IBV) is prevalent in Pakistan causing enormous economic losses. To date no clear data are available on circulating genotypes and phylogeographic spread of the virus. Hence current study assessed these parameters for all available IBV Pakistani isolates, based on the 9 new sequences, with respect to other Asian and non-Asian countries. Results indicated that all Pakistani isolates belonged to genotype I (GI), with more than half of them (16/27) belonging to the GI-24 lineage, against which no vaccine is available. Three possible introduction events of the GI-13 IBV lineage into Pakistan, based on the estimated IBV population using isolates from this study, were observed possibly from Afghanistan, China, and/or Egypt. These events were further analyzed on the S1 amino acid level which showed unique alterations  in 1 isolate  when compared to GI-1 lineage. Both GI-1 and GI-13 Pakistani strains showed close homology with homologous vaccine strains that are used in Pakistan. For GI-24 strains, none of the used vaccines showed substantial homology, necessitating the need for further exploration of this lineage and vaccine design. In addition, our findings highlight the importance of genomic surveillance to support phylogeographical studies on IBV in genotyping and molecular epidemiology.Infectious bronchitis virus ( IB) is one of the most important infectious diseases of poultry industry due to its contagious nature and significant economic losses (IBV), which is part of the Gammacoronavirus genus and Coronaviridae family. This group of viruses is featured by a positive-sense RNA genome of about 27.6 kb in size  at the 5′ and 3′ ends, along with 2 overlapping open reading frames (ORFs), encoding for the polyproteins 1a and 1ab (S), envelop (E), matrix (M), and nucleocapsid (N) proteins. Also, 2 accessory genes, ORF3 and ORF5, resulting in proteins 3a and 3b and 5a and 5b, respectively, have also been described (HVRs) (RDRP) which lacks proof-reading activity (The IBV genome comprises 2 untranslated regions ( and 1ab . In addiescribed . The S pescribed . The S1 d (HVRs) . This isd (HVRs) . This isactivity . Hence, activity . In geneactivity , making activity . More thactivity  and circactivity . Based oactivity . Commonlactivity . The emeactivity .RT-PCR) and quantitative reverse transcriptase PCR techniques have been developed in last 3 decades for this purpose  and understand the spread of the virus . Reverse purpose . Through purpose . In a de purpose . Another purpose . NeverthBEAST) analysis to assess evolutionary rate, phylogenetic relatedness, and map potential introduction events into Pakistan. Isolation source of the isolates were also analyzed to map possible links between different circulating genotypes.This study is the first to deliver data on the phylogeography of all the reported IBV isolates in Pakistan. Here, existing IBV sequences from Pakistan, neighboring Asian, and some non-Asian countries were supplemented with 9 new Pakistani sequences from IBV field isolates. These were used for genotyping and a Bayesian Evolutionary Analysis Sampling Trees (n = 18) and oviduct (n = 7) from 25 suspected cases of IBV were taken during a disease outbreak period between October 2017 to February 2018 from various districts of Punjab. PureLink RNA Mini Kit  along with TRIzol Reagent  was used for RNA extraction according to company guidelines. Extracted RNA was stored at −80°C.Tissue samples from trachea/lungs  was used for reverse transcription with gene-specific primer (SX2−) according to the company's guidelines. Briefly, incubation was done at 42°C for 60 min, 25°C for 10 min and again at 42°C for 2 h. Finally, mixture was incubated at 70°C for 10 min to inhibit reverse transcriptase. cDNA was stored at −20°C.2; 0.5 μL of 10 mM dNTPs; 2.5 U DreamTaq DNA Polymerase  and nuclease-free water to make final reaction volume 25 μL. For nested PCR, 8 μL of first PCR product was used with SX3+ and SX4− primers; the rest of the ingredients were same. Nuclease-free water was adjusted to make final reaction volume 25 μL. The PCRs were performed using the following conditions: denaturation , annealing (48°C for 1.5 min), extension  for a total of 30 cycles. The expected 393 bp product was purified and used for sequencing.RT-PCR primers and conditions were used as previously described for partial amplification of S1 gene . The seqPurified cDNA samples were sent to Macrogen, Inc. (South Korea) for sequencing along with internal primers SX3+ and SX4−. Sequencing results were used to generate a consensus sequence by Geneious v10.2.5 on which 2 sets of primers were designed  for furtAll sequences were used in a phylogenetic analysis to allow genotyping based on previously published genotyping schemes . All seqn = 27) and sequences from other Asian countries (n = 77). Also, some non-Asian isolates (n = 16) were included in the analysis. A multiple sequence alignment was performed using MAFFT  tree was constructed using IQtree2  measures was observed in Tracer , M41 (X04722), and H52 (AF352315); GI-13: 4/91 (KF377577); GI-17: KM91 (FJ807946)) and other main GI vaccine strains as described by Guzmán and Hidalgo  GI-1: M. These sNine samples  were declared positive by nested RT-PCR and were used for sequencing showing a 393 bp band . These in = 16) belonged to the GI-24 lineage. Interestingly, 1 isolate  was shown to be a GI-1 lineage strain. Among our 9 new sequences, 1 belonged to the GI-24 lineage , while the other 8 belonged to the GI-13 lineage. GI-13 lineage strains are associated with samples primarily isolated from the respiratory and reproductive tracts while GI-24 lineage includes isolates from liver and kidney in addition to the respiratory tract.Our 9 new sequences were subjected to genotyping, using a reduced phylogenetic inference, including most relevant genotypes. As shown in −3 nucleotide substitutions per site per year with a 95% highest posterior density (HPD) interval of . Also, the most recent common ancestor (mrca) was estimated at 1962 . As shown in mcc) tree. By inferring geographical locations on the tree nodes, putative introductions into Pakistan could be traced. This highlighted at least 3 introduction events that took place in Pakistan  showed an N278I mutation. This indicates that IBV is rapidly undergoing genetic alterations in Pakistan which is resulting in the spread of unique genotypic variants like GI-24 which was not reported before.The independent GI-13 lineage introductions of IBV into Pakistan were further confirmed by looking at the amino acid differences within the sequenced data. This showed unique amino acid changes to be present in the protein of the isolate with putative origin from Egypt . Unique alternations were observed as S250H, T270K, and Q298S (as compared to the GI-1 UAF8 Pakistan strain) (n = 1), GI-13 (n = 10), and GI-24 (n = 16) lineages. Key difference between these lineages was recently highlighted by a study on cleavage recognition motifs of S1 gene, with GI-1 and GI-24 having Arg-Arg-Phe-Arg-Arg (RRFRR) while GI-13 having Arg-Arg-Ser-Arg-Arg (RRSRR) motifs  motifs .Mass) vaccine strain and is also widely present in IBV endemic countries  in 2015, more introduction events from China can be expected. This enhances the importance of phylogeographical studies as shown here, which are based on accurate sequencing data and associated metadata. More studies are needed on the genetic characterization of circulating field isolates in Pakistan to get a more accurate picture on the diversity and phylogeographic relationship of IBV strains.Based on the phylogeographic analysis in the BEAST software, estimations on the tree node origins could be made to trace 3 possible introduction events of IBV into Pakistan, 2 of which showed a very high probability (Egypt and China). First introduction event to and from Pakistan has low probability and could be due to the geography of the area. A selection of isolates with sufficient and accurate metadata  was performed. Inclusion of a broader range of isolates from more (neighboring) countries and time points might improve the estimations here. Still, more sequencing efforts are thought to be required in South Asia to facilitate this type of analyses. Migratory birds may play a possible role in this over a long period of time , traveliThis analysis also allowed to estimate the actual IBV population size over time, which highlighted a reduction of the population, which coincided with the 2 GI-13 introduction events in Pakistan. The GI-13 strain likely became the predominant lineage in Pakistan in this period as it is known to be widely circulating across the world . After tIn our study, IBV 17 of GI-24 lineage showed alterations as Q255E, H277T, S282P, E319T, P319T, S330K, and L335P . These differences can result in certain mutations which allow GI-24 lineage strains to evade the vaccines targeted against GI-23 lineage. Other amino acid differences within the sequenced data also highlighted the key changes in the selected S1 protein changes. This information suggests that GI-24 is taking over as dominant lineage in Pakistani field IBV isolates over the last years (2017–2020). As there are no vaccination strains currently being used in this lineage, it can allow further spread. It also opens doors toward manufacturing of live attenuated and killed vaccines based on this lineage in the Indian subcontinent.nsp) 2, 3, and 16 coding regions also showed high variability , with the GI-24 lineage being of particular interest as no closely related vaccine is currently available in Pakistan and in the world. Our study also highlighted a minimum of 3 independent IBV introduction events into Pakistan over the last 5 decades and estimated the circulating IBV population in Pakistan and its neighbors over this period. Amino acid changes in the strains also highlighted the differences between genotypic lineages, showing that the IBV isolates acquired key mutations over the years resulting in spread of novel genotypes not reported before. The results highlight the importance of IBV genotyping and phylogeographic analyses to trace the spread and introductions of IBV lineages, which will also aid in the design of new vaccines and setting up eradication campaigns.


