other panel. A further sub‐categorisation selection field is included between the “Protein” and “Site” fields to select for sites that may or may not be in common with the 85‐min dataset Fig . The “Stet al, Saccharomyces cerevisiae and human. The analysis was performed using the enrichment analysis tool from the PANTHER classification system  biological processes Tables  and 2 aset al, The analysis was performed on the 49 conserved proteins from the 83 proteins that were identified to contain TORC1‐dependent phosphosites. Using the STRING database (Szklarczyk TM and PN conceived the experiments. TM designed and performed the experiments. AWJ performed mass spectrometry and processed raw data. TM analysed processed data. TM and PN wrote the manuscript.The authors declare that they have no conflict of interest.Review Process FileClick here for additional data file.Expanded View Figure PDFClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Table EV3Click here for additional data file.Table EV4Click here for additional data file.


In addition, potent butyrylcholinesterase (BChE) inhibitory activity with IC50 values of 14.91 to 60.95 µM was observed in some compounds. Enzyme kinetic study revealed the most potent compound (6c) as a mixed-type AChE inhibitor. No cytotoxicity from the quinoxaline derivatives was noticed in the human neuroblastoma cell line (SHSY5Y). In silico study suggested the compounds preferred the peripheral anionic site (PAS) to the catalytic anionic site (CAS), which was different from AChE inhibitors . We had proposed the molecular design guided for quinoxaline derivatives targeting the PAS site. Therefore, the quinoxaline derivatives could offer the lead for the newly developed candidate as potential acetylcholinesterase inhibitors.A quinoxaline scaffold exhibits various bioactivities in pharmacotherapeutic interests. In this research, twelve quinoxaline derivatives were synthesized and evaluated as new acetylcholinesterase inhibitors. We found all compounds showed potent inhibitory activity against acetylcholinesterase (AChE) with IC Dementia is defined as a group of progressive neurological disorders that deteriorate memory, thinking, behavior and emotion. Dementia can occur in diverse forms, including Alzheimer’s disease (AD), vascular dementia, dementia with Lewy bodies and frontotemporal dementia. As Alzheimer’s disease contributes almost 70 percent of the cases, especially in older adults, it is considered the major form of dementia. In 2020, Alzheimer’s Disease International (ADI) reported over 50 million cases of dementia worldwide. With over 10 million new cases globally each year, the number of dementia cases was anticipated to rise to 131.5 million by 2050 . Graphs were plotted by means of GraphPad Prism 7.03 for Windows  [Kinetic studies of ay 2021) .m) and Michealis constant (Km) were calculated. Using non-linear regression (selected panel: enzyme kinetic inhibition/ mixed model inhibition) the values of Ki and alpha were determined. The alpha value defines the mechanism. Its value defines the degree to which the binding of inhibitor changes the affinity of the enzyme for substrate. Its value is always greater than zero. When alpha = 1, the inhibitor does not alter the binding of substrate to the enzyme, and the mixed model is identical with non-competitive inhibition. When the alpha is very large, the binding of the inhibitor prevents the binding of the substrate, and the mixed-model inhibition becomes competitive inhibition, and when alpha is very small (but greater than zero), the binding of the inhibitor enhances substrate binding to the enzyme, and the mixed model becomes nearly identical with an uncompetitive inhibition.From obtained equations of regression curves in the Lineweaver-Burk plot, the values of maximum velocity (Vhttp://www.swissadme.ch (accessed on 16 May 2021) [Mw, consensus log P, number of hydrogen bond donors and acceptors, rotatable bonds, and topological polar surface area (tPSA), GI absorption and BBB permeation of the quinoxaline derivatives were calculated by the SwissADME software accessed from ay 2021) ,50.2. Cells  were seeded in a 96-well plate and incubated at 37 °C, 5% CO2 for 24 h. The cells were then treated with or without various concentrations  of each compound for 48 h. After an incubation period, 40% (w/v) trichloroacetic acid (TCA) was added to the cells, and the cells were then incubated at 4 °C for 1 h. A total of 0.4% (w/v) SRB solution (100 μL) was added to each well, and the cells were incubated for 1 h at room temperature. The SRB solution was removed, and then the cells were washed three times with 1% (v/v) acetic acid, and they were allowed to dry at room temperature. The protein-bound dye was dissolved with 10 mM Tris base solution, and the absorbance was measured at 492 nm using a microplate reader.The cytotoxicity assay of the tested compounds against human SH-SY5Y neuroblastoma cells was measured using a sulforhodamine B (SRB) assay ,52. The www.rcsb.org, accessed on 20 April 2021), [The x-ray crystal structure of Recombinant Human Acetylcholinesterase in complex with (−)-huperzine A, PDB ID: 4EY5, was obtained from the RCSB Protein Data Bank (l 2021),  in a PDBl 2021), . The prowww.pccdb.org, accessed on 6 April 2021), and saved in mol2 format, which then converted into the PDB format using Obabel [The 3D structure file of the quinoxaline structure used was obtained from the PubChem database. The quinoxaline structure was taken from a structure with CID 7045, and other ligands were created by Public Computational Chemistry Database Project (g Obabel . Hydroge3), with the grid spacing of 0.375 Å at the center of the protein structure . The last grid size was considered as the blind docking process. Other parameters followed the default values in ADT. Fifty genetic algorithm (GA) runs with a population size of 200 were performed for conformational sampling. Ligands are arranged by the calculated ΔG value; lower ΔG values correspond to more desirable ligand binding, while higher ΔG values are less desirable [Molecular docking studies were performed using the AutoDock4 program, similar to previous studies ,57. Duriesirable . The docesirable ,59.3 with the grid spacing of 0.375 Å from the nitrogen atom the indole ring . The docking protocol was identical to the blind docking process mentioned above.To perform the site-specific docking, the PAS site was then defined. The PAS site grid box was based on Trp286. The grid was set at 40 × 40 × 40 ÅThe molecular docking study was carried out using AutoDock4 program in Ubuntu 18.04. The ligand-protein interaction was visualized and analyzed using the Visual Molecular Dynamic (VMD) package . The hydIn this work, we presented the design and synthesis of quinoxaline-based compounds as new acetylcholinesterase inhibitors. Drug-likeness and BBB permeabilities were predicted from the synthesized compounds. The potency in acetylcholinesterase inhibition from the synthesized compounds was observed within the same range of known AChE inhibitors. In addition, the peripheral anionic site (PAS) was proposed for the potential binding moiety, leading to the further efficient modification on the quinoxaline scaffold.


