Antifreeze proteins are site-specifically conjugated onto polymer-stabilised gold nanoparticles, resulting in hybrid materials capable of modulating ice growth processes. Antifreeze proteins (AFPs) have many potential applications, ranging from cryobiology to aerospace, if they can be incorporated into materials. Here, a range of engineered AFP mutants were prepared and site-specifically conjugated onto RAFT polymer-stabilized gold nanoparticles to generate new hybrid multivalent ice growth inhibitors. Only the SNAP-tagged AFPs lead to potent ‘antifreeze’ active nanomaterials with His-Tag capture resulting in no activity, showing the mode of conjugation is essential. This versatile strategy will enable the development of multivalent AFPs for translational and fundamental studies. The PFPR and IR  or tris- were synthesized by NaBHEscherichia coli, and purified by nickel affinity chromatography and HPLC. Three mutants were produced bearing N-terminal modifications of hexa-histidine (His-AFP), SNAP (SNAP-AFP) and cysteine (Cys-AFP). Direct conjugation of the SNAP-AFP was achieved by incubation with the BG-pHEA45@AuNP4 in buffer. NTA-pHEA17@AuNP4 was first activated by addition of NiCl2 followed by His-AFP. Direct Cys-AFP immobilization onto AuNPs was unsuccessful (see ESIRecombinant AFP type III (from ocean pout) were produced by expression in l see ESI. Particll see ESI.–1. There was no significant difference between the two mutants, showing the fusions were tolerated in terms of retention of function. His-AFP-Ni-NTA-pHEA17@Au4 was found to be surprisingly inactive as an ice growth inhibitor with no activity up to 1 mg mL–1 (AFP concentration). This could be due to the dynamic nature of the His-NTA interaction or incorrect orientation of the AFP on the surface. This also shows that designing a multivalent AFP is non-trivial, with the correct chemistry being essential. Previous studies suggest excess charge does not affect AFP activity and was ruled out as the cause.45@Au4 was found to be a very potent IRI, et al. for dendrimer AFPs–1. As with the IRI measurements, this confirms that all activity is retained despite the major structural modifications and that the placement of the AFPs via-polymeric linkers is tolerated. As the effective AFP concentration in solution when immobilized on NPs is lower, this suggests that the particles can bind multiple planes of ice simultaneously. To probe this dynamic ice shaping experiments were conducted in sucrose solution  moiety to be installed. The polymer coating is essential to enable conjugation in buffer, avoiding nanoparticle aggregation. It was found that the SNAP-tag  approach lead to the most active particles compared to oligohistidine capture. The observed ice recrystallisation inhibition and thermal hysteresis activity was comparable to free antifreeze proteins on a valency-corrected basis confirming retention of activity in this format. Other conjugation methods based on ionic interactions did not give stable particles or resulted in loss of activity. These results demonstrate that antifreeze protein can be incorporated into more complex assemblies to facilitate their application in colloid science or for fundamental studies exploiting nanoparticle cores for bioimaging and tracking.In conclusion, we report the synthesis of hybrid nanoparticle–AFP conjugates, making use of polymer-stabilized gold nanoparticles and protein engineering to enable precision-conjugation. Hetero-telechelic polymers were obtained by RAFT, enabling a nanoparticle immobilization moiety (thiol) and distal protein capture (nickel NTA or Ohttp://wrap.warwick.ac.uk.The research data supporting this publication can be found at There are no conflicts to declare.Supplementary informationClick here for additional data file.


The in ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (TLR)21 signaling pathway. Although the delivery of CpG ODNs in ovo at embryo day (ED) 18 has been shown to reduce infectious bronchitis virus (IBV) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered CpG ODNs are capable of protecting chickens against a post-hatch challenge is unknown. Thus, our objectives were to determine the protective effect of the in ovo delivery of CpG ODNs at ED 18 against IBV infection encountered post-hatch and, then, to investigate the mechanisms of protection. We found significantly higher survival rates and reduced IBV infection in the chickens following the pre-treatment of the ED 18 eggs with CpG ODNs. At 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (CD)8α+ and CD4+ T lymphocytes, and an up-regulation of interferon (IFN)-γ mRNA in the respiratory tract of the chickens. Overall, it may be inferred that CpG ODNs, when delivered in ovo, provide protection against IBV infection induced morbidity and mortality with an enhanced immune response. Coronaviridae [Infectious bronchitis (IB) is mainly an acute and severe disease of the respiratory system of chickens . The cauaviridae . There iaviridae ,4. Althoaviridae ,6,7.Ever since the first record of IB in the early 1930s , periodiEscherichia coli [Salmonella Typhimurium [Toll-Like receptor (TLR)s are a family of germ line encoded pattern recognition receptors (PRRs) expressed on the surface or within the endosomal compartments of cells . These rhia coli  and Salmhimurium , and virhimurium  and infehimurium ,22, in chimurium ,24,25. Thimurium .nuclear (N) gene mRNA expression in various embryonic tissues pre-treated with CpG ODNs, compared with the control, highlighting the value of CpG ODN treatment in IBV control. However, they did not demonstrate whether the in ovo CpG ODN delivery is effective against the IBV challenge encountered post-hatch. In this study, we determined whether the CpG ODNs delivered in ovo could provide protection against a post-hatch IBV challenge. Furthermore, we looked into several cytokines and immune cells that may be activated with such protection. We found that in ovo delivery was protective against IBV challenge post hatch, suggesting a potential lasting protective effect of CpG ODNs towards IBV infection, which could be exploited for developing control measures.A study that pre-treated chicken embryos with class B CpG ODNs at embryo day (ED) 18 in ovo, and then challenged them with IBV Ark99 strain the day after (ED 19), showed an increased up-regulation of IFN-γ, IL-1β, IL-6, IL-8, and oligoadenylate synthetase (OAS) A in the embryonic spleen . AdditioThe specific pathogen free (SPF) eggs from white leghorn layer hens were obtained from the Canadian Food Inspection Agency (CFIA), Ottawa, and were incubated according to the manufacturer’s instructions in digital egg incubators , located at the Health Research Innovation Centre (HRIC) 53, University of Calgary. All of the animal care protocols as well as the use of live chickens, embryos, and SPF eggs in our experiments, have been reviewed and approved by the Health Science Animal Care Committee . At ED 11, the incubated eggs were candled in order to select viable eggs for further incubation, and the hatched birds were transported and housed in high containment poultry isolators at the Prion/virology animal facility, HRIC, University of Calgary, with access to ad libitum food, water, and necessary veterinary care.50) [The IBV Massachusetts (M)41 strain was purchased from the American Type Culture Collection  and was used in all of the experiments. Nine day old SPF viable eggs were used to propagate M41 strain of IBV, and the allantoic fluid was harvested at 3 dpi by careful aspiration. The end point dilution assay was employed to assess the viral titers using ED 9 SPF eggs, and was expressed as a 50% embryo infectious dose (EID50) .The synthetic CpG ODNs, class B CpG motifs recognized by chicken TLR21 (5′-TCG TCG TTG TCG TTT TGT CGT T-3′), and the negative control ODN 2007 (5′-TGC TGC TTG TGC TTT TGT GCT T-3′) were purchased from Cedarlane , and were used in our experiments.n = 7). The control ODNs were diluted in PBS to the same concentration (50 µg in 200 µL per egg), and were delivered via the same route (n = 6). The in ovo TLR ligand delivery was carried out as described previously [4 EID50 per bird, and were monitored for 11 days post-infection (dpi) for disease progression and outcome. The humane end point of the birds was determined based on the clinical score of each bird . The clinical score of 5 was considered the humane endpoint.Fifty µg of class B CpG ODN 2007 was diluted in 200 µL of phosphate buffered saline (PBS), and was inoculated per SPF egg on ED 18, via the in ovo route (n = 21), control ODNs , and PBS alone , as described earlier. The eggs were then incubated for 3 days until hatching. On the day of hatching, a subset of CpG ODN-treated chickens (n = 12) were infected with IBV M41 strain intra-trachealy at a dose rate of 2.75 × 104 EID50 per bird, while maintaining the rest of the birds in that treatment group as uninfected controls (n = 9). Similarly, a subset of the control ODN-treated chickens was infected with IBV M41 strain with the same dose (n = 18), with the remaining birds being the control ODN treated birds (n = 11), and all the PBS treated birds (n = 3) were kept as controls. The birds were weighed, wing tagged and after infection, were placed in separate isolators until the subsets of the animals were euthanized at 3 (n = 5–9 per group) and 7 (n = 3–9 per group) dpi. The clinical signs were observed and recorded daily as described, and the oro-pharyngeal and cloacal swab samples obtained using Puritan®UniTranz-RT® Media Transport Systems  at 3 and 7 dpi, and the IBV genome load were quantified following the RNA extraction. Simultaneously, the lung tissue was collected at 3 and 