Activation-by-inhibition is a biochemical paradox seldom observed in exosite enzymes, wherein active site-bound inhibitors unexpectedly lead to enzyme activation. This intriguing phenomenon occurs at low, undersaturating substrate concentrations, posing a significant challenge in drug discovery, especially when targeting enzymes such as protein kinases, proteases, and other posttranslational modification enzymes. These enzymes often rely on accessory recognition sites known as exosites, which contribute to complex substrate binding mechanisms and unique kinetic behaviors. This study aims to provide a theoretical kinetic explanation for nonallosteric mechanism-based activation-by-inhibition, shedding light on the complexities of inhibiting exosite enzymes solely through active site targeting. Notably, the dual activator-inhibitor behavior of active site-bound inhibitors manifests in a nonmonotonic biphasic dose—response, emphasizing the importance of understanding the role of the inhibitor concentration at low substrate levels. Our findings underscore the potential widespread occurrence of activation by inhibition, a phenomenon that may have been overlooked in the past, and thus advocate for novel strategies in drug design that consider the impact of exosites on enzyme behavior to effectively target exosite enzymes.  Catalyzed by a diverse array of posttranslational modification enzymes, more than 700 different types of PTMs have been identified that critically contribute to maintaining cellular homeostasis Dysregulation of these enzymes has been implicated in a plethora of pathological conditions, including inflammatory diseases, neurodegenerative and cardiovascular disorders, diabetes and cancer Notably, kinase and protease inhibitors currently account for one-third of all drug discovery research and development efforts. Efforts that, however, are often frustrated by the onset of cardiotoxicity, hypertension, hypothyroidism and other off-target effects that are frequently associated with these inhibitors and that prevent their progress to clinical trials or reduce their therapeutic window Here, we propose an alternative perspective, suggesting that the high failure rate of drug discovery campaigns targeting proteases and kinases may stem, at least in part, from the paradoxical activation of exosite enzymes triggered by active site-directed competitive inhibitors.Enzyme activation-by-inhibition is among the most intriguing and counterintuitive behaviors exhibited by enzymes. The crucial characteristic common to all types of linear reversible inhibition is, by definition, the cessation of catalytic turnover: when an inhibitor binds, it hinders the enzyme-substrate interaction in the case of competitive inhibition, or it halts the progress of the catalytic cycle toward the release of the reaction product in the case of uncompetitive inhibition. Either way, at the single-molecule level, enzyme activity comes to a complete standstill. According to conventional theory, this should inevitably lead to a reduction in the reaction rates observed in the bulk. Surprisingly, however, this is not always the case, and over the last decade, numerous examples of enzymes that are nonallosterically activated by active site-bound inhibitors have emerged, challenging traditional assumptions Attempts to explain the occurrence of this activation-by-inhibition have been made, but they have exclusively revolved around the formal mechanism of mixed inhibition Interestingly, enzymes that have been reported to undergo activation-by-inhibition all share a common feature: they possess either multiple substrate binding subsites, as observed in β-glucosidases Enzymes that catalyze modifications of large macromolecular substrates, such as endonucleases The study of serine proteases involved in the coagulation cascade provides a compelling example of the interplay between active sites and exosites during substrate recognition. This interaction leads to distinct kinetic behaviors, as illustrated by the cases of active site-bound inhibitors of prothrombinase and intrinsic Xase, resulting in mixed-type inhibition instead of the expected competitive inhibition Building upon this intriguing observation, we propose a kinetic mechanism that offers a general rationale for the activation of exosite enzymes by active site-bound inhibitors and that might help to explain the occurrence of unexpected side effects caused by inhibitors targeting exosite-containing proteases, protein kinases and other PTM enzymes.22.1While the actual mechanism of substrate recognition by exosite enzymes can be multifaceted Based on the idea that enzymes exist as populations of random-fluctuating conformers Km), this alternative path can paradoxically result in enzyme activation , the binding of an inhibitor to the active site of an exosite enzyme results in an inhibition of the mixed or noncompetitive type Keq) is set to 0 (which implies that the free enzyme exists solely as the E conformer), the steady-state analysis of an inhibitor with a Kd of 1 µM gives an observed competitive inhibition constant (Kic) of 104 µM. Thus, a first notable singularity of exosite enzymes is that they tend to neutralize the inhibitory effect of active site-directed ligands, turning, as in this case, relatively strong binders into poor inhibitors.For the enzyme modeled in our theoretical example, if the E-F equilibrium constant  and the substrate concentration is below saturation, the inhibition pattern becomes tangled up by the occurrence of activation-by-inhibition (c). This phenomenon occurs because the free enzyme E is now engaged in a triple equilibrium, and the binding of the inhibitor recruits enzyme molecules not only from the E pool through the E↔EI and SE↔E↔EI equilibriums, as in the previous case but also from the F pool through the F↔E↔EI equilibrium.However, when an equilibrium is established between conformers E and F  is lower than koff, activation-by-inhibition remains feasible, provided that [S] remains sufficiently low. The extent of maximum activation observed at a given [S] increases with greater Keq and a higher k-1(SEI)/ koff ratio.While an analytical description of this mechanism would be overly complex and of limited practical utility, it is worth noting that, under the assumption of active and exosite independence, the inhibition-to-activation transition critically requires the presence of an E↔F equilibrium. Moreover, the onset of activation is contingent on both the dissociation rate of the inhibitor (EI→E + I) and the dissociation of S from the ternary SEI complex (SEI→EI+S). It is indeed the fate of SEI that governs the possibility of activation-by-inhibition. Hence, irrespective of the EI dissociation rate, as long as 2.2a. Under these circumstances, the binding of I only leads to inhibition, and no activation can occur . In this case, it is the binding of S that has no impact on the E↔F equilibrium, resulting in a sort of noncompetitive-like inhibition that causes a significant increase of the apparent Km. Unlike the scenario described in section 3.1, in this case even at high [S], the substrate is unable to shift the E↔F equilibrium toward E, causing the E + I→EI binding to exert a quasi-constitutive enzyme activation that is observed even at [S]≫Km . Here, Keq was again set to 1. Because of the competition between the E↔F and E↔SE steps, if Keq is allowed to assume larger values, a sharp activation-by-inhibition anomaly occurs even if the Km of the F conformer is only slightly higher than that of E. This example demonstrates that activation-by-inhibition can arise whenever different forms of the free exosite enzyme are in thermodynamic equilibrium and possess varying affinities for the substrate. Therefore, this mechanism holds general validity and extends its applicability beyond the specific assumption made in the simplified model.To simplify the description of the activation model, we have assumed that the F conformer is completely inactive. However, it is important to recognize that such a strict requirement is not necessary for activation-by-inhibition to take place. 2.4Kd range or higher and leads to the paradoxical increase in apparent substrate affinity.In operational terms, the key distinction between allosteric and nonallosteric activation-by-inhibition lies in the substrate and inhibitor concentrations needed to trigger the activation process. In cases driven by allostery, such as the protease ClpP, the heat shock protease DegP or the human protease HTRA1, the binding of the inhibitor triggers structural modifications that alter the kinetic properties of the enzyme The literature offers several instances of enzymes whose activation-by-inhibition conforms to our nonallosteric model. Among the four most prominent examples, we can list the inhibitor-induced activation of NAD-dependent histone deacetylase sirtuins γ-Secretase is a transmembrane protein complex comprised of four essential components: NCT, APH-1, PEN-2, and the catalytic subunit presenilin. This enzyme belongs to a family of intramembrane-cleaving proteases responsible for controlled proteolysis of transmembrane peptides and generation of biologically active signaling fragments. Notably, γ-secretase plays a critical role in the pathogenesis of Alzheimer’s disease through its implication in the processing of the β-amyloid precursor protein (APP). The initial step in this process involves the extracellular cleavage of APP by BACE, resulting in the generation of a C-terminal 99-residue fragment known as C99-APP. Subsequently, γ-secretase further processes C99-APP, yielding various lengths of Aβ peptides. The accumulation of these Aβ peptides is closely associated with the formation of β-amyloid plaques, a hallmark feature of Alzheimer's disease. Over the past three decades, extensive endeavors have been dedicated to the design of γ-secretase inhibitors aimed at the development of medications that could alter the progression of Alzheimer's disease. Regrettably, these initiatives have resulted in repeated failures, as illustrated by the notable examples of presenelin inhibitors semagacestat and avagacestat, whose clinical trials were prematurely halted due to their association with accelerated cognitive decline and exacerbation of Alzheimer's symptoms In accordance with these findings, numerous studies have consistently documented that γ-secretase inhibitors can elevate Aβ production. This Aβ-rise, observed in rat brain, cell-based assays and experiments with purified solubilized enzyme, has been demonstrated to manifest only at low enough concentrations of the C99-APP substrate Deeper insights drawn from the structural and mechanistic characteristics of γ-secretase further underscore the parallels between our activation-by-inhibition model and this specific case. For instance, recognition between γ-secretase and C99-APP involves an exosite located between the NCT and PEN-2 subunits. The substrate is then translocated to the active site via an intermediate second exosite situated between the NCT and PS subunits 3The activity of a prototypical exosite enzyme, adhering to the mechanism expounded in the Results k1= 107 mol−1s−1, k-1= 10 s−1, k2= 103 mol−1s−1, k-2= 1 s−1, kcat= 100 s−1, which gives Km= 10 µM. kon= 109 mol−1s−1, koff= 103 s−1, corresponding to a Kd of 1 µM. The equilibrium constant Keq is defined as the ratio between the forward E→F conversion and the backward E←F conversion (kfwd= kbkwd=103 s−1).The enzyme concentration was 100 pM, the substrate concentrations were between 0.1 and 100 µM, and the inhibitor concentrations were between 0 and 100 µM.Km) and inhibitor concentrations (expressed as [I]/Kd).The computed initial reaction velocities have been reported as a function of substrate concentrations  into the catalytic complex (ES), making the SE→ES step kinetically irrelevant. Moreover, allosteric cross-talk between the active site and exosite can either accentuate or abolish the onset of activation. In some circumstances, the physiologic substrate concentration might fall within or exceed the Km and [I]/Ki combinations on PTM enzyme activity can enhance our comprehension of the conditions needed for effective inhibition, both in vitro and in vivo, of vital pharmacological targets. These insights may contribute to optimizing resources in drug research, preventing wasteful efforts on hits and lead compounds that may never progress to approved medications.Nevertheless, this study illuminates unexplored potential in enzyme behavior and suggests that should this activation mechanism be actively searched for, the prevalence of PTM enzymes that are activated by inhibitors might significantly increase. In addition, our theoretical model offers a conceptual framework that can assist in making sense of seemingly paradoxical evidence and may stimulate targeted research endeavors. Investigating a wide range of [S]/As the only author of the present paper, Alessandro Pesaresi has taken care of all aspects of this work.Authors declare no conflict of interests.