Hydrothermal treatment at 150 °C for 28 d induces a phase transition in the Al-stabilized compound from the commonly observed cubic Iad structure to the acentric Id subtype. LiI ions at the interstitial octahedrally (4 + 2-fold) coordinated 48e site are most easily extracted and AlIII ions order onto the tetrahedral 12a site. Deep hydration induces a distinct depletion of LiI at this site, while the second tetrahedral site, 12b, suffers only minor LiI loss. Charge balance is maintained by the incorporation of HI, which is bonded to an O atom. Hydration of Ga-stabilized LLZO induces similar effects, with complete depletion of LiI at the 48e site. The LiI/HI exchange not only leads to a distinct increase in the unit-cell size, but also alters some bonding topology, which is discussed here.Single crystals of an Li-stuffed, Al- and Ga-stabilized garnet-type solid-state electrolyte material, Li X3Y2Z3O12, has been well described mineralogically and crystallographically in recent decades , have raised particular interest as promising materials for use as solid-state electrolytes in all solid-state Li batteries due to their superior Li-ion conductivity 3 , La2O3, ZrO2 and Al2O3 or Ga2O3 were carefully mixed in the required stoichiometric proportions for the nominal compositions Li6.55La3Zr2Al0.15O12 (Al15-LLZO) and Li5.8La3Zr2Ga0.4O12 (Ga40-LLZO). The mixtures were pressed into pellets and preheated at 850 °C for 4 h for decarbonatization. The samples were subsequently milled in a high-energy ball mill under alcohol using ZrO2 balls, then dried, pelletized and sintered at 1230 °C for 6 h. This yielded a dense ceramic consisting of large individual crystallites of up to 150 µm. Structural refinements were carried out on the fresh LLZOs within 24 h of synthesis to assess the unaltered structural state. Parts of the pellets were crushed carefully and aged in a 45 ml Teflon-lined autoclave (25 mg sample and 250 ml distilled water) at 150 °C for a period of 28 d. The pH value of the liquid, in which the crystals were submerged, was measured after the experiment; the pH value was ∼13 for both compositions. The hydrated single LLZO crystallites were then filtered off and the remaining liquid was left to evaporate. The resulting precipitate was identified as Li2CO3 using powder diffraction (PXRD), i.e. proving that LiI was extracted from the LLZO material.Single crystals of Al- and Ga-stabilized LLZO were obtained using a solid-state ceramic sintering method, which has been described in detail elsewhere  analysis on a similar material synthesized using an identical experimental setup  is possible for all atoms using the same strategy as that applied by Wagner et al.  to that obtained for the untreated sample. The Li2 and, in particular, the Li3 sites are distinctly depleted in Li and no anisotropic atomic displacement refinement is possible. Thus, the isotropic adps are adjusted and fixed to the Ueq value refined for the Li1 site; anisotropic adps could be obtained for Li1. Protons are located close to the O1 atom using residual electron-density maps. Fixing the Ueq value of hydrogen yields reliable occupation factors for this site and an almost charge-balanced chemical formula (with a slight surplus of 0.35 negative charges).For pristine Ga-stabilized LLZO, indexing of the diffraction data yields the space-group symmetry l. 2016a,b ▸. ThIad to acentric Id. The structure of this compound is refined using the model of the Ga-stabilized LLZO, as described above for the La-, Zr- and the two O-atom positions. The electron densities at Li1 and Li2 were modelled first with only Li+ ions. In this case, the Li1 position is distinctly overpopulated, while the occupation of the Li2 position is low, and there is no indication that the Li3 position is occupied at all. Consequently, all AlIII is assigned to the Li1 site and its occupation is fixed to the value used in the unaltered sample, consistent with the assumption that no Al left the structure during hydration. Refinement of the anisotropic adps is not possible for the Li1 site, and the isotropic adp is very small, so the isotropic adp of Li1 was adjusted in such a way that it has a similar value to the Ueq value of the Li2 site (where anisotropic adp refinements were possible) and fixed as such in subsequent refinements. Two distinct residual electron-density peaks are identified in residual electron-density maps: one high (2.5 e Å−3), very close to the Zr-atom position, and another at ∼0.8 Å from the O1 atom. This latter  position is close to the proposed H-atom positions given by Larraz et al.  Å. As depicted in Fig. 1d tetrahedral site (Li1) and at the interstitial 96h position (Li2), often denoted to have a distorted octahedral coordination . AlIII substitutes into the 24d position and the four equivalent Li—O lengths are 1.9044 (17) Å; both the 24d and the 96h positions show distinct vacancies.In the pristine state, Al-LLZO shows the typical garnet structure with k = odd and l = odd that obey Iad symmetry. Calculated precession images of the hk0 plane for unaltered and altered Al-LLZO are compared in Fig. 2Iad symmetry are marked. Indexing of the observed data definitively yield the space-group symmetry Id, in accordance with the findings of Larraz et al.  with site symmetry Iad) to the 16c position  with site symmetry 3 (Id). Some positional disorder is observed at the Zr position, where around 20% of the ZrIV is displaced to a general 48e position, with a Zr—Zr offset of ∼0.39 (3) Å. Free refinement of the site occupancies of these two Zr positions total 1.96 ZrIV atoms per formula unit, i.e. very close to the expected value of 2.0. Whereas in Iad, the six Zr—O bond lengths are equivalent to a value of 2.1076 (16) Å. In the hydrothermally altered structure, ZrIV is in a very distorted octahedral environment at 16c, with Zr1A—O1xi bond lengths of 2.007 (4) Å (×3), and Zr1A—O2xii of 2.217 (4) Å (×3) (see Table 2B—O bonds range between 1.85 (3) and 2.36 (3) Å (Table 26La3ZrTaO12 (LLZTO). Pristine LLZTO shows Iad space-group symmetry but deep hydration induces a symmetry reduction to Id. A disorder at the Zr/Ta site is also observed in hydrated LLZTO similar to that in hydrated Al-stabilized LLZO in this study. It would appear that the symmetry reduction, induced by LiI/HI exchange, causes a large distortion of the O-atom environment around the 16c position and induces some positional disorder.The hydrothermally altered sample of Al-LLZO shows additional Bragg peaks of type  al. 2013 and Orer al. 2016 for LiI/Å Table 2. It is id tetrahedral site of the Iad garnet structure splits into two different sites, 12a (Li1) and 12b (Li2), upon symmetry reduction to Id. AlIII is ordered onto the 12a (Li1) site but a distinct number of vacancies are observed on both sites. While ∼4.5 apfu LiI occupy the interstitial 96h position in the pristine state, this position (Li3 at 48e) is completely unoccupied in the deeply hydrated form, i.e. all the LiI ions have vacated the interstitial octahedral site.The regular 24et al. (2021bd and 12a) occupation in Al-stabilized LLZO during continuous LiI/HI exchange in a humid atmosphere and under mild hydrothermal conditions. The shift and ordering of LiI from the interstitial site to the regular tetrahedral site, and the preference of LiI and AlIII for the 12a position, are described as triggers for the symmetry reduction from Iad to Id  apfu], but there is only a moderate change in site occupation at 12b [∼1.1 to 0.82 (2) apfu]. As LiI is extracted from the structure, HI is incorporated and bonds with the O1 atom. The LiI ions in the 12a tetrahedron are thus coordinated by four OH groups, with the O—H vector pointing towards the empty Li3 site 3·8H2O within ∼30 d of exposure.Recently, Redhammer l. 2021b observedl. 