7 dpi in RNA Save® , in order to determine the viral genome loads in lungs. To evaluate the IBV N antigen in the tracheal mucosal epithelium, the tracheal tissues from the 3 dpi birds were collected and preserved in an optimum cutting temperature (OCT) compound , and were snap frozen in dry ice until use in immunofluorescent assay. To observe the histopathology, the tracheal tissues of the 3 dpi birds were fixed in 10% neutral buffered formalin  and sent to the Histopathology Diagnostic Services Unit at the University of Calgary, Faculty of Veterinary Medicine, for hematoxylin and eosin (H and E) staining. Additionally, the trachea and lung tissues were collected in an OCT compound , snap frozen, and subjected to immunofluorescent assay so as to quantify the key innate and adaptive immune cells. Another portion of the tissues were collected iusing RNA Save®  for the cytokine mRNA expression analysis. The animal numbers represent the total number of animals in two independent experiments.In order to evaluate the mechanisms of protection of the in ovo delivered CpG ODNs, the ED 18 SPF eggs were injected with class B CpG ODN 2007  protocol was adopted as per manufacturer’s guidelines. The concentration of extracted RNA was measured using Nanodrop1000 spectrophotometer , with the absorbance at a 260/280 nm wavelength. Two µg of total RNA from the tissue samples and 200 ng of total RNA from the swab samples were used to synthesize the cDNA with the use of the High Capacity cDNA Reverse Transcription Kit , as per manufacturer’s guidelines.The total RNA from the lungs collected at 3 and 7 dpi was extracted using a Trizol reagent , according to the manufacturer’s guidelines. For the RNA extraction of oro-pharyngeal and cloacal swabs, the E.Z.N.A.® Green Master Mix  in order to quantify the IBV N gene and cytokine mRNA expressions. RT-PCR assays were conducted in a 96 well un-skirted, low profile PCR plate , where the final reaction volume of the qPCR was maintained at 20 µL. Each qPCR run consisted of samples of interest, a positive control/s (gene specific plasmid), negative reverse transcriptase (NRT) control (cDNA construct without the multiscribe reverse transcriptase enzyme), and negative template (NTC) control. All of the cDNA samples originating from the tissues, along with the plasmid dilution series used to generate the standard curves, were run in triplicate. The target genes were quantified in relation to the β actin housekeeping gene. The target gene and the housekeeping gene for each sample was run on the same plate. Five picomolar (pM) of different gene specific primers (Forward and Reverse primers) were used in each reaction (A RT-PCR assay was carried out using Fast SYBRreaction . The cha® 550 (red fluorescence)  was then used in a 1:500 in 5% goat serum for 1 hour (h), followed by adding Vectashield® mounting medium with 4′, 6-Diamidine-2′-phenylindole dihydrochloride  (Blue fluorescence), placing cover slips and edges sealed with lacquer as the final step.For the cluster of differentiation (CD)8α+ cell and macrophage (KUL01+) of the lung and trachea, 5 μm thick sections were cut from the OCT preserved tissues and were fixed using cold acetone for 5 minutes (min). The tissues were then blocked by adding 5% goat serum diluted in a Trizma buffered saline (TBS) buffer  at room temperature for 30 min. After tipping off the excess blocking buffer, as the primary antibodies, the mouse monoclonal antibody specific for chicken macrophages/monocytes, KUL0+ , CD8α , was used in a 1:200 dilution in a 5% goat serum for 30 min. The secondary antibody, goat anti-mouse IgG (H+L) conjugated with Dylight® 488 (green fluorescence) streptavidin in a 15:1000 dilution was added for 30 min, followed by a final step of mounting the slides with a Vectashield® mounting medium with DAPI . All of the incubations were performed in a humidifying chamber at room temperature. Each incubation with an antibody was followed by washing the slides in a TBS-T buffer for 3 min twice and in PBS for 3 min once.For the CD4+ T cell staining, before blocking the tissues with 5% goat serum, sections were blocked with avidin followed by biotin , each with 15 min incubation periods, in between washing with TBS-T for 3 min twice and with PBS for 3 min once. After blocking with 5% goat serum for 30 min, a primary antibody, CD4  was added in a 1:200 dilution in 5% goat serum for 30 min. Next, biotinylated goat anti-mouse IgG (H+L)  was used as a secondary antibody in a 1:250 dilution in a 5% blocking buffer, and was incubated for 30 min. Then, DyLight® 550  and DyLight® 488 (CD4+ cells) positive signals were expressed relative to the total area (as estimated by nuclear staining with DAPI), and were given as a percentage.For the quantification of the tissue KUL01+, CD4+ cells, and CD8α+ cells, five areas with maximum positive