Large-bore arteriotomies can be percutaneously closed with suture-based or plug-based vascular closure device (VCD) strategies. The efficacy of both techniques remains controversial.We conducted a meta-analysis of comparative studies between both VCD strategies, focusing on the most commonly applied VCDs (MANTA and ProGlide).We searched MEDLINE, the Cochrane Central Register of Controlled Trials and Google scholar for observational studies (OS) and randomized controlled trials (RCT) comparing vascular closure with the MANTA-based and the ProGlide-based technique. The principal endpoint of this analysis was access-site related vascular complications. Both study types were analyzed separately.p = 0.01, I2 = 0%), but more frequent in the analysis of RCT data . Both data sets provided no significant difference between the VCD techniques in terms of overall bleeding events . RCT data showed that endovascular stenting or vascular surgery due to VCD failure occurred more often after MANTA application .Access-site related vascular complications were less frequent after vascular closure with the MANTA technique in the analysis of OS (RR 0.61 [95%CI 0.43–0.89], While OS point to favorable outcomes for large-bore vascular closure with the MANTA-based technique, RCT data show that this strategy is associated with more access-site related vascular complications as well as endovascular stenting or vascular surgery due to device failure compared with the ProGlide-based technique.The online version contains supplementary material available at 10.1007/s00392-022-02145-5. Suture-based vascular closure devices (VCDs) have been the sole option for non-surgical percutaneous large-bore arterial access-site closure for several years. The Perclose ProGlide VCD  is most commonly used, and appears to be superior to other suture-based techniques , 2. It iThe MANTA VCD  is the most studied of the recently introduced newer generation large-bore VCDs. It relies on access-site closure using a collagen plug, an approach that is similar to smaller plug-based VCDs . A growiConsequently, meta-analyses comparing both VCDs have been recently performed –18, but We searched PubMed, Google Scholar, and the Cochrane Central Register of Controlled Trials for reports published between January 1, 2014, and November 1, 2021. We set the start of the search period two years before the CE certification of the MANTA VCD. Three term groups were used, of which at least one term of each group was required to match: (1) vascular closure; vascular closure device; vascular; large-bore arteriotomy; large bore arteriotomy; large-bore arteriotomies; large bore arteriotomies; percutaneous closure; percutaneous AND (2) MANTA; plug-based; plug based; plug AND (3) ProGlide; Perclose; suture-based; suture based; suture.We included peer-reviewed RCTs as well as peer-reviewed OS comparing the plug-based MANTA and the suture-based ProGlide vascular closure techniques after large-bore vascular access. Only studies reporting vascular complications according to the Valve Academic Research Consortium (VARC)-2 criteria were included . StudiesTwo independent investigators  performed the literature search using the previously defined search terms. Studies that did not meet the eligibility criteria were excluded. Discrepancies were resolved by consensus after discussion. Data extraction from available full-text articles was also carried out independently by two investigators (OD and DO) using piloted spreadsheets. Once again, discrepancies were resolved by consensus after discussion. In addition, unpublished data from the CHOICE-CLOSURE trial  and the The principal endpoint of this analysis is access-site related vascular complications defined according to VARC-2. OS only partially differentiated between access-site related and non-access-site related vascular complications. Therefore, we also assessed the occurrence of overall vascular complications in this data set.Secondary endpoints included all-cause mortality, overall-, life-threatening or major and minor bleeding events (VARC-2), VCD failure (VARC-2), as well as endovascular stenting and vascular surgery due to VCD failure.As the MASH trial did not provide information on the distribution of life-threatening, major and minor bleeding events, only the rates of overall and access-site related bleeding events were reported in the RCT group. The two RCTs used different definitions of VCD failure. Therefore, we only matched the need for endovascular stenting and vascular surgery due to VCD failure. In addition, mortality data in the MASH trial have not been reported. A detailed list of all outcome definitions, inclusion and exclusion criteria for each study is provided in supplementary tables 3 and 4.Q statistic and Higgins and Thompsons I2 were calculated to assess heterogeneity. We used R (version 4.1.2) and its package meta (version 5.1-1) for all statistical analyses.OS and RCTs were analyzed separately. For the principal and secondary outcomes, risk ratios (RR) were calculated based on the number of events and the number of patients per group. These study-level results were pooled by means of a random effects meta-analysis using the Mantel–Haenszel method as primary analysis. Between-study variance was estimated using the Paule-Mandel estimator. The result of a fixed-effect meta-analysis is reported in addition. In case of evidence for a small-study effect and a more conservative result of the fixed-effect meta-analysis as compared to the random effects meta-analysis, primary interpretation was based on the result of the fixed-effect meta-analysis. Cochran’s n = 587) and ProGlide (n = 819) techniques after TAVI   were significantly less frequent after application of the MANTA technique. The individual events access-site related major  and access-site related minor vascular complications  did not differ significantly, as well as overall major  and minor vascular complications   and endovascular stenting or surgery due to device failure  between both techniques. The analysis of overall bleeding events  and the subtypes of life-threatening or major bleeding  and minor bleeding  showed a trend but no significant difference between the plug-based and the suture-based techniques. There was also no significant difference in mortality between both VCD strategies  .In the OS group, overall access-site related vascular complications  , but not that of major access-site related vascular complications . Endovascular stenting or vascular surgery due to VCD failure had to be performed more frequently after MANTA VCD application . No significant difference was observed between the two techniques in terms of overall bleeding  and access-site related bleeding   Overall access-site related vascular complications appear to be more common in the MANTA cohort of the RCTs; (2) patients in the MANTA cohort of RCTs were more likely to undergo vascular stenting or vascular surgery due to VCD failure; (3) in contrast to the RCT data, the results of the OS meta-analysis show better outcomes with the MANTA VCD in terms of overall access-site related vascular complications.In contrast to our analysis, none of the previously published meta-analyses could find a difference between the two VCDs in terms of vascular complications –18. MostDifferent event rates were observed in both RCTs and OS, particularly with regard to vascular complications. Presumably, these differences are a consequence of different assessment, adjudication and follow-up strategies. An important example is the structured ultrasound follow-up in the CHOICE-CLOSURE trial. Compared with clinical follow-up alone, routine ultrasound may be able to diagnose vascular complications more accurately, particularly minor ones.Plug-based vascular closure is a commonly applied technique for small-sized arterial access and is associated with favorable outcome , 25. TheAnother procedural advantage of the ProGlide technique is the fact that wire access to the vessel is maintained during vascular closure. This enables the operator to apply additional VCDs even if the device does not achieve full hemostasis. In contrast, once the plug-based VCD fails, there are only a few bail-out options such as endovascular stenting or vascular surgery. Nevertheless, the MANTA VCD has already been successfully used in several cases as a bail-out device after ProGlide failure , 15, 30,Plug-based VCDs could represent an additional option for other procedures requiring large-bore vascular closure such as thoracic endovascular aortic repair (TEVAR), EVAR and va-ECMO. Initial registry studies and case reports have already shown that plug-based VCDs could be a safe alternative to the ProGlide VCD –33. HoweThe results of our study reinforce the importance of adequately powered RCTs for the evaluation of new procedures and devices. Even OS with a large number of included patients Below is the link to the electronic supplementary material.