2021b, it is o al. 2016 for purel. 2021a,b ▸, whI/HI exchange is accompanied by a large increase in the a unit-cell parameter from 12.9637 (2) to 13.0738 (2) Å, which is among the largest values yet recorded for hydrated LLZO, cf. 13.06245 (4) Å at 77 K for hydrated LLZO with an Li2.3H4.7La3Zr2O12 composition  site, both require more space and are considered responsible for the lattice expansion.The LiId symmetry. A section of this crystal structure is illustrated in Fig. 3a and 12b positions appear to be fully occupied, with the GaIII ions ordered onto the 12a position. In contrast to the Li-stuffed garnets with Iad symmetry, the decreased number of vacancies at the regular tetrahedral sites are considered to be a characteristic feature of the Id garnet structure. The 48e interstitial site [Li3 in Fig. 3a)] is occupied by 3.71 LiI apfu, equivalent to being ∼62% full. All of the tetrahedral faces of the fourfold oxygen coordination around the Li1 and Li2 sites are shared with neighbouring Li3 sites, with interatomic contacts of 1.645 (8) (Li1—Li3) and 2.340 (9) Å (Li2—Li3), thereby forming a three-dimensional network that is responsible for the good Li-ion conductivity in this compound. The dodecahedral site has a small number of vacancies, while the regular octahedral positions are fully occupied with ZrIV, resulting in a composition of La2.94Zr2.00Ga0.28Li6.43O12 for the unaltered Ga-stabilized LLZO material.Pristine unaltered Ga-stabilized LLZO shows a) site, which hosts GaIII, is slightly smaller than the Li2 (12b) site, whereas the Li3 site, as in Al-LLZO, shows a very distorted 4 + 2-fold coordination, with bond lengths between 1.878 (4) and 2.639 (8) Å; the average of the four shorter bonds is 2.073 Å, i.e. slightly smaller than in the pristine Al-LLZO [see Fig. 3a)].A smaller and larger set of La—O1/O2 bonds are observed at the dodecahedral site [2.4935 (19)–2.5264 (19) and 2.587 (2)–2.5975 (19) Å] and the octahedron is regular with two independent Zr—O bonds ranging between 2.0823 (18) and 2.1346 (18) Å. Therefore, the octahedra are much less distorted than those in the altered Al-LLZO with the same symmetry Table 2. The Li1I/HI exchange. Again, a large increase of the lattice parameter to 13.06720 (12) Å is close to the value in hydrated Al-LLZO, suggesting that values around 13.07 Å represent an upper limit for lattice-parameter increase due to hydration. From site-occupation refinements, very minor LiI ions are found at the interstitial 48e position, i.e. this site is almost completely depleted. One key difference with Al-LLZO is that no LiI is lost from the 12a position. It seems that GaIII ions pin LiI at this site. However, LiI ions are extracted from the 12b site where the amount of LiI is reduced from 1.49 apfu in the pristine to 0.50 apfu in the altered sample. Protons are again located close to the O1 atom [Fig. 3b)], giving rise to a fully hydrated Li1(OH)4 coordination around the 12a site. Some differences to Al-LLZO are, however, evident: there is no significant reduction in the La-site occupation and no residual electron density is observed close to the ZrIV ions, i.e. there is no indication of disorder at the 16c position in altered Ga-LLZO. Nevertheless, the ZrO6 octahedron is much more distorted in the altered sample, with Zr—O bond lengths ranging between 2.03 (3) and 2.187 (4) Å; the difference between the two independent Zr—O bonds increases with prolonged LiI/HI exchange. This was outlined by Redhammer et al.  global, LLZO-Al15-pristine, LLZO-Al15-hydro-150C, LLZO-Ga40-pristine, LLZO-Ga40-hydro-150C. DOI: 10.1107/S2053229621012250/ep3020LLZO-Al15-pristinesup2.hklStructure factors: contains datablock(s) LLZO-Al15-pristine. DOI: 10.1107/S2053229621012250/ep3020LLZO-Al15-hydro-150Csup3.hklStructure factors: contains datablock(s) LLZO-Al15-hydro-150C. DOI: 10.1107/S2053229621012250/ep3020LLZO-Ga40-pristinesup4.hklStructure factors: contains datablock(s) LLZO-Ga40-pristine. DOI: 10.1107/S2053229621012250/ep3020LLZO-Ga40-hydro-150Csup5.hklStructure factors: contains datablock(s) LLZO-Ga40-hydro-150C. DOI: 2122845, 2122844, 2122843, 2122842CCDC references:


The self-assembled monolayer (SAM) is the most common organic assembly utilized for the formation of the monolayers of alkane-thiolates on gold electrode, resulting in a wide range of applications for the modified SAM on gold in various research areas. This study examined the desorption of a SAM that was developed on the gold surface of an interdigitated chain-shaped electrode  with the goal of determining the most efficient strategy of SAM removal for the ICE to be re-used. A simple and proficient solution-based cleaning procedure was applied for the removal of a SAM on the gold surface of the ICE by using a sodium borohydride solution within short-term treatment, resulting in efficiency for the recovery of the originally electrochemical characteristic of ICE of 90.3%. The re-use of ICE after the removal process was confirmed by the successful re-deposition of a SAM onto the electrode surface, resulting in the high efficiency percentage of 90.1% for the reusability of ICE with the SAM modification. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used as tools to investigate the changes in the electrode interface at each stage of the SAM removal and the electrode recycling. X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy were employed, being powerful spectrum techniques, for the characterization of the bonding structure and chemical state of the bare ICE and the modified ICE at each treatment step. Based on the comprehensive discussion of analytical chemistry from the obtained EIS and CV data in this study, we confirmed and proved the effectiveness of this promising method for the removal of a SAM from the ICE and the re-use of ICE in the field of material deposition, with the aims of saving money, improving experimental handling, and protecting the environment. Potassium chloride  was bought from Daejung Chemicals and Metals Co., Ltd., . Phosphate-Buffered Saline  was purchased from Welgene Inc. . De-ionized (DI) H2O was obtained from Water Purification System, PURESCIENCE . Ethanol  was provided by OCI Company Ltd., .6-Mercaptohexanoic acid , sodium borohydride , and a 50 nm thick Au electrode layer . Next, the lift-off process was used to generate the paired electrode finger, including the space of 5 μm and the width of 5 μm, for the working and reference electrodes, respectively. The ICE was manufactured on a glass slide substrate of (14 mm × 3.5 mm). The electron beam evaporator system  was used to deposit a 25 nm thick adhesive Ti layer  to create the SAM on the Au surface of ICE (ICE/SAM) through the bonding between gold (Au) of ICE and sulfur (S) at the head group of the SAM, as shown in 4 solution for 10 min at RT to remove the SAM on the ICE surface (re-ICE) and return the ICE to its original state.To re-use the ICE, the ICE/SAM was immersed in 0.5 M NaBH2O. This procedure is similar to the deposition of the SAM on bare ICE, as described above, which is a quality control process for the successful formation of the SAM on the gold surface of ICE, as shown in the Results and Discussion section.To approve the re-use of ICE, the SAM was again deposited on the re-ICE (re-ICE/SAM) by dipping the re-ICE in a 100 mM MHA solution for 2 h at room temperature (25 °C), and then it was washed with extra pure ethanol and DI Hα, hν = 1486.6 eV) gun and a monochromatic gun was used to characterize the chemical state and the electronic structure of the bare electrode and the electrode modification.X-ray photoelectron spectroscopy  with X-ray sources of a twin anode  was used to characterize the functional and bonding groups on the electrode surface. The FT-IR result was obtained by measuring three consecutive runs for each sample.Fourier-transform infrared spectroscopy  was used to investigate the electrochemical impedance spectroscopy (EIS) and the cyclic voltammetry (CV) measurements of the ICE and the ICE modification. EIS was performed in a two-electrode configuration with an input sinus amplitude of 50 mV and the applied frequency range of 1 MHz to 100 mHz, and CV was performed in a three-electrode configuration with an external Ag/AgCl electrode, in 1 mM K[Fe(CN)4 solution to remove dust and impurities existing on the surface. 6]3−/4− containing 0.1 M KCl in a Nyquist plot of the image impedance (−Im(Z)) vs. the real impedance (Re(Z)), as shown in s, corresponding to the solution resistance which can be found by reading the real axis (Re(Z)) value at the high frequency intercept; Rct, corresponding to the interfacial charge-transfer resistance which equals the semicircular diameter of the Nyquist plot; Cdl, corresponding to the double-layer capacitance which is in parallel with the Rct.Cleaning ICE is an important step to remove numerous contaminants on the electrode surface and enhance the quality of the active Au surface of ICE for the chemical modification. Therefore, the bare ICE was cleaned with 0.5 M NaBHct value of ICE decreased significantly to 86 kΩ after the cleaning step, as compared to the high Rct value (390 kΩ) of ICE before the cleaning step. This decrease in Rct was due to the easy transport of the K[Fe(CN)6]3−/4− redox probes toward the electrode surface; this result indicated that the movement obstruction of redox probes was prevented by elimination of the impurities that existed on the electrode surface, demonstrating the successful removal of contaminants on the electrode surface, and that the original electrochemical behavior of the ICE had been brought back. CV of the ICE after cleaning (−1 in a solution of 0.05 M H2SO4) 3−/4− containing 0.1 M KCl, and results are expressed through Nyquist plots, as shown in ct, Cdl, and Rs were obtained, and are displayed in ct value of the ICE was 86 kΩ. Significant enhancement in Rct was observed after the addition of the SAM to the ICE surface 6]3−/4− redox couple to the ICE surface. As seen in The EIS tests of ICE, ICE/SAM, re-ICE, and re-ICE/SAM were performed in 1 mM K3−/4− redox couple toward the electrode surface; the hindrance of the transfer of the redox probe to the surface of electrode was removed, which was caused by the elimination of the SAM on the ICE surface. It is observed that the Rct value of the re-ICE (87 kΩ) approximately equals the Rct of the bare ICE (86 kΩ), demonstrating that removal of the SAM from the ICE was successfully completed by the treatment with NaBH4 solution. The re-confirmation of the elimination of the SAM from the ICE’s surface was also performed by CV measurement. The CV result of the re-ICE after removing SAM in ct in the Nyquist plot of the re-ICE/SAM showed a remarkable increment (1870 kΩ in ct of the re-ICE (87 kΩ) and the ICE (86 kΩ). This increment in Rct confirmed the formation of the SAM insulator on the surface of re-ICE, leading to delaying the transfer of the K[Fe(CN)6]3−/4− redox couple to the electrode surface. The re-ICE/SAM showed the approximate Rct value, as compared to the Rct of the ICE/SAM, as seen in After the successful removal of the SAM from the ICE, the re-ICE was used to immobilize the SAM for the purpose of demonstrating the ICE’s reusability, as described in To evaluate the possible factors for the recovery of ICE’s characteristic after the removal of the SAM and the re-use of ICE with the SAM modification, the efficiency percentage was determined by calculation, as described in the following evaluation:ICE is the Rct of the bare ICE, and Rre-ICE is the Rct of the re-ICE after the removal of the SAM.The efficiency percentage of the ICE recovery after SAM removal can be calculated as follows:ICE/SAM is the Rct of the ICE/SAM with the addition of the SAM to the ICE, and Rre-ICE/SAM is the Rct of the re-ICE/SAM with the re-modification of re-ICE with a SAM.The efficiency percentage of the reusability of ICE with the SAM modification on the surface can be calculated as follows:Therefore, as shown in The aim of this work was to study the most effective method for removing the modified SAM on an ICE’s gold surface in order to determine the plausibility of a reuse procedure for the electrode.4 with the purpose of reusing ICE to save money, protect the environment, and expeditiously manage experiments.Therefore, in this study, a convenient, simple, and efficiency strategy to remove SAM on the gold surface of ICE was explored by treatment for 10 min with a solution of NaBHThe full removal of the SAM and the good reusability of ICE with re-addition of the SAM were evaluated and confirmed by analyzing the electrochemical measurements, such as EIS and CV, and by using microscopy techniques such as XPS and FT-IR.The high-efficiency percentages for the recovery of the electrochemical characteristics of ICE, and for the re-deposition of the SAM on the ICE surface, were also determined, and found to be 90.3% and 90.1%, respectively, indicating a well-founded approach for the regeneration of ICE’s gold surface in applications of material deposition, providing advantages in terms of cost savings, experimental control, and environmental protection.4 solution was also presented in this paper.A new and concise route for cleaning the bare electrode surface by applying a NaBH