fluorescent signals of KUL01+, CD4+ cells, and CD8α+ cells per tissue section were captured at X 20 magnification, along with the corresponding nuclear stained (DAPI) areas. The images were then subjected to fluorescent intensity quantification using Image J software . The fluorescent intensities for the Dylightt test. One-way analysis of variance (ANOVA) followed by the Students–Newman–Keuls test were used to identify the group differences in all of the other experiments. The Grubbs’ outlier test was performed in order to identify the outliers before the data was analyzed. The data in the graphs are shown in the original scale of the measurements. However, because of the non-normality and inability to satisfy the model assumptions of data belonging to the cell counts and cytokine mRNA expression, a natural log transformation was applied to these data sets prior to analysis. Model statistics were performed using GraphPad Prism Software 5, La Jolla, CA, USA. A normality test, generation of histograms, box plots, and Q–Q plots were performed in R statistical software, R studio version 1.0.153, Boston, MA, USA. * = significant at p ≤ 0.05, ** = significant at p ≤ 0.01 *** = significant at p ≤ 0.001.A log-rank test was used to identify the differences in survival percentage. The Kruskal–Wallis test followed by the Mann–Whitney U test were used to identify the group differences in the clinical score data for each time point. The differences among the two groups were identified using othe student’s p < 0.05) when compared with the control ODN-treated chickens, as seen in p < 0.05, We observed a significant increase in the survival rate of the CpG ODN-treated chickens .In general, the in ovo CpG ODN pre-treated IBV infected group recorded higher macrophage and CD4+ and CD8α+ T numbers in the trachea compared with the uninfected and control ODN pre-treated groups, although not all of the specific comparisons reached statistical significance a–c. Simip < 0.01) and uninfected (p < 0.05) groups , when compared to the in ovo delivered control ODNs in both the IBV infected and uninfected groups. Previously, we saw that the in ovo delivered CpG ODNs increased the macrophages in the trachea at one day of age ,22. ThisOur observation of the increased recruitment of CD4+ and CD8α+ T cells in the lungs following in ovo CpG ODNs delivery indicated that CpG ODNs could act as a mitogen, as has been shown previously . This CpOf the examined immune mediators, IFN-γ, a dominant product of the T helper (Th)1 type cells, was upregulated in the CpG ODN pre-treated IBV infected and uninfected groups, when compared with the control ODN pre-treated IBV infected and uninfected groups. The source of the lung IFN-γ mRNA could be the CD4+ T cell lymphocytes and CD8α+ cytotoxic lymphocytes ,34, and Two other immune mediators that were induced by the CpG ODNs and originated from the innate immune cells, such as the macrophages in the lungs, are the chemoattractant, IL-1β, and the NO production inducer, iNOS . In the The significance of the observations described in our study are two-fold. First, we found that the in ovo administration of CpG ODNs is capable of limiting IBV replication in the lungs and trachea, leading to an increased survival and reduced morbidity in the early post-hatch birds. Second, in our study, the early recruitment and maintenance of key immune cells, such as CD8α+ and CD4+ T cells and macrophages, and the up-regulated IFN-γ mRNA, exhibited not only an initiation of the early innate response, but also an effective and early adaptive host response mediated by the CpG ODNs, which would facilitate protection against the IBV infections encountered in birds in their immediate post-hatch life.Further experiments elucidating the mechanisms of the CpG ODN-mediated adoptive response, such as cell- and antibody-mediated immune responses in chickens and the duration of protection provided by this ligand against IBV, would be greatly beneficial in order to better understand the protective effects of CpG ODNs, and may aid in the development of more effective IBV control measures.To conclude, we show that the CpG ODN-mediated protective response against post-hatch encountered IBV infection is associated with the up-regulation of IFN-γ mRNA expression (in the lungs) and the enhanced recruitment of macrophages (in trachea) and CD4+ and CD8α+ T cells (in the lungs). Our findings, although preliminary, may provide a basis for developing novel control strategies in the long term against IBV infection in chickens.