Moisture condensation, fogging, and frost or ice formationon structuralsurfaces cause severe hazards in many industrial components such asaircraft wings, electric power lines, and wind-turbine blades. Surface-acoustic-wave(SAW) technology, which is based on generating and monitoring acousticwaves propagating along structural surfaces, is one of the most promisingtechniques for monitoring, predicting, and also eliminating thesehazards occurring on these surfaces in a cold environment. Monitoringcondensation and frost/ice formation using SAW devices is challengingin practical scenarios including sleet, snow, cold rain, strong wind,and low pressure, and such a detection in various ambient conditionscan be complex and requires consideration of various key influencingfactors. Herein, the influences of various individual factors suchas temperature, humidity, and water vapor pressure, as well as combinedor multienvironmental dynamic factors, are investigated, all of whichlead to either adsorption of water molecules, condensation, and/orfrost/ice in a cold environment on the SAW devices. The influencesof these parameters on the frequency shifts of the resonant SAW devicesare systematically analyzed. Complemented with experimental studiesand data from the literature, relationships among the frequency shiftsand changes of temperature and other key factors influencing the dynamicphase transitions of water vapor on SAW devices are investigated toprovide important guidance for icing detection and monitoring. However,sensing, monitoring, and/or removal of these hazardous formationsin a cold environment are challenging due to the interplay of manyenvironmental factors including temperature, RH, pressure, etc., whichinfluence the phase transitions of water .11 Frost or ice formation rates are also significantly increased withan increase of the RH values in air and affected by atmospheric pressure,wind speed, cold rains, and ice formation at different temperatures.13Cold environments, especiallythose under high relative humidity(RH) levels, can lead to moisture condensation and the formation offrost, snow, or ice on the structural surfaces, imposing severe safetyissues for aircraft wings,16 Most techniques for monitoring ice/frost formationare based on in situ measurements of parameter changes for the propertiesof a specific sensor due to mass loading and changes of the dielectricconstants, capacitance, inductance, optical properties, or vibrantfrequencies .These methods all have their merits and limitations,32 as presented in Various techniques have been developed to monitor moisturecondensationand the formation of frost, snow, and ice, among which ice monitoringis the most studied.34 Another key advantage of these techniquesis that they can also be used as active deicing or antiicing methods.For example, recent studies clearly show that, for ice mitigation,surface-acoustic-wave (SAW) devices can generate both acoustic wavevibrations and thermal effects on the device surface,36 thus offering great potential for both antiicing and deicing witha reasonably high efficiency.40 Therefore, it could be applied as one of the appropriatetechniques for effectively tackling icing issues on structural surfaces.However, the conventionally used bulk piezoelectric ceramic-basedultrasonic or SAW devices have critical issues such as brittlenessof the piezoelectric substrates , rigidity, and noncompatibilitywith structural surfaces or microelectrics-based mass production technologies.On the contrary, thin-film-based SAWs have the advantage that theycan integrate multiple functions into a single structure on differentsubstrates, such as silicon, metals, glass, or polymers,16 and also flexible and wearable ones.41 This provides one of the best solutions to integratingthin-film SAW devices directly onto structural component surfaces for both monitoringand mitigation functions.As seen from 3945 the hybrid effects of multifactors in complex cold environments have neverbeen systematically studied in the literature.Various phenomena such as moisturecondensation and water, fog,frost, snow, and ice formation will cause different changes in thephysical or electrical properties  of the SAW devices.v of the SAWs induce changes in the resonantfrequency f, i.e., f = v/λ, where λ is the SAW wavelength. In the cold environment,frequency shifts of the SAW device could be affected by a combinationof temperature changes, electrical effects , and mass loading effects (generated by the adsorption ofwater molecules or coverage of frost/ice at various temperatures)as well as changes in the RH values  of SAW sensorsis affected by their physical, electrical, or electromagnetic propertiesdue to mass loading, electrical loading, temperature, and/or elasticloading,64 as revealed from 39f0 is thecentral frequency of the SAW device, v is the phasevelocity, Δm is the change in mass, Δσis the change in conductivity, ΔT is the changein temperature, Δc is the change in mechanicalconstant, Δε is the change in the dielectric constant,ΔP is the change in pressure, Δηis the change in viscosity, and Δρ is the change in density.The frequencyshift  and accessibility of saturation water vapor, the water vapor willbe condensed into a water droplet or a layer of fog, causing a significantmass loading effect. As the temperature continues to drop , the saturation water vapor or fog condensed from the saturationwater vapor are then changed into ice/frost on the surface of theSAW device, as shown in In a cold environment above the freezing temperature of the water,apart from the temperature change of ΔConsidering the complex influences of various parametersin a coldenvironment on the SAW device, in the following sections, we willfirst analyze each single factor and then discuss the influences ofthe combined or integrated factors using multivariate logistic regressionanalysis.2.142T, f, v, λ, and α are the temperature, frequency,phase velocity, designed wavelength, and temperature expansion coefficientof each material of the multilayer structure. The change of frequency(ΔfT) due to changesof temperature can be simply calculated using In a coldand dry environment, if only the temperature has been changed, thetemperature coefficient of frequency (TCF) is often applied to showthe rate of frequency changes versus temperature relative to the originalresonant frequency of the SAW device. TCF is defined using 2.250 For example, when a layer of material is depositedonto a substrate that is vibrating synchronously with the waves, thechanges of the average kinetic energy per area of the wave, ΔUK, can be described asvx0, vy0, and vz0 are the SAW particle velocitiesat the surface, f0 is the central frequency, v0 and U0 denotethe unperturbed wave propagation velocity and energy density, w is the angular frequency w = 2πf0, ρs is the density of theperturbation film, and P is the power density ofthe SAW device.A mass loadingon the SAW device’s surface arising from perturbations, suchas the formation of a surface layer or adsorption of foreign objects,will change the wave propagation velocity, which is fundamentallyrelated to the change in the wave energy density. A fundamental relationshipbetween the wave velocity and energy density for the SAW device excitedat a given frequency is equal to the negative fractional change in the wave energy density.52f0 is thecentral frequency, k1 and k2 are material constants of the SAW substrate  of the ice, f. Therefore, thechanges in the SAW frequency arising from the ice layer perturbationcan be calculated using f is a function of K and G of the ice. It should be emphasized that the bulk andshear moduli of ice changes with the density of ice and ambient temperature.53However, it should be addressedthat, in a cold environment, themass loading effects arising from the adsorption of water molecules(or a fog layer formation) and the ice frozen on the surface of theSAW device are dramatically different, as illustrated in 2.354When an acoustic wave propagates in the piezoelectric material, itgenerates a layer of bound charges at the surface that arises fromthe generated strains. This induces an electrical polarization inthe surface-normal direction of the crystal, accompanied by the propagatingmechanical waves. Therefore, surface charges are sinusoidally variedwith the charge density accompanying the mechanical waves, as shownin 56 on the surface of theSAW device. There are transfer phenomena of protons among water moleculesthat produces H3O + and OH– under the applied electrical fields, as shown in formula  and surface conductivity (σs) of the material can be described using 58K02 is the electromechanical coupling coefficient, Cs = ε0 + εs, where ε0 and εs are the dielectric constants of airand the substrate, and σs = hρ,where ρ is the resistivity and h is the thicknessof the material. In the literature, there are reports that the electricalloading effect was caused by the adsorption of water molecules fornonspecific coatings such as Al2O3 nanotubes59 and nanocrystalline barium titanate,60 which influenced the phase and insertion lossof the SAW sensors.50The hydrogen ions (protons) are transferredfrom one water moleculeto another, thereby leading to changes of the electrical resistances.2.455 When the saturated air comes in contact with a cold surface whosetemperature is less than the dew-point temperature  or the freezing temperatureof water (0 °C or 273 K), the water vapor can be directly changedfrom a gaseous state to a solid state. It is well-known that the relationshipbetween the temperature and saturation water vapor pressure is describedby the following equation:61Pr is thereduced vapor pressure, PV (Pa) is thesaturated pressure at temperature TA (°C), TA is the absolute temperature, PC (Pa) is the critical saturated pressure, Trp is the reduced temperature, TC is the critical temperature, TP is the vapor temperature corresponding to a pressure of zero, and a0 and a1 are theparameters that are determined from the experimental data.62 For water, TC =647.14 K, PC = 21898.54 kPa, TP = 52.6 K, a0 = −3.506674,and a1 = 0.456054.In a coldenvironment, the phase transition of water vapor is related to thetemperature and vapor pressure. When the saturated air is cooled,the moisture is released in the form of fog.63PA (Pa)denotes the actual water vapor pressure value that is used to characterizethe atmospheric water vapor content.The conditionsof a cold environment, such as water vapor, fog, and frost or ice,can be modulated by the ambient temperature and vapor pressure, whereasthe vapor pressure is linked to the RH. RH is the ratio of the actualwater vapor pressure to the saturation water vapor