This review describesthe mechanisms of ribosome rescue thanks to  Fig. 1)[In a bacterial cell, protein synthesis involves the 70S ribosome that consistsof the small 30S and large 50S subunitsFig. [1, 2,3Fig. 1). Movement of theribosome can also stop on an intact template; e.g., during translation of“rare” codons and polyproline sequences [Unlike eukaryotic cells, where translation is preceded by mRNA processing,bacteria are unable to control the quality of the template before proteinbiosynthesis. Translation in a bacterial cell occurs simultaneously withtranscription. This coupling of the two most important processes in time andspace, on the one hand, is an advantage: it not only enables the cell toproduce proteins at a higher rate, but also underlies the regulatory mechanismof attenuation. On the other hand, the absence of any control over the mRNAbefore translation inevitably leads to ribosome stalling during the proteinsynthesis on a template damaged by various factors. The most common cause ofthese occurrences is ribosome stalling on a damaged mRNA and the formation ofthe so-called non-stop complex . Thelisequences  or underequences . Of courequences . This reThe factors that solve the problem of stalled translation may be divided intotwo types:1. Factors causing emergency termination of translation, first and foremost,with subsequent hydrolysis of the peptidyl-tRNA and release of theribosome’ and2. Factors causing the reactivation of translation in emergency conditions.Let us consider in more detail the causes behind translation stalling and therescue systems operating in each specific case.One of the most common problems that the ribosome may encounter during mRNAtranslation is the absence of a stop codon . This erTrans-translationtrans-translation performed by transport-messenger RNA(tmRNA), which is encoded by the ssrA gene, and the SmpBprotein. The tmRNA structure and the trans-translationmechanism are described in detail in a number of papers [The most common mechanism for the rescue of ribosome complexes is thef papers , 13,14.To perform its function, tmRNA requires the SmpB protein . SmpB sttrans-translation, the tmRNA–SmpB–EF-Tu–GTP complex binds to the A site of the ribosome. Unlikea ternary complex that interacts with mRNA at the A site, thetmRNA–SmpB–EF-Tu–GTP complex interacts with an empty A site.In this case, the codon–anticodon interaction is replaced by theinteraction between SmpB and a ribosome site that binds mRNA on the3’-side of the P site during normal translation. In this case, thetmRNA–mpB–F-Tu complex triggers GTP hydrolysis. If the mRNA channelis empty, then the tmRNA remains in the A site to continue the translation ofthe tmRNA coding part. If mRNA is present in the channel, the interaction isprevented because of steric overlap. Thus, thetrans-translation mechanism does not affect translatingribosomes [At the first step of ibosomes . Trans-translation continues until astop codon of the tmRNA is reached, which is recognized by the canonicalrelease factor RF1 or RF2 that terminates translation and releases thepolypeptide with a tmRNA-encoded tag. Further, the polypeptide is recognized byseveral proteases, including ClpXP, ClpAP, HflB, and Tsp13, which leads to itsrapid degradation[Entry of the tmRNA–SmpB complex into the A site leads to the transfer ofa polypeptide chain to Ala-tmRNA and is accompanied by subsequent translocationof deacylated tRNA from the P site to the E site, and thepeptidyl-tmRNA–SmpB from the A site to the P site. During translocation,the tmRNA reading frame enters the mRNA channel, such that its first codon,known as the “resume codon,” displaces the C-terminal tail of SmpBfrom the decoding center..Escherichiacoli cells, this process is carried out by the RNase R that isrecruited by tmRNA–SmpB [The interaction between the protease and the ssrA tag is provided for by theSspB adaptor protein. The original mRNA involved in the non-stop complex isalso degraded to avoid repeated translation and a recurrence of emergencysituations . In EschRNA–SmpB .Thus, tRNA–SmpB .Reserve pathways of ribosome rescue involving ArfA and BrfAtrans-translation activity, theribosome is rescued through an alternative pathway using the Arfa  protein. ArfA recruits RF2 to the ribosome, which inturn hydrolyzes the peptidyl-tRNA in non-stop complexes[In the case of limited , 21. trans-translation, ArfA activity leads only to the releaseof ribosomes but is not accompanied by a subse quent degradation of nascentpolypeptides or mRNA [ArfA compensates for the absence of a stop codon at the A site and promotespeptidyl-tRNA hydrolysis by RF2 .Therefo or mRNA ,23, 24. or mRNA  through or mRNA .trans-translation system [E. coli, the ArfAmRNA adopts a hairpin structure and contains an RNase III cleavage site; RNaseIII removes the stop codon and the final 18 codons of the open reading frame.The arfA gene of Neisseria gonorrhoeae lacksan RNase III cleavage site; however, the hairpin facilitates transcriptiontermination before the stop codon, thereby providing inhibition of ArfAsynthesis [trans-translation, and theprotein undergoes rapid proteolysis [trans-translation is limited or impaired, then a truncated ArfAlacking the ssrA degradation tag is formed. This truncated product replaces thetmRNA–SmpB system. This regulation mechanism makes ArfA a true reserveribosome rescue system that operates only whentrans-translation activity is low or absent [should be noted that ArfA is synthesized from non-stop mRNA, and its expressionis directly regulated by the n system . In E. cynthesis . Ribosomteolysis . In somer absent .Bacillus subtiliscells [trans-translation. However, theArfA and BrfA proteins lack structural similarity and are evolutionarilydistant from each other. In addition, despite the fact that both proteinsrecruit RF2, the interaction of each of these proteins with RF2 is different[trans-translation system.The ribosome rescue mechanism involving the ArfA protein is used by onlygram-negative bacteria. In gram-positive bacteria, other mechanisms arepresent, and, for a long time, the canonical release factors were believed notto be involved in them. However, a mechanism of ribosome rescue similar to theaction of ArfA was recently described in iscells . The proifferent. ProbablArfB: an alternative rescue systemarfB gene wasfirst identified as a lethality suppressor in an E. colimutant lacking both trans-translation and the ArfAprotein [arfB gene were found in 34% of the sequenced genomes of bothgram-positive and gram-negative bacteria [An alternative way to rescue stalled ribosomes is provided by the protein ArfB. The protein . Homologbacteria . Unlike bacteria .E. coliand C. crescentus is able to hydrolyze thepeptidyl-tRNA in non-stop complexes in vitro in the absence ofthe RF1 and RF2 release factors[The ArfB N-terminal domain is homologous to the catalytic domains of RF1 andRF2. This domain contains the GGQ motif that plays a crucial role inArfB-mediated peptidyl-tRNA hydrolysis. In this case, several important aminoacid residues necessary for the recognition of the retained complex and bindingof the stalled ribosome are located not in the N-, but in the C-terminal domainof the protein. ArfB lacks a domain capable of interacting with a stop codon. PurifieFig. 