Accurate division of cells into two daughters is a process that is vital to propagation of life. Protein phosphorylation and selective degradation have emerged as two important mechanisms safeguarding the delicate choreography of mitosis. Protein phosphatases catalyze dephosphorylation of thousands of sites on proteins, steering the cells through establishment of the mitotic phase and exit from it. A large E3 ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) becomes active during latter stages of mitosis through G1 and marks hundreds of proteins for destruction. Recent studies have revealed the complex interregulation between these two classes of enzymes. In this review, we highlight the direct and indirect mechanisms by which phosphatases and the APC/C mutually influence each other to ensure accurate spatiotemporal and orderly progression through mitosis, with a particular focus on recent insights and conceptual advances. From the outset, the process of cell division might seem mired in extraordinary complexity. Following a series of distinct steps, a eukaryotic cell is able to duplicate its genetic content and partition it among its two daughters along with other organelles with remarkable precision. Decades of research has sought to understand the molecular and physico-chemical underpinnings of the ‘cell division cycle’: from DNA replication, transitory structures such as the mitotic spindle devoted to its distribution, to control systems that monitor the fidelity of these processes. The realization that reversible protein modifications such as phosphorylation and ubiquitylation are at the heart of the cell cycle engine has demystified many of its aspects.The primary focus of this review is to highlight the roles of two classes of protein modifying enzymes that are central to accurate mitosis and the complex interplay between them: protein phosphatases and the E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C), with a focus on recent advances. These two modifiers act in concert with numerous mitotic kinases to orchestrate various events of mitosis.The discovery of phosphatases, then called ‘prosthetic group’-removing or PR enzymes, predated that of kinases . AbundanThe years following the discovery of PP1 witnessed the identification of many different classes of phosphatases which can be broadly divided into two groups: one is Protein Tyrosine Phosphatases (PTPs), which comprises classical PTPs and Dual Specificity Phosphatases (DSP) that catalyze dephosphorylation of p-Tyr, or p-Tyr, p-Ser and p-Thr residues, respectively . The oth5R (PTP loop). The sulfur atom of the catalytic cysteine initiates a nucleophilic attack on the phosphorylated substrate  subfamily which comprises of PP1, PP2A, PP2B/calcineurin, PP4, PP5, PP6 and PP7. These phosphatases have been implicated in diverse functions in the cell cycle and beyond. Their catalytic sites adopt similar folds and essentially follow the same mechanism of catalysis . Two div binding ,9. The m binding . PP1 and binding . They me binding ,13,14. AThe examples of PP1 and PP2A highlight a leitmotif in phosphatase function, including ones controlling cell division: in vivo they tend to function as obligate multimeric enzymes. The catalytic subunit typically associates with one or more additional subunit. These additional subunits can act as scaffolds, regulators or inhibitors, thereby fundamentally altering the properties of the phosphatase. Since early experiments were often performed with the catalytic subunit, phosphatases appeared to show little discrimination towards substrates. This observation, coupled with the relatively few numbers of known phosphatases in comparison to kinases, led to the erroneous yet resilient view that phosphatases are promiscuous housekeeping enzymes working in the background as their kinase counterparts orchestrate major cellular events. In theory, this could indeed serve as an organizational principle in cellular control. For instance, it is well established that oscillating activity of cyclins in complex with cyclin-dependent kinases (Cdks) propels the cell through various transitions by orderly phosphorylation of substrate proteins ,16, withThousands of unique phosphorylation events on almost three-quarters of the proteome have been reported, regulating division and other cellular functions ,28,29.In addition to selective phosphorylation, selective degradation of proteins provides a major avenue for control of the cell cycle. A few decades following the discovery of phosphatases, it was found that cells selectively target proteins for degradation by marking them with covalently-attached chains of ubiquitin, a small, heat-stable protein ,31. ThesThe attachment of ubiquitin, ‘ubiquitylation’, is a multi-step process with sequential actions of three enzymes (E1-E2-E3) ,38,39: TDeubiquitylating enzymes (DUBs) disassemble ubiquitin chains on substrates, providing another avenue for cellular control and homeostasis. There are about 93 different DUBs in the human genome, compared to about 600 E3 ubiquitin ligases ,41, drawDrosophila [The anaphase-promoting complex/cyclosome (APC/C) was found to be the