pressure at theprevailing temperature, calculated using the following equation:m/A can be related to the RH value usingthe water absorption constant β, which is the mass of the adsorbedwater molecules per unit RH and per unit area, typically expressedin units of kg/m2.For the resonant frequencyshifts of the SAW device, ΔT is the change of the temperature and Td is the dew-point temperature. Combining Combining 33.12 flow ratio of 10/13 sccm, and a gas pressure of 6 ×10–4 mbar. The distance between the target and substratewas 100 mm, and the substrate was rotated during deposition to improvethe uniformity of the deposited film without intentional substrateheating.In thisstudy, we selected ZnO thin-film SAW devices as an example for demonstrationsfor ice monitoring. The SAW devices were fabricated by depositinga piezoelectric ZnO film (∼3.5 μm thick) on aluminumplates (1.5 mm thick) and silicon and glass wafer substrates (both500 μm thick) using radio-frequency magnetron reactive sputtering.During the deposition process, a zinc target with 99.99% purity wassputtered with a direct-current target power of 420 W, an Ar/OFigure S1. A network analyzer (Agilent E5061B) was used tocharacterize the reflection (S11) spectra of the SAW devices, withone example shown in Figure S2.The interdigitated transducers (IDTs) of the SAW deviceswere fabricated by using a conventional photolithography and lift-offprocess, with a bilayer of 20 nm Cr and 100 nm Au. SAW devices withdifferent IDT wavelengths  wereprepared and used in different experiments .First the frequency changes of SAW devices 1–4 were recordedby cooling them from +25 to −20 °C in an open environmentwithout controlling the humidity, the method of which was previouslyintroduced in ref . In the To monitor the frequency shiftsof ZnO thin-film SAW devices in a controlled laboratory environment,a cold chamber  with adjustabletemperatures via the current input and precise humidity control wasused 6. TheZnTo evaluate the temperature effects of the ZnOthin-film SAW devices(devices 1–4 were chosen) with a minimized humidity effect, the cold chamberwas cooled from temperature ∼80 ± 0.3 °C (a dry andhot environment) to a set low temperature of ∼−20 ±0.1 °C (a dry and cold environment) and then gradually increasedfrom −20 to +80 °C at a rate of 10 °C/min. In thistest, both the frequency and temperature were recorded simultaneously.To evaluate the humidity effects on the ZnO thin-film SAW devices,we conducted experiments about the humidity effects in the cold chamberby performing tests using device 2 at different RH values from 10%to 90% but at four different temperatures, e.g., −4 °C(a wet and cold environment) and 0, 4, and 10 °C (a wet and warmenvironment), respectively. The RH values were controlled by flowingthe chamber with different ratios of humid air and nitrogen carriergas. Five readings of the frequency values were in situ recorded afterthe SAW device was kept in a stable humidity value for ∼20min, and the average values were taken.f) of the SAW device asa function of both the temperature and RH were also performed. BothSAW devices 1 and 4 were kept in the environmental chamber with ahumidity of ∼0% by cooling the temperature from +30 to −45°C at a rate of 3 °C/min and then maintained at −45°C for ∼1 min. The chamber was then opened, and the ambientair (which had a room temperature of 20 °C and a humidity of65%) was introduced to the chamber for 1 min. The temperature of thecontrolled chamber was then naturally increased to 25 °C whenthe door was opened. During all of these procedures, the frequenciesof the SAW devices were continuously recorded.To evaluate a combinedeffect of humidity and temperature on theZnO thin-film SAW devices, we first examined the relationship betweenthe temperature and RH values in the enclosed cold chamber to reduceany analysis errors of the independent factors  and then studied their combined influences on the frequencyshifts of the SAW devices. In the test, the starting RH value wasadjusted to 60% and 80%, and the starting temperature was adjustedto +60 °C  and −10 °C. Then the RH values were recordedwith the temperature decreased at a rate of 3 °C/min. The dynamicfrequency shifts (Δf) of SAWdevices with varied ice thicknesses and ice types  were monitored. For the ice thickness tests, SAW device1 was cooled along with the chamber at RH = 100%  and different temperatures of −10, −20,−30, and −40 °C. The thickness of rime ice 38 was estimated byweighing the mass of the device before and immediately after the icingprocess at 1 min intervals. To do different ice type tests, SAW devices5 and 6 were cooled in the chamber with RH = 80% and the temperaturewas set at −10 °C, and then the glaze ice 38 was formed by freezing a deionized water dropleton the SAW devices’ surface within a poly(dimethylsiloxane)chamber  and further frozenfor 10 min to increase the adhesion process. In these tests, the frequencyreadings were recorded continuously . The TCF values were calculated basedon these linear lines, and the results are summarized in To study the real principle for this phenomenon, thechanges ofthe frequencies for SAW device 4 as a function of the temperatureduring the cooling and healing stages with a controlled RH value ofnearly 0%  were measured. The obtained frequency–temperaturecurve is shown in 4.2PV) of the watervapor decreases as the temperature decreases.The experimental data thus far indicate that combinedeffects of the temperature and RH have strong influences on the frequencychanges of the SAW devices. PA) is 60%, the decrease of the temperaturefrom T = −10 to −22 °C (ΔT = 12 °C) results in a decrease of the saturated vaporpressure (PV), and then the RH (RH = PA/PV) is increasedto full saturation (RH = 100%). As shown in PA) is 60%, as the temperature is decreasedfrom T = −10 to −22 °C (ΔT = 12 °C) in a cold environment, the RH value is increasedto full saturation (RH = 100%). In order to achieve full saturationfor an initial PA of 80%, the temperatureonly needs to drop by 7 °C from T = −10to −17 °C. However, in a warm environment , when the initial PA is 80%, in order to achieve full saturation, the temperatureneeds to decrease from T = 60 to 47 °C, thatis, requiring a temperature reduction of 13 °C. In the warm environmentwith an initial PA of 60%, the temperatureneeds to be reduced by 22 °C (from T = 60 to38 °C) for the RH to reach saturation (RH = 100%).Figure S6a, no apparent ice formation was observedon the surface of devices. However, during the heating stage, whenambient air (20 °C and RH of 65%) was suddenly introduced intothe controlled chamber and the chamber temperature was quickly increasedfrom −45 to 0 °C, frost began to form on the SAW devices’surfaces . Thethickness of this frost layer was gradually increased as the freezingtemperature was continuously increased in the cold environment . With frost formed on surfaces and a continuous increaseof the frost thickness, the frequency of the SAW devices was foundto decrease significantly with increases in temperature .This dramatically changes for the frequency curves of the SAW devicesand could be explored to monitor evolutions between frost/ice andmoisture formation. After the surfaces were gradually dried, the resonantfrequencies of both devices 1 and 4 were decreased linearly with anincrease of the temperature.4.3Partsa and b of 5T = 0 (the temperature was maintained constantat 6 °C), the surface conductivity (σs) ofthe adsorption of water molecules can be written as σs = hρ = (βRH)ρ. The influenceof humidity on the frequency of the SAW device can be obtained using k1 = −5.47× 10–8 m2·s/kg, k2 = −2.06 × 10–8 m2·s/kg, f0 = 14.25 MHz, K02 = 1.2%, ρ = 1900 Ω/m, v0 = 2649 m/s, and Cs = ε0 + εs = 8.5 + 1 = 9.5 Pf/cm.66 Based on the above formula  is fixed, the saturated vapor pressure (PV) will be decreased with a decrease of the temperature.However, because the water vapor content remains unchanged, the RHwill be increased with a decrease of the temperature. When the temperaturedrops below the dew point (TA ≤ T) and the supersaturated condition of water vapor (RH ≥100%) is reached, the water vapor is condensed quickly onto the surfacesof SAW devices. The mass loading effect becomes more pronounced forchanging the frequencies of the SAW devices. When the temperatureis continuously decreased (TA ≤ T), more water vapor in the air is condensed and frozenas frost or ice, and thus the frequency will be decreased even faster.From the results of the dynamic experiments10, tempeK and G) of ice cannotbe precisely obtained, the change trend cannot be analyzed quantitatively.The frequency shifts of SAW devices in the wet and cold environmentsdue to the coverage of frost/ice are due to the results of the jointeffects of temperature (ΔT) and RH (ΔRH).As the external warm and wet air is slowly introduced in the coldchamber, the temperature is gradually increased (TA < 0 °C). The changes of ΔT cause changes of the device’s frequency. Simultaneously,due to the mass loading effect, the mass change Δm is also continuously increased. The absolute value of frequencyΔf increases with an increase of ΔT and also with the mass loading Δm; therefore, the frequency of the SAW device shows a substantiallydecreasing trend. As the temperature is continuously increased (TA ≥ 0 °C), frost/ice begins to meltand evaporate, and the frequency of the SAW device is gradually increaseddue to the significantly reduced mass loading effect. Once all wateror vapor on the SAW device is removed, a linear trend will be seendue to the dominant temperature effect (ΔT).Qualitative analysis of the theoretical model is consistent with thecurve trend fitted with the experimental data, as shown in m) due to an increasein the ice thickness are superimposed with the nonlinear changes inthe frequency caused by the changes of the f term due to the bulk and shear moduli(K and G) of the ice, the finalfrequency changes of the SAW devices are decreased nonlinearly withthe ice thickness. Because the density of the glaze ice (exceeding900 kg/m3) is much higher than that of the rime ice (200–600kg/m3),68 the mass loading effect due tothe glaze ice at the same thickness is much more significant thanthat for the rime ice. The rime ice is in loose contact with the surfaceof the SAW device, and the ice particles cannot effectively vibratealong with the SAW device. A positive frequency shift effect for theSAW device during the initial stage of frost and ice formation willweaken the mass loading effect for SAW devices. Therefore, the frequencyof SAW devices decreases slowly at the beginning stage, and then thisdecrease rate becomes much larger due to mass accumulation