4A), 31.The ribosome with a free A site serves as a substrate for tmRNA and ArfA; asimilar arrangement was suggested for ArfB, but ArfB was found to interact withribosomes even when a small mRNA segment extends from the P site. In thisFig. 4B)[Substrates of ArfB also include ribosomes with a rather extended mRNA fragment. In thisFig. 4B).arfB in C. crescentus does notaffect viability, but it is lethal in combination with deletion of ssrA[trans-translation, becausethe ΔssrA C. crescentus strain has a pronounced growthdefect [trans-translationactivity and it most probably does not act exclusively as a reserve system fortrans-translation [Deletion of  of ssrA. Howeverhdefect . In addinslation ,31. Thenslation .ArfT releases ribosomes through a different mechanismFrancisella tularensis. Francisellatularensis lacks ArfA and ArfB, but inactivation of the ssrA/SmpBsystem is not a lethal mutation for this bacterium. Transposon mutagenesisfollowed by deep sequencing revealed a new alternative ribosome rescue factorcalled ArfT [An unusual mechanism of ribosome rescue was found in the causative agent oftularemia, led ArfT .arfT gene was found to lead to a loss ofviability only in F. tularensis mutants incapable oftrans-translation. Overexpression of ArfT, on the contrary,promotes the intensive growth of these cells [in vitro. The KKGGSTNKK sequence near the C-terminus of ArfTcontains, like ArfA, a number of positively charged residues; therefore, ArfTcan probably use this sequence to bind the ribosome [invitro modeling of abnormal translation, the addition of ArfT and RF1from F. tularensis to the non-stop complex led to thehydrolysis of the peptidyl-tRNA with an efficiency of 95%, and the addition ofArfT and RF2 from F. tularensis led to the same hydrolysiswith an efficiency of 84% [Deletion of the se cells . ArfT isribosome . ArfT cay of 84% .Despite the similarity of the C-terminal sequence of ArfT and ArfA, the abilityof ArfT to activate both RF1 and RF2 may mean that ArfT interacts with releasefactors differently than ArfA does. In addition, it is worth noting that ArfTformation is not regulated by translation termination.HflXE. coli HflX protein.Heat shock is another cause of translation stalling. In this case, rescuesystems interact with the 70S ribosome containing a peptidyl-tRNA in the P siteand intact mRNA in the A site. One of the factors that can recognize thissubstrate is the Fig. 5A)[Fig. 5B)[There are several potential mechanisms of HflX activity. According to one ofthem, Hf1X can bind to a free E site. A peptiFig. 5A). HflX waFig. 5B). In thisHflXrhflXgene in the pathogenic bacterium Mycobacteriumabscessus was recently found to increase sensitivity to macrolideantibacterial agents. The product of this gene is capable of disassembling theribosomes blocked by macrolides and, as thus, plays an important role in thedevelopment of antibiotic resistance in some pathogens [The action mechanism of numerous antibacterial agents is based on translationsuppression. Many of them bind to the PTC, thereby inhibiting the peptidyltransferase reaction . Resistaathogens .Listeria monocytogenes[E. coliHflX whose function is to disassemble a stalled ribosome [hflXr gene renders bacteria moresensitive to erythromycin and lincomycin, the sensitivity phenotype manifestsitself only upon simultaneous deletion of another gene, lmo0919[A nontrivial mechanism of resistance, which is probably related to the activityof the HflXr protein, was described in ytogenes, 43. Thiribosome . Althoug lmo0919.Release of the ribosome by RqcH and RqcPFig. 6)[Among the causes behind the abrupt arrest of protein biosynthesis, there is arather unusual one–premature dissociation of ribosomal subunits. Therelease of the 50S subunit from a complex with the peptidyl-tRNA occurs usingseveral mechanisms. One of them involves the RqcH and RqcP proteins. TheactB. subtilis is a homologue ofthe eukaryotic translation quality control factor Rqc2. In a model shown inFig. 6,the RqcP protein binds to the 50S ribosomal subunitand stabilizes tRNA on the P site [The Rqc2 homolog (RqcH) found in e P site ,45. Rqce P site . Furthere P site .Hsp15E. coli Hsp15 protein [Actinobacteria and gamma-proteobacteria lack the RqcH and RqcP proteins.However, it should be noted that the RqcP protein is a homologue of the protein . Like Rq protein .PrfHE. coli K-12 gene encoding an amino acid sequencewith high similarity to the RF1 and RF2 sequences was identified [In 1992, the entified . The eleentified .There are several suggestions regarding the function of PrfH and which ribosomecomplex may constitute its substrate. The most plausible hypothesis is thatPrfH is a ribosome rescue factor .prfH overexpression was found to increase theresistance of Pseudomonas aeruginosa bacteria to azithromycin[prfH overexpression was shown to decrease the numberof stalled ribosome–model mRNA complexes formed in the presence ofazithromycin.For example, hromycin. In addiHowever, the role of PrfH is unknown and requires further investigation.Elongation factor PIt should be noted that template damage is not the only reason behind ribosomestalling during translation. Ribosomes are often stalled on intact mRNAs. Thissituation can develop in two scenarios: either elongation resumes, or mRNA iscleaved to form a non-stop complex. Ribosome profiling studies havedemonstrated that this ribosome pausing is short-term, because it does notblock the movement of other ribosomes translating the same template and doesnot disrupt gene expression , 52. ManE. coli EF-P contains a lysinyl-hydroxylysine moiety generatedby the YfcM [P.aeruginosa contains a rhamnose moiety [B. subtilis is5-aminopentanol [Stalled ribosomes are capable of spontaneous elongation resumption ortranslation termination, but specialized translation factors often help inthese processes. One of them, EF-P, is a highly conserved protein, a eukaryoticeIF5A homologue, that promotes the synthesis of polyproline sequences , 53,54.the YfcM , YjeK, athe YfcM , 57,58 e moiety , 60, andpentanol . In eukapentanol .The formation of a peptide bond between proline residues is