E3 ubiquitin ligase responsible for degradation of Cyclin B during mitosis ,43,44 byosophila , etc. Coosophila ,53. Someosophila . PerhapsRecent structural studies have illuminated the overall architecture and subunit arrangement of the APC/C holocomplex. It adopts a hollow triangular shape, with the co-activator binding into this nook. Together with the core subunit Apc10, a co-activator forms the substrate recognition module of the enzyme and binds short linear degradation signals, or ‘degrons’ such as the destruction box (D-box) or the KEN-box present within intrinsically unstructured regions of target proteins ,56,57,58In humans, it has been reported that 170 proteins are ubiquitylated by the APC/C, likely to be an underestimate . Due to Unidirectional and orderly transitions through cell cycle phases are critical to our initial question of how cells divide in an error-free manner. Concerning mitotic exit, studies in both yeast and vertebrates show that APC/C-mediated proteolysis and dephosphorylation work in concert to ensure irreversible transition through this phase and establish a biochemically G1 state ,73.The APC/C and phosphatases exhibit considerable crosstalk during mitosis; they influence each other directly and indirectly. Broadly, APC/C activation during mitosis requires phosphorylation and is opposed by phosphatase action. Association of co-activators with the APC/C, however, requires them to be dephosphorylated by phosphatases. Phosphatases are also essential to silence the SAC and further promote APC/C activation. Active APC/C catalyzes mitotic cyclin degradation (inactivation of Cdk1) which then leads to phosphatase reactivation. These observations outline the interlinked nature of an extensive network of phosphorylation and ubiquitylation throughout M-phase and will be explored in greater detail in the following sections. Readers interested in detailed kinase and phosphatase crosstalk governing kinetochore-microtubule attachments and the SAC that are beyond the scope of this review are pointed to recent excellent comprehensive reviews ,76,77,78Xenopus indicate that preceding Cdk1-Cyclin A-mediated phosphorylation sets in motion mitotic commitment [As mentioned previously, a quantitative increase in Cdk activity from G1 to M-phase triggers sequential passage through the cell cycle. Transition from G2 to M-phase requires an explosive, switch-like activation of Cdk1-Cyclin B. During G2, although the synthesis of Cyclin B increases due to transcriptional activation, concomitant increase in associated kinase activity requires removal of two inhibitory phosphates on the Cdk1 subunit: p-Thr14 and p-Tyr15. Phosphorylation of these residues prevents catalysis by causing ATP misalignment , and is mmitment .In humans, three Cdc25 paralogues exist; Cdc25A, Cdc25B and Cdc25C and perform distinct but overlapping functions in Cdk activation. Active Cdk1-Cyclin B first emerges at centrosomes, catalyzed by Cdc25B. In fact, centrosome splitting proceeds a few minutes after emergence of active kinase in unperturbed cells . In a spThe activity and subcellular localization of these phosphatases are themselves modified through phosphoregulation and proteolysis. For instance, phosphorylation of Cdc25C by Cdk1 and Polo-like kinase (Plk) increases its activity. Additionally, Cdc25C phosphorylation by a number of kinases at a 14-3-3 consensus sequence leads to its cytoplasmic retention and catalytic inactivation by these adaptor proteins ,92. An iThousands of proteins are phosphorylated during mitosis by Cdk1, Plk, Aurora A and Aurora B, to name a few. They drive the dramatic cytological changes one associates with this phase: reorganization of cortical cytoskeleton to induce cell rounding and dissolution of the nuclear envelope in vertebrates, compaction of chromosomes to aid segregation and formation of a spindle structure to capture and biorient chromosomes at the metaphase plate. A coherent forward step towards this phase would dictate that opposing phosphatase activity be dampened, swinging the balance towards phosphorylation. Downregulation of many phosphatases occurs at this juncture through direct Cdk1-dependent phosphorylation or binding of various small, heat-stable inhibitors (see below).Increasing Cdk1 kinase levels phosphorylate the highly conserved AGC kinase Greatwall. The two phosphorylated residues lie within the activation loop of the kinase domain and produce a partially activated enzyme. Intramolecular autophosphorylation of its C-terminal tail leads fully active Greatwall by stabilization of its active conformation ,97.Active Greatwall phosphorylates two small, heat-stable, intrinsically unstructured proteins: ENSA (α-endosulfine) and Arpp19 (cAMP-regulated phosphoprotein 19) ,99. Phoscatk two-three orders of magnitude lower than that of a generic PP2A-B55 