and compactionof the ice. However, for the glaze ice, the bonding between the glazeice and the substrate is stronger, so the interface can be consideredto be a rigid contact. Thus, the bulk and shear moduli (K and G) of the ice become more important, whichcan cause large strains and nonuniform displacement across the glazeice/substrate to consume the energy of SAW transmission and reducethe propagation speed.69 Therefore, therigid contact and mass loading effect work together and result inthe frequency of the SAW device decreasing sharply at the beginningstage and then much less sharply later for glaze ice.From theresults of the ice thickness and ice type experiments,the frequency changes of SAW devices are sensitive to the ice typesand ice thickness. The frequency decreases nonlinearly with increasingice thickness . Accordingto the theoretical model i.e., 16, becau6TA ≤ T), water molecules precipitate to form fogwater, which intensifiesthe decreasing trend of the SAW frequency. Compared with those forthe rime ice, the effect of the bulk and shear moduli (K and G) of the glaze ice on the shift of the SAWfrequency is more significant at the initial stage of formation. Moreimportantly, the frequency changes of SAW devices are sensitive tothe ice types and ice thicknesses, which can be used to monitor differentice types and ice thicknesses. Therefore, all of the theoretical andexperimental results in this study lay a solid foundation for developinghigh-precision SAW icing detection sensors.In this study, we systematicallystudied the resonant frequencychanges based on the adsorption of water molecules, moisture condensation,and frost/ice formation by considering the temperature, electricalloading, and mass loading effects for SAW devices in cold environments.Experiments were then carried out to study the influences of the temperatureand/or humidity as well as their hybrid effects on the evolution ofthe frequencies of SAW devices. The mechanisms and influential processesof the phase transition caused by water vapor in dynamic environmentsof dry and cold, dry and hot, warm and wet, and cold and wet on thefrequency of SAW devices were systematically investigated. The theoreticalmodels that qualitatively predict the production of phenomena suchas moisture condensation and formation of fog, frost, and ice in acold environment are obtained. Both the experimental results and theoreticalanalysis demonstrated that the absorption of water molecules is themain reason for the nonlinear changes of the SAW frequency with thetemperature at room temperature. As the temperature drops below thedew point (


DDT26 demonstrated significant anti-proliferative activity against both TNBC cell lines and MCF-7 cells. Intriguingly, the phthalazinone moiety of DDT26 mimicked the PAPR1 substrate, resulting in DDT26 displaying a moderate inhibitory effect on PARP1 with an IC50 value of 4.289 ± 1.807 μM. Further, DDT26 was shown to modulate the expression of c-MYC and γ-H2AX, induce DNA damage, inhibit cell migration and colony formation, and arrest the cell cycle at the G1 phase in MCF-7 cells. Our findings present potential lead compounds for the development of potent anti-breast cancer agents targeting BRD4.BRD4 inhibitors have demonstrated promising potential in cancer therapy. However, their therapeutic efficacy in breast cancer varies depending on the breast cancer subtype, particularly in the treatment of TNBC. In this study, we designed and synthesized 94 derivatives of 4-benzyl)phthalazin-1(2H)-one to evaluate their inhibitory activities against BRD4. Notably, compound DDT26 exhibited the most potent inhibitory effect on BRD4, with an IC In 2022, breast cancer remained a significant health concern, with China and the United States reporting 429,105 and 259,827 new cases, respectively . In bothBromodomain-containing protein 4 (BRD4) possesses two bromodomains at its N-terminal, enabling it to bind acetylated histones and non-histones, thereby influencing gene transcription, cell cycle regulation, and apoptosis . As a trAs illustrated in Given the multifaceted roles of BRD4, combinations of BRD4 inhibitors with other drugs have showcased advantages, including potent therapeutic effects, wide safety index and reduced susceptibility to drug resistance in cancer treatments . Dual-ta7, which was proven to be a modest BRD4 inhibitor with an IC50 value of 0.544 μM against the BD1 domain of BRD4 benzyl)phthalazin-1(2H)-one scaffold. We further investigated their anti-breast cancer efficacies and underlying mechanisms. Our findings offer valuable insights for drug research and development aimed at breast cancer.In 2013, Hewings’s group designed and synthesized compound  of BRD4 . After a7 and BRD4 through molecular docking revealed several key interactions. As shown in 7 engaged in hydrophobic interactions with the “WPF shelf”, a hydrophobic region in the binding pocket which was composed of Trp81, Pro82, and Phe83. Interestingly, the hydroxyl group of compound 7 did not interact with BRD4 and was oriented toward the ZA channel, a hydrophobic channel region which was composed of Pro82 to Leu91.Analysis of the binding modes between compound 7 to BRD4, we employed several strategies. First, we replaced the benzene ring in compound 7 with phthalazinone. The lactam moiety of phthalazinone, acting as both hydrogen bond donor and acceptor, could form hydrogen bonds with BRD4. Second, we substituted the carbonyl group of compound 7 with a methylene group. This modification was aimed at increasing the compound’s flexibility, thereby facilitating a more favorable conformation for phthalazinone to bind with BRD4. Finally, we replaced the hydroxyl group of compound 7 with various fragments to interact with ZA channel of BRD4.As shown in DDT01 displayed no inhibitory activity against BRD4. This observation prompted us to introduce various substituents on the nitrogen atom to enhance the inhibitory potential. When the methyl group in DDT02 was substituted with cyclopentyl or cyclohexyl, there was a marked improvement in inhibition (as seen in DDT03 and DDT04). Compounds DDT05-DDT07 exhibited weak inhibitory effects, suggesting that the aromatic structure might be detrimental to potency. Modifying the methylene group in the cyclohexane ring of DDT04 with either oxygen or sulfone led to decreased inhibitory effects (as observed in DDT08 and DDT09). Introducing hydroxyl groups into the cyclohexane ring of DDT04 yielded two optical isomers, DDT10 and DDT11. Notably, DDT10 demonstrated a higher inhibitory rate than DDT11, indicating the stereochemistry of the chiral carbon influenced the compound’s potency. Interestingly, both DDT13 and DDT14, derived from the reduction of the carbonyl group in DDT12, exhibited enhanced inhibitory effects on BRD4. However, the stereochemistry of DDT13 and DDT14 did not seem to influence their inhibitory potential. Lastly, most compounds synthesized by substituting cyclohexylamine with either piperidine or piperazine lost their inhibitory effects on BRD4, with the exceptions being DDT15 and DDT16.The inhibitory effects of all synthesized compounds on BRD4 were assessed at a concentration of 1 μM, and the structure-activity relationship (SAR) will be discussed in detail. As presented in DDT05 generally displayed high inhibitory rates against BRD4. The positions of the substituents had a more pronounced effect on inhibitory activity than their electronic properties. Specifically, ortho-substituted derivatives exhibited the most potent inhibitory activity. Upon removal of the carbonyl group’s oxygen in DDT22, a series of benzyl-substituted amine derivatives were synthesized. However, most of these derivatives showed weak inhibitory effects against BRD4, with the exceptions being DDT47-DDT49.As depicted in DDT50, alkyl-substituted sulfonamides did not exhibit inhibitory activity against BRD4. A subset of benzene sulfonamide compounds was then evaluated. While the 3-methyl and 4-methyl substituted compounds demonstrated potent inhibitory effects, the 3,4-dimethyl and 3,5-dimethyl derivatives showed reduced activity (refer to DDT58-DDT61). Introducing larger substituents in place of the 4-methyl group led to decreased inhibitory effects (see DDT62-DTT65). DDT66, derived by substituting the benzene ring of DDT59 with pyridine, showed a marked reduction in inhibitory activity. Among the methoxy-substituted derivatives, the 4-methoxy compound DDT69 exhibited the highest inhibitory rate. Compounds DDT76-DTT78 displayed low inhibitory rates, suggesting that inserting a methylene group between the benzene and sulfonyl groups hindered BRD4 binding. Compounds DDT80-DDT83 and DDT94, with electron-withdrawing groups, showed weak inhibitory activities. Among halogenated derivatives, only the ortho and para chlorine-substituted compounds demonstrated potent inhibitory effects (refer to DDT87 and DDT89). These findings suggested that, for benzene sulfonamide derivatives, electron-donating groups and para substituents enhanced the inhibitory potency against BRD4.50 values of several synthesized compounds for their inhibitory effects on BRD4. As presented in DDT26 exhibited the most potent inhibitory activity against BRD4, with an IC50 value of 0.237 ± 0.093 μM. In subsequent investigations, we observed that DDT26 could induce DNA damage in MCF-7 cells. This observation led us to hypothesize that DDT26 might interact with targets other than BRD4, potentially interfering with cellular DNA damage repair mechanisms. Notably, the 4-benzylphthalazin-1(2H)-one structure in DDT26 bore resemblance to Olaparib, a known PARP1 inhibitor. Supporting this hypothesis, biochemical assays revealed that DDT26 indeed possessed a modest inhibitory effect on PARP1, with an IC50 value of 4.289 ± 1.807 μM. Interestingly, both DDT14 and DDT49 demonstrated stronger inhibitory activities against PARP1 than DDT26 when tested at a concentration of 0.5 μM.We determined the ICDDT26 displayed robust inhibitory effects on all three breast cancer cell lines, and its inhibitory potency on MCF-7 cells surpassed that of JQ1.Several compounds exhibiting strong inhibitory effects against BRD4 were evaluated for their anti-proliferative activities on three breast cancer cell lines, using JQ1 and Olaparib as positive controls. As detailed in DDT26 against both BRD4 and cell proliferation, we undertook a comprehensive analysis of this compound. Through molecular docking, we examined the binding modes of some compounds with BRD4 in DDT26 with BRD4 was quite similar to that of compound 7. The 3,5-dimethylisoxazole moiety of DDT26 snugly fit into the acetylated lysine binding pocket. The isoxazole’s oxygen