complicated andoften leads to protein synthesis arrest . Similarin vivo andin vitro when ribosomes are stalled on polyproline sequences.EF-P is believed to promote the stabilization of thePTC substrate conformation productive for the peptidyl transferase reaction.Despite the fact that EF-P eliminates a small number of abnormalities, it isimportant enough to the physiology of a bacterial cell. For example, E.coli and S. enterica strains lacking EF-P havemembrane integrity defects and exhibit increased sensitivity to someantibacterial agents [Structural studies of EF-P on the ribosome have shown that EF-P binds betweenthe E site and the P site on the 50S subunit in close proximity to peptidyl-tRNA. Binding of EF-P stimulates elongation l agents .EF-4 (LepA)The well-known conserved translation factor EF-4, also known as LepA, wassuggested as a promoter of elongation by catalysis of reverse translocation ofstalled ribosomes . HoweverEttAE. coli [ATP-binding cassette (ABC) type F proteins that bind to ribosomes and promotedissociation of the ribosome– antibiotic complex are capable ofprotecting the ribosome against antibiotics , 67, 68.E. coli . EttA doE. coli , 71. At Pseudomonasaeruginosa, Staphylococcus aureus,Enterococcus faecalis, and B. subtilis, makethese organisms resistant to a wide range of antibiotics [Also, some ABC-F proteins underlie the mechanisms of antibiotic resistance. Adetailed review of the ABC-F proteins that protect the ribosome fromantibiotics is presented in . TheseAibiotics .Table).Most bacteria need at leastone ribosome rescue mechanism to survive. In this case,trans-translation has become the most widespread system: thessrA and smpB genes are found in more than99% of bacterial species [trans-translation system are present inalmost all bacterial genomes, and mutations in the genes encoding theseproteins reduce cell viability, the proteins involved in this system areconsidered as attractive targets for new antibacterial agents. Theseconsiderations have also been confirmed by the fact thattrans-translation is specific to bacterial cells, whichreduces the risk of possible side effects. Several compounds, potentialinhibitors of the release of non-stop complexes throughtrans-translation, have been selected by high-throughputscreening [The ability to release the ribosomes stalled on mRNA during translationmarkedly increases the chance of survival and, therefore, has been retainedduring natural selection( species . Becausecreening . The mectrans-translation contain an alternative releasefactor [ssrA deletion is maintained by arfAin E. coli, brfA in B.subtilis, arfT in F. tularensis, andarfB in C. crescentus. Shigellaflexneri and N. gonorrhoeae cannot survive withouttrans-translation [E. coliArfA homologue capable of replacing the tmRNA–SmpB system[F. tularensisRF1/2 but is unable to bind E. coli RF1/2. The BrfAfactor interacts exclusively with RF2 of B. subtilis. Thus,the described ribosome rescue systems are not interchangeable in differentspecies [trans-translation. Deletion of ssrA orsmpB results in many different phenotypes. For example,mutants lacking ssrA may exhibit increased sensitivity to antibiotics andtemperature fluctuations and should have virulence defects[Trans-translation is preserved in allbacteria, but no species has adapted to the exclusive use of the ArfA, ArfB, orother system. Activity of tmRNA/smpB not only releases stalled ribosomes, butalso promotes the removal of nascent polypeptides and damaged mRNAs, which alsoprovides a significant advantage to the system over reserve rescue systems. Apartial analogue of trans-translation is the RqcH–RqcPsystem, whose activity leads to the degradation of an incorrect polypeptide.The cells of almost all studied bacterial species capable of surviving in theabsence of efactor . For exanslation .This maB system, 73.Notspecies . In this defects, 74.Tratrans-translation; there is a suggestion thatribosome rescue is the primary mechanism in translation stalling. Of course,trans-translation is the most beneficial of the mechanisms,because it relieves the cell of unwanted and potentially toxic molecules.However, when it is limited or absent, the central need is still implemented– the rescue of stalled ribosomal subunits for subsequent rounds ofprotein synthesis. Thus, bacteria have acquired a variety of translation rescuesystems aimed mainly not at controlling the quality of mRNA but at releasingribosomal subunits.The additional ribosome rescue systems, both reserve and independent, mayhardly be called quality control mechanisms in protein biosynthesis. Thesesystems do not target damaged mRNAs, or the polypeptides synthesized on theirbasis, for degradation. Despite a variety of reserve mechanisms, none of themduplicates


Porphyromonas gingivalis, a keystone pathogen in periodontal disease, possesses an extensive phosphotyrosine signaling network. The phosphorylation reaction is catalyzed by a bacterial tyrosine (BY) kinase, Ptk1, and a Ubiquitous bacterial Kinase UbK1. Dephosphorylation is mediated by a low-molecular-weight phosphatase, Ltp1 and a polymerase and histidinol phosphatase, Php1. Phosphotyrosine signaling controls exopolysaccharide production, gingipain activity, oxidative stress responses and synergistic community development with Streptococcus gordonii. Additionally, Ltp1 is secreted extracellularly and can be delivered inside gingival epithelial cells where it can override host cell signaling and readjust cellular physiology. The landscape of coordinated tyrosine kinase and phosphatase activity thus underlies the adaptive responses of P. gingivalis to both the polymicrobial environment of bacterial communities and the intracellular environment of gingival epithelial cells.Tyrosine phosphorylation modifies the functionality of bacterial proteins and forms the basis of a versatile and tunable signal transduction system. The integrated action of tyrosine kinases and phosphatases controls bacterial processes important for metabolism and virulence.  