substrate. PP2A-B55—p-ENSA mK is about four orders of magnitude lower than that for a phosphosubstrate, indicating very tight binding of the inhibitor. Strong inhibitor binding coupled with low rate of catalysis has been termed ‘inhibition by unfair competition’ [In keeping with a paradigm often governing enzyme-inhibitor interactions, PP2A-B55 itself is able to dephosphorylate p-ENSA/p-Arpp19, albeit very slowly, with a etition’ . This imPP2A-B56 is present at kinetochores, where it phosphoregulates kinetochore-microtubule interactions, and is opposed by Aurora B. Phosphorylation of another small, heat-stable protein Bod1 recruited by Ndc80 causes it to inhibit PP2A-B56 complexes. This inhibition is proposed to maintain the correct balance of PP2A-B56 and Aurora B activities ,103, altDirect phosphorylation of PP2A subunits has also been reported to regulate enzyme activity by affecting assembly. Human PP2A catalytic subunit phosphorylation at its C-terminal tail at Thr307 prevented binding of B55 . A recenCdk1 also directly phosphorylates PP1 near its C-terminus at Thr320 to inhibit its activity down to 30–40% of its peak levels ,108,109.As previously mentioned, PP1 binding proteins often employ one or more short docking motifs to interact with the enzyme. Of its 200 known interactors, at least 90% employ the RVxF motif . When thPP6 is a phosphatase belonging to the PPP superfamily that also forms a heterotrimeric enzyme. It dephosphorylates the regulatory T-loop of Aurora A thus inhibiting its activity at centrosomes . During Drosophila PP4 is targeted to centromeres via interaction of its regulatory subunit R3 with the centromere protein CENP-C, where it regulates centromeric integrity during mitosis [Drosophila [C. elegans [Another ubiquitous phosphatase of the PPP family, PP4, forms heterodimeric or heterotrimeric complexes in vivo ,117, pre mitosis . It has osophila , C. eleg elegans  and huma elegans . At the  elegans .During metaphase/anaphase, APC/C-mediated degradation of Cyclin B and various mitotic kinases sets in motion cascade of events that lead to reactivation of phosphatases . Thus, tIt is important to note that while there is a general swing towards kinase activation, some phosphatase activity is preserved throughout mitosis. Indeed, it is essential for mitotic progression. As is discussed in the next few sections, PP1 and PP2A-B56 activity at kinetochores plays an important role in stabilizing amphitelic kinetochore-microtubule linkages and silencing the SAC. These phosphatases have also been implicated in dephosphorylating the APC/C co-activator Cdc20 and initiating anaphase ,123,124.Successful execution of chromosome segregation requires capture of kinetochore by microtubules emanating from spindle poles or centrosomes at the opposite ends of the cells. This ensures that each daughter cell gets one copy of the genome. Until all sister chromatids have correctly attached, kinetochores activate the SAC and hold cells in metaphase by inhibiting the APC/C. Thus, phosphatase mediated error correction and SAC inactivation are intimately linked to APC/C activation. In turn, APC/C degrades many proteins involved in these processes. Kinetochore capture is a stochastic process; many rounds of incorrect attachments precede mature, end-on attachments ,127,128.How does the cell recognize correct attachments? On a molecular level, this occurs through warring activities of kinases and phosphatases at the centromere and kinetochore. Central to error correction is the kinase Aurora B, which is recruited to inner centromeres in two different ways. First, the mitotic Haspin kinase phosphorylates histone H3 at Thr3, which directly binds the Aurora B-containing Chromosome Passenger Complex (CPC) subunit Survivin . Second,A collaboration between PP1 and PP2A-B56 phosphatases, recruited through many mechanisms, leads to direct dephosphorylation of Aurora B substrates to strengthen kinetochore-microtubule interactions. PP2A-B56 directly binds the outer kinetochore, and serves to stabilize initial microtubule-kinetochore contacts, enabling them to form mature, end-on attachments . B56-bouAs previously mentioned, Cyclin A is degraded during prometaphase by APC/C action, even though the spindle checkpoint is active. Timely degradation of Cyclin A at this juncture is essential for formation of stable kinetochore-microtubule (KT-MT) attachments . The queOnce correctly attached, the two sister chromatids are pulled to opposite poles of the cell, thereby generating tension that is thought to place the kinetochore proteins outside the zone of influence of inner centromere localized Aurora B, more than 100 nm away . Here, pDuring mitotic exit, BubR1  and Bub1 are targeted for degradation by the APC/C ,144, shuUntil all kinetochores are correctly captured and bioriented at the metaphases plate, the APC/C activity