formed a hydrogen bond with Asn140, its nitrogen engaged in a water-mediated hydrogen bond with Tyr97, and its methyl group participated in hydrophobic interactions with Pro82. Consistent with our design strategy, the 2-trifluoromethyl benzamide extended into the ZA channel where its benzene and trifluoromethyl groups established T-shaped pi-pi stacking and hydrophobic interactions with Trp81, respectively. Intriguingly, the phthalazinone moiety of DDT26 did not align with the WPF shelf but formed a hydrogen bond with Met149.Given the impressive inhibitory performance of DDT26 was a PARP1 inhibitor, which prompted us to analyze the binding mode of DDT26 with PARP1. As shown in DDT26 with PARP1 closely resembled that of Olaparib. The phthalazinone moiety of DDT26 formed hydrogen bonds with Gly863 and Ser904. Its aromatic ring engaged in face-to-face pi-pi stacking interaction with Tyr907. The isoxazole’s oxygen formed a hydrogen bond with Arg865, and the fluorine of trifluoromethyl group established a hydrogen bond with Arg878.Biochemical experiment showed that DDT26 could downregulate the expression of c-MYC in MCF-7 cells in a concentration-dependent manner. γ-H2AX is recognized as a marker for DNA double-strand damage. Western blot results also indicated that DDT26 induced the accumulation of γ-H2AX in MCF-7 cells in a concentration-dependent fashion.c-Myc, a member of the Myc family of oncogenes, is known to promote cancer cell proliferation, metabolism, and protein synthesis. BRD4, functioning as a transcriptional regulator, plays a role in modulating the expression of c-MYC. As illustrated in DDT26 at concentrations of 0.3, 1, and 3 μΜ for 48 h displayed distinct cell cycle effects. As depicted in DDT26-treated groups were 53.8%, 59.8%, and 64.5%, respectively. This indicated that DDT26 induced a dose-dependent G1 phase cell cycle arrest in MCF-7 cells. At a concentration of 1 μΜ, the JQ1-treated group exhibited a more pronounced cell cycle arrest compared to the DDT26 group. In contrast, the Olaparib-treated group did not influence the MCF-7 cell cycle. The combination treatment group displayed a slightly reduced cell cycle arrest capability compared to the JQ1-treated group, suggesting that concurrent inhibition of BRD4 and PARP1 did not synergistically enhance cell cycle arrest.MCF-7 cells treated with compound DDT26 and Olaparib. DDT26 inhibited colony formation in MCF-7 cells in a dose-dependent manner. The results of colony formation assay showed that DDT26 could effectively inhibit the proliferation of MCF-7 cells at concentrations above 3 μM.Colony formation assay is an effective method to directly observe the anti-proliferation effect of compounds on cancer cells. As shown in DDT26, JQ1, and Olaparib on the migratory capability of MCF-7 cells using a wound scratch assay. As depicted in DDT26’s inhibitory effect on MCF-7 cells migration was slightly less pronounced, while Olaparib showed no inhibitory activity. The migration-inhibitory effect of DDT26 on MCF-7 cells was further amplified at a concentration of 3 μM, underscoring DDT26’s concentration-dependent inhibitory action.Enhanced migratory capability can lead to the metastasis of cancer cells from the primary site to other tissues via the blood or lymphatic system, culminating in the formation of new tumor lesions. This is a critical indicator of the progression of many cancers. We evaluated the effects of DDT26’s potential to induce DNA damage in MCF-7 cells. As illustrated in DDT26 induced DNA damage in MCF-7 cells in a dose-dependent manner. At a concentration of 3 μM, DDT26 inflicted more pronounced DNA damage than the combination treatment. These findings suggested that DDT26’s ability to inhibit MCF-7 cell proliferation was intrinsically linked to its DNA damage-inducing properties.PARP1 acts as a sensor for DNA single-strand damage, while BRD4 plays a role in regulating the transcription of homologous recombination related proteins. Given the pivotal roles of both PARP1 and BRD4 in DNA damage repair, we employed a comet assay to investigate Breast cancer imposes a significant psychological and economic burden on patients. Consequently, the quest for effective targeted therapies for breast cancer remains at the forefront of drug research and development. Given the intricate classifications of breast cancer, not all types are responsive to BRD4 inhibitors. This underscores the need to identify novel BRD4 inhibitors with distinct mechanisms of action to broaden their therapeutic applicability in breast cancer treatment .DDT26 emerged as the most potent, exhibiting robust inhibitory activity against BRD4 at sub-micromolar concentrations. Furthermore, biochemical analyses revealed that DDT26 also mildly inhibited PARP1, a critical target for treating breast cancers with BRCA1/2 mutations. Molecular docking studies highlighted the phthalazinone moiety in DDT26 as pivotal, facilitating interactions with Met149 of BRD4 and Gly863, Ser904, and Tyr907 of PARP1.In this study, we designed and synthesized a series of novel 4-benzyl)phthalazin-1(2H)-one derivatives as potential BRD4 inhibitors. Among them, DDT26 demonstrated a more pronounced inhibitory effect on MCF-7 cells proliferation compared to JQ1, even though its direct inhibitory activity against BRD4 was somewhat inferior. We postulate that DDT26’s enhanced cytotoxicity in MCF-7 cells may stem from its dual-targeting capability, simultaneously inhibiting both BRD4 and PARP1 , it was coupled with boric ester (compound 9) to yield intermediate 10 through Suzuki reaction. This was followed by a palladium-catalyzed cyanation of intermediate 10 to produce intermediate 11. A subsequent Horner−Wadsworth−Emmons reaction between intermediate 11 and phosphonate resulted in olefin 12. Refluxing olefin 12 with hydrazine hydrate in ethanol led to the formation of the phthalazinone in intermediate 13. The pivotal intermediate 14 was synthesized by hydrolyzing the nitrile group of intermediate 13 under alkaline conditions. Ultimately, condensing intermediate 14 with the appropriate amines under standard conditions yielded amides DDT01-DDT21.The synthetic pathways for the target compounds are depicted in DDT22-DDT94 are illustrated in 18 was synthesized following a procedure analogous to that used for intermediate 13. The essential intermediate 19 was derived by reducing the nitro group of intermediate 18 using zinc powder. This intermediate 19 was then reacted with the respective benzoyl chlorides to produce target compounds DDT22-DDT32. Additionally, NaBH3CN-mediated reductive amination reactions between intermediate 19 and the relevant benzaldehydes yielded target compounds DDT33-DDT49. Lastly, reactions between intermediate 19 and sulfonyl chlorides produced target compounds DDT50-DDT94. Detailed synthesis procedures and spectroscopic characterizations for compounds DDT01-DDT94 can be found in the The synthetic pathways for target compounds Crystal structures of BRD4 (PDB ID: 4J0S) and PARP1 (PDB ID: 5DS3) were chosen for molecular docking. The molecular docking studies were performed by the software Accelrys Discovery Studio (version 2016). The PARP1 protein structure was processed using the Protein Preparation module in this software, with all default parameters. The BRD4 protein structure was processed similarly to PARP1, except that a conserved water molecule (Water 311) was retained. The active sites were defined as spherical regions with a 10 Å diameter, centered on the ligand molecules. The Full Minimization module was employed to minimize the poses of compounds based on the CHARMm forcefield. Docking simulations were conducted using the CDOCKER module, in which twenty random conformations of per compound were generated. The top ten poses of per compound were saved based on CHARMm energy and the poses with the most favorable interactions were chosen for subsequent analysis.The inhibitory effects of target compounds on BRD4 and PARP1 were assessed at Huawei Pharmaceutical Co. Ltd .50 values were determined through nonlinear regression with a normalized dose-response fit in Prism GraphPad software.The BRD4 activity assay utilized TR-FRET technology, employing a recombinant BRD4 (BD1 + BD2) and its corresponding ligand. The TR-FRET signal generated in the assay is proportional to the ligand’s binding to the bromodomains. All reactions contained a final DMSO concentration of 1%. All binding reactions were conducted at room temperature. Each 20 µL reaction mixture, in assay buffer, comprised bromodomains, the BET Ligand, and the specified inhibitor amount. For the negative control, 5 µL of assay buffer replaced the BET ligand. After a 120-min incubation with the ligand, the TR-FRET signal was captured using a Tecan Infinite M1000 plate reader. IC+ (100 μM), biotinylated NAD+ (25 μM), and slDNA (200 nM) in 30 μL of reaction buffer were added to each well. This was followed by the addition of 5 μL of either the compound at varying concentrations or a solvent control. The reaction was initiated by adding 20 μL of PARP (50 ng/well) and incubating at 30°C for 1 h. Post-reaction, 50 µL of streptavidin-conjugated HRP was introduced, and the assay continued at 30°C for an additional 30 min. Finally, 100 μL of a solution containing H2O2 and luminol in 0.1 M citrate buffer (pH 5.4) was added, and the luminescent signal was recorded using a Molecular Devices SpectraMax M5 microplate reader. The inhibition rate of PARP1 enzymatic activity was determined as: (Lu control −Lu treated/Lu control) ×100%. IC50 values, representing the concentration required for 50% inhibition of PARP1 enzymatic activity, were determined using nonlinear regression with a normalized dose−response fit in Prism GraphPad software.The inhibition of PARP1 enzymatic activity by the tested compounds was gauged using an ELISA in 96-well plates. Each well was pre-coated with histone (20 μg/mL) in 100 μL of PBS buffer and incubated overnight at 4 °C. Subsequently, NAD2.MDA-MB-468, MDA-MB-231, and MCF-7 cell lines were sourced from the National Collection of Authenticated Cell Culture, China. Cells were maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin, and incubated at 37°C in a humidified atmosphere containing 5% CO3 cells per well in a 96-well plate and treated with varying concentrations  of the compounds for 72 h. Post-incubation, 100 µL of MTT solution (0.5 mg/mL) was added to each well and further incubated for 4 h. The resulting formazan crystals were dissolved in 150 µL DMSO, and the absorbance was measured at 570 nm using a microplate reader.The viability of cells was assessed using the MTT assay. Cells were seeded at a density of 3×10DDT26, JQ1, and