Signal transduction mediated by coordinated and reversible protein phosphorylation events regulates a wide variety of biological processes in all living cells. In bacteria, phospho-dependent signaling can occur through two component systems (TCS) which rely on His/Asp phosphorylation, as well as through phosphorylation of Ser/Thr or of Tyr residues , 2. Moreubk genes are present in most bacterial genomes, but not in Archea or eukaryotes [Substrate phosphorylation is initiated upon autophosphorylation of the BY kinase which dissociates octamers of inactive enzyme, and releases the YC of one monomer from the catalytic site of an adjacent monomer which is then available for substrate recognition . More rekaryotes . There akaryotes .Regulation of BY-kinases by phosphatases occurs through dephosphorylation of the YC. Nevertheless, BY kinase activity is not dependent simply on the phosphorylation state of one key regulatory residue, as is the case for a majority of eukaryotic kinases. Rather, activity varies between a state where the YC motif is highly phosphorylated, to a state where YC phosphorylation is significantly reduced , and theE. coli [The phosphotyrosine proteome in bacteria is quite extensive, with over 500 unique phosphotyrosine sites identified on almost 350 proteins in E. coli . TyrosinPorphyromonas gingivalis, a keystone pathogen in periodontal disease, is an obligate asaccharolytic anaerobe. In the ecosystem of the subgingival compartment, P. gingivalis is a constituent of a complex polymicrobial community and it is this community that is the etiological unit in periodontal disease [P. gingivalis is geared toward acquisition of peptides through the action of gingipain proteases on host proteins, resistance to oxidative stress, and synergistic interactions, including metabolic communication, with community partners. In addition to interbacterial interactions, P. gingivalis engages gingival epithelial cells in an intricate molecular dialogue which can disrupt tissue and immune homeostasis, to create a microenvironment conducive to survival and persistence. As will be discussed, tyrosine (de)phosphorylation based signaling plays a key role in these processes. First, however, an exposition of the tyrosine kinases and phosphatases produced by P. gingivalis. disease , 14. MucP. gingivalis has been performed mainly in strain 33277, although homologs are present in other sequenced strains.Study of kinases and phosphatases in P. gingivalis contains the canonical Walker A, A' and B domains, and the YC motif [(i) Tyrosinse kinases. (A) The Ptk1 BY kinase of YC motif , 16. In YC motif . The Wal5R. The nucleophile cysteine within this phosphotransfer loop is required for phosphatase activity, while the arginine is necessary for substrate binding [php1 gene is located immediately downstream of the ptk1 gene, whereas ltp1 is remotely located, an uncommon arrangement in Gram-negative bacteria.(ii) Tyrosine phosphatases. (A) Ltp1 is a LMW-PTP which contains the conserved catalytic motif C(X) binding , 19. (B) binding . ConservE. coli, the Wzc BY-kinase together with its cognate tyrosine phosphatase, Wzb, are responsible for group 1 capsule and colonic acid polysaccharide biosynthesis [P. gingivalis; however, deletion of the ptk1 gene results in loss of EPS secretion in strain 33277, and failure to produce a capsule in strain W83 [E. coli [Staphylococcus aureus [php1 mutant of P. gingivalis produces significantly less EPS [P. gingivalis, through impacting transcriptional regulation of multiple genes involved in biosynthesis and transport [P. gingivalis will depend on a balance of the activities of Ptk1, Php1 and Ltp1, a theme that pervades many of the tyrosine kinases/phosphatases signaling events.(i) Extracellular polysaccharide (EPS) production. One of the first functions described for tyrosine (de)phosphorylation in bacteria is extracellular polysaccharide production. In ynthesis . The curynthesis . Detailerain W83 . Ptk1 carain W83 , as also[E. coli  and Staps aureus , indicatless EPS . Interesransport . Hence, P. gingivalis. These enzymes degrade host proteins, including immune effectors, to provide peptides for metabolism [(ii) Gingipain activity. The arginine  and lysine (Kgp) specific gingipain proteases are critical for growth and virulence of tabolism , 26. Thetabolism . The efftabolism , consistP. gingivalis, Ubk1 phosphorylates and modulates the activity of the orphan response regulator (RR) RprY [P. gingivalis includes genes involved in oxidative stress responses, suggestive of a role for UbK in the process. However, this contribution of Ubk1 to P. gingivalis physiology has yet to be confirmed, a problematical task given that ubk1 is an essential gene in the organism [(iii) Oxidative stress. UbK enzymes can control stress responses in other organisms , 29. In RR) RprY . The Rprorganism .P. gingivalis with Streptococcus gordonii. Co-adhesion between the organism is mediated by the FimA and Mfa1 fimbriae of P. gingivalis engaging the SspA/B and GAPDH surface proteins, respectively, of S. gordonii [mfa1 gene expression [ptk1 are deficient in P. gingivalis-S. gordonii community development [ltp1 mutants show enhanced accumulation on substrata of S. gordonii [S. gordonii such as hydrogen peroxide  and para-amino benzoic acid (pABA) [S. gordonii metabolites through inactivation of Ltp1 thus leads to increased fimbrial adhesin production and primes P. gingivalis for community development [P. gingivalis may be able to sample the environmental milieu and use phosphotyrosine signaling to promote adherence and co-colonization with metabolically compatible synergistic organisms.(iv) Heterotypic community development. Tyrosine phosphorylation dependent signaling plays a major role in the development of synergistically pathogenic communities of gordonii –36. Ptk1pression , 37    Epithelial cell manipulation. Eukaryotic LMW-PTPs have emerged as signaling hubs in a number of physiological processes and are drivers of carcinogenesis and cancer aggressiveness . Bacteriachinery , 40, 41.achinery , 43, and nucleus . As Ltp1 nucleus . ReducedP. gingivalis, the presence of two phosphatases may allow additional calibration of the Ptk1 BY kinase. P. gingivalis exploits the efficiency and precision of phosphotyrosine dependent signaling to impose layers of control over functions important for in vivo colonization and persistence. Sensitivity of the Ltp1 LMW-PTP to extracellular metabolites creates a sensing system for detection of potential community partners and upregulation of relevant adhesins. Delivery of Ltp1 within gingival epithelial cells, where it can reprogram host signal transduction pathways, has consequences for host cell, tissue and inflammatory homeostasis. The emergence of phosphotyrosine signaling as a key device in the control of numerous cellular functions in P. gingivalis creates access points for novel therapeutics designed to maintain the organism in a state of eubiosis and prevent dysbiotic interactions with the host.Signaling coherence is enhanced when circuitry is tunable and can be insulated from, or interconnected with, other transduction mechanisms. Bacterial protein tyrosine phosphorylation/dephosphorylation illustrates this principle. Extending beyond simple on/off switches, the kinases and phosphatases are structurally geared to cycle between high and low activity states by reciprocating phosphorylation and dephosphorylation reactions. In RL and DM wrote the manuscript and approved the submitted version.We thank the NIH for supporting the original research that is the basis for this review through DE023193, DE012505, DE011111 (RL), and DE028346 (DM). The funding sources had no input in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.