Olaparib for a duration of 24 h. Cells were then lysed using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using the BCA Protein Assay Kit. Equal amounts of protein (20–30 µg) were separated by SDS-polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked using 5% milk in PBS prior to incubation with primary antibodies. Primary antibodies against p-H2AX , c-MYC, and β-actin  were used. After overnight incubation at 4°C, membranes were washed and incubated with HRP-conjugated secondary antibodies for 1 h. Protein bands were visualized using ECL reagents.For Western blotting, cells were treated with compound DDT26, JQ1, Olaparib, or their combinations for 48 h. After treatment, cells were harvested, fixed in ice-cold 70% ethanol overnight at 4°C, and subsequently washed with PBS. Cells were then treated with 100 μL RNase at 37°C for 30 min and stained with 400 μL PI for 30 min at 4°C in the dark. The cell cycle distribution was analyzed using an Accuri C6 Plus flow cytometer , and data interpretation was performed with FlowJo software.The cell cycle distribution was assessed using the DNA Content Quantitation Assay Kit . MCF-7 cells were seeded in six-well plates and exposed to different concentrations of MCF-7 cells were seeded in 6-well plates at a density of 500 cells/well and allowed to grow for 8 days to facilitate colony formation. After incubation, cells were washed with PBS, fixed in methanol for 15 min, and stained with 1% crystal violet for an hour. Colonies were then imaged and counted.5 cells/well and incubated overnight. A scratch was made in the cell monolayer using a 10 µL pipette tip. After creating the scratch, cells were washed with PBS and treated with DDT26, JQ1, or Olaparib. Images were captured at 0, 12, 24, and 48-h intervals.MCF-7 cells were seeded in 6-well plates at a density of 5×10DDT26, JQ1, Olaparib, or their combinations for 24 h, they were harvested and mixed with 0.7% low melting point agarose at a 1:7 (v/v) ratio. This mixture was spread onto microscope slides, which were then submerged in lysis solution for an hour. Electrophoresis was conducted in a horizontal apparatus at 25 V and 300 mA. Post-electrophoresis, slides were stained with PI for DNA visualization, and images were captured using a fluorescence inverted microscope.The comet assay was performed under alkaline conditions using the Comet Assay Kit  as per the manufacturer’s instructions. Briefly, after treating cells with


Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes.The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the ‘insulin responsive vesicles’ are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16  The regulation of glucose transport is a central facet of insulin action . Upon intrans Golgi network (TGN) and IRVs. Many of the molecules involved in these sorting pathways have been defined, but their regulation is less well understood (Key to this effect of insulin is the ability of fat and muscle to sequester GLUT4 in tubules and vesicles throughout the cytoplasm which are collectively referred to as the GLUT4 storage compartment . A subfrderstood .Patients with Type two Diabetes Mellitus (T2DM) exhibit impaired insulin-stimulated glucose transport in muscle and fat; many studies have supported the hypothesis that GLUT4 sorting and/or trafficking are impaired in these patients . For exaa-SNARE involved in GLUT4 trafficking towards IRVs (a-SNAREs are regulated by interaction with members of the Sec1/Munc18 (SM) family  family ; Sx16 bi) family , a prote) family . This in protein . In the omplexes .Previously published data suggested that Sx16 and mVps45 act together in GLUT4 sorting . How thein vitro and show over-expression of Sx16 containing the phosphomimetic T7D mutation in 3T3-L1 adipocytes significantly impaired insulin-stimulated glucose transport. These and other data argue that the N-terminus of Sx16 is a potential regulatory site for the control of GLUT4 sorting in adipocytes. Finally, we show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site suggesting that T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes.Here we show that interaction between Sx16 and mVps45 is required for the effective sorting of GLUT4 in adipocytes and that this interaction may be regulated by phosphorylation of Sx16 in the N-terminal regulatory domain. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 α1/ α2 knockout mouse embryonic fibroblasts (MEFs) and control MEFs were cultured as described previously (HEK293T cells (Cat# CRL-3216 RRID:CVCL_0063) and 3T3-L1 fibroblasts  were obtained from the American Tissue Culture Collection and grown and differentiated into adipocytes as outlined in . HeLa ceeviously . Antibodeviously .HA-GLUT4-GFP in the pRRL-PGK plasmid was from Cynthia Mastick  and pCOGE. coli and purified on IgG-Sepharose . Protein levels were determined using Micro-BCA kits .Sx16 lacking its transmembrane domain, or the T7A or T7D mutants, were sub-cloned in-frame with dual protein-A tags, expressed in 2-Deoxy-D-glucose transport was assayed in cells exactly as outlined  in excepg for ten minutes and the fat cake carefully aspirated. The supernatant was used in a series of in vitro kinase assays; supernatant was stored at −80 °C prior to use. In brief, 30 µg of cytosol extract was incubated in 50mM HEPES pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 0.2 mM DTT, 50µM ATP.Mg2+ (±[γ-32P]-ATP) in the presence of 5 µg of recombinant Protein A-tagged Sx16 , protein A alone or myelin basic protein at 30 °C for 2 h. Samples were analysed by autoradiography.Adipocyte cytosol was prepared from day-10 adipocytes. Cell plates were washed three times in ice-cold buffer with protease inhibitors  and homogenised by passing through a needle twenty times. The homogenate was centrifuged at 12,000 Analysis of the phosphorylation sites was performed at the FingerPrints facility, University of Dundee. Phosphorylation reactions were carried out exactly as outlined above but using non-radioactive ATP; control reactions lacking cytosol and/or ATP were performed. Bands corresponding to Sx16-PrA were excised and trypsinised, followed by liquid chromatography-electrospray ionisation mass spectrometry on an ABI QTrap 4000 instrument. Potential phospho-peptide ions present in samples but absent from controls were submitted to ESI-MS/MS to determine their amino acid sequence and locate the phosphorylated residue.2, 0.1 mM [γ-32P]-ATP (approx. 0.5 × 106 cpm/nmol) and kinase buffer  Brij-35, 0.1% (v/v) β-mercaptoethanol) at 30 °C in a total volume of 50 µl. A 20 µl aliquot of reaction mix was taken at the times indicated on the figure legends and the reactions terminated by addition of 4xNovex SDS PAGE loading buffer and heating at 70 °C for 15 mins. The Sx16 was separated from [ γ-32P]-ATP by SDS-PAGE on Novex 4–12% gradient gels using a MOPS buffer. After staining with Coomassie Brilliant Blue to visualise the protein bands, the gels were dried and subjected to autoradiography. Finally, the Sx16 bands were excised, and the incorporated radioactivity was determined by scintillation counting.A total of 2 µg of purified ProteinA-Sx16 or Protein A-Sx16-T7A was incubated with the indicated purified recombinant protein kinases  in the presence of 10 mM MgClin vitro kinases assays typically contained 0.5 mg of bacterially expressed Sx16 incubated with 30 µg of cytosol in the presence or absence of 25 mM ATP for 60 min at 30 °C as indicated. Samples were then immunoblotted with anti-Sx16 antibodies or anti-phospho-T7 antibodies as described in the figure legends.In experiments using anti-phospho-T7 for detection, t-test or ANOVA as indicated in the figure legends, with p < 0.05 being deemed as significant. Statistical analysis was carried out using GraphPad Prism software.Result are expressed as ± SD with the N-number indicated. Significant differences were determined using either via its N-terminal peptide which is necessary for productive SNARE complex formation  and found that over-expression of wild-type HA-mVps45 did not change the cell surface GLUT[HA staining in non-permeabilised cells]/GFP ratio compared to non-transfected cells, but over-expression of (V107R)-mVps45 consistently increased this ratio by 20%  that might phosphorylate this site. The peptide sequence around T7 resembles the proposed consensus sequence for the AMP activated protein kinase (AMPK) and several of its related kinases . AMPK acssay mix . Sx16 wassay mix  and the ssay mix ; MARK1  impaired insulin stimulated glucose transport in 3T3-L1 adipocytes . Althougin vitro kinase assays, we show that both AMPK and SIK2 phosphorylate Sx16 in vitro  did not phosphorylate this or indeed other sites in Sx16  may be potential regulators of this site. The peptide sequence around T7 resembles the proposed consensus sequence for the AMP activated protein kinase (AMPK) and several of its related kinases . Using iin vitro ; however in Sx16 .Given the importance of T7 in regulating interaction with mVps45, we used our phospho-specific antibody to ascertain whether there were insulin- or AICAR-driven changes in Sx16 phosphorylation at this site , 7B. No etc.In an effort to ascertain whether AMPK was the predominant kinase for T7, we prepared lysates from MEFs engineered to lack AMPK (‘AMPK KO cells’) and found that phosphorylation of Sx16 was modestly reduced when using MEFs from AMPK KO cells compared to their wildtype counterparts. While supporting the hypothesis that AMPK phosphorylates Sx16, clearly other potential explanations for this result should be kept in mind, such as inadequate activation of a distinct down-stream AMPK, These caveats notwithstanding, our data raise the interesting possibility that activation of AMPK (a known regulator of glucose transport ) or SIK2In sum, our data confirm an important role of Sx16/mVps45 in GLUT4 sorting in adipocytes and reveal that phosphorylation of Sx16 might offer a novel target for therapeutic intervention in Type-2 diabetes.10.7717/peerj.15630/supp-1Supplemental Information 1Original MS spectra of the three species shown in Click here for additional data file.10.7717/peerj.15630/supp-2Supplemental Information 2in vitro kinase assay using wild-type Sx16 or Sx16-T7A incubated with MARK1 or BRSK1 kinases as described. MARK1 phosphorylatedSx16, but this was not reduced in the T7A mutant and thus suggests this kinase targets a distinct site. BRSK1 was an example of a kinase that did not phosphorylate Sx16.Shown are data from an Click here for additional data file.10.7717/peerj.15630/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15630/supp-4Supplemental Information 4Click here for additional data file.