is held in check by a highly potent, diffusible complex that acts as a stoichiometric inhibitor—the Mitotic Checkpoint Complex (MCC). Even one unattached kinetochore can sequentially recruit an array of proteins to form the MCC, holding the entire cell in metaphase and safeguarding genome segregation.The MCC is a complex composed of Mad2, BubR1, Cdc20 and Bub3. At unattached kinetochores, the Ndc80 complex, unfettered by microtubule binding, instead recruits the kinase Mps1 and drives a phospho-dependent signaling cascade. By virtue of being present at the outer kinetochore, Mps1 phosphorylates various proteins, including the MELT motifs of Knl1. This is the most upstream event in the MCC formation. As seen during error correction, Knl1 again acts as a scaffold and recruits Bub1-Bub3 complexes via its phosphorylated MELT motifs, which then recruit BubR1-Bub3 complexes. At this juncture, Mps1 directly phosphorylates Bub1 and leads to Mad1-Mad2 heterodimer binding . Next, Mcomet adopts a Mad2-like fold, and extracts c-Mad2 from its complex with Mad1 and from within the MCC by directly dimerizing with it [comet also acts as an adaptor protein and delivers c-Mad2 to the AAA+ ATPase TRIP13, which then catalyzes its conversion to inactive o-Mad2 by ATP hydrolysis and inactivates the MCC [comet activity in Xenopus and humans [It was initially thought that the MCC functions by sequestering Cdc20 away from the APC/C. Recently, however, it has become clear that the MCC employs a multitude of mechanisms to target numerous aspects of APC/C function to obstruct its activity. Cryo-electron microscopy structures have revealed molecular details of APC/C-MCC interactions ,64. The  with it ,158. p31 the MCC . Phosphod humans ,161,162.Finally, in addition to MCC dissociation, further SAC signaling must also be extinguished at the source to ensure timely anaphase entry. Indeed, kinetochores proteins are in a state of flux due to presence of both kinases (Aurora B and Mps1) and recruited phosphatases (PP2A-B56-BubR1 and PP1-Knl1), which enables quick responsiveness of the system to changes in the input. Once the Ndc80 complex is bound to kinetochores, Mps1 can no longer bind and initiate its signaling cascade and MCC formation . Direct These molecular interactions underlie the highly responsive nature of MCC-mediated inhibition and explain how cells commence Cyclin B degradation as soon as the last chromosome is aligned .One of the hallmarks of the mitotic phase is the appearance of discrete, rod-shaped ‘condensed’ chromosomes, a prerequisite for accurate chromosome segregation to daughter cells. This is mediated by actions of two different SMC  protein complexes, condensin and cohesin ,169. TheCondensin I and condensin II are ring-shaped multisubunit complexes that perform distinct functions in chromosome organisation and segregation in mitosis. In vitro reconstitution of mitotic chromatids has revealed that Cdk1 phosphorylation of condensin I is the sole mitosis-specific modification required for chromatid reconstitution . NonetheCohesin is a ring-shaped proteinaceous complex that holds together duplicated sister chromatids by physically entrapping them. Cell cycle-dependent acetylation of one of the cohesin subunits serves to ‘lock’ the ring in both yeast and vertebrates. Sororin, a vertebrate-specific protein is specifically recruited to acetylated cohesin. It protects cohesin from the ‘anti-establishment’ activity of a negative regulator, Wapl. Following entry into mitosis, vertebrate cells witness a removal of majority of cohesin complexes due to extensive phosphorylation of both Sororin and Wapl catalyzed by Cdk1, Plk1 and Aurora B. This occurs concomitant to condensin activity. Sororin phosphorylation destabilizes its interaction with cohesin, allowing Wapl activity to predominate ,181. CenAnaphase onset liberates separase by APC/C-Cdc20-mediated degradation of its inhibitor, securin. Centromeric cohesin is cleaved by this protease and sister chromatids commence their movement to opposite poles of the cell. A collaboration with PP2A-B56 phosphatase ensures that this step ensues in a switch-like fashion and precedes later events such as cytokinesis, which can damage any lagging chromosomes. CaMKII phosphorylates securin and makes it more amenable to APC/C-mediated degradation by increasing its degron-APC/C affinity. Separase-bound securin, however, is kept in a hypophosphorylated state by recruitment of PP2A-B56. Thus, free securin is targeted by the APC/C before separase-bound securin, ensuring that liberated separase is not re-inhibited by remaining securin molecules. Once activated, separase self-cleavage precludes PP2A binding and therefore re-inhibition by securin ,187,188.In budding yeast, securin degradation and decisive anaphase onset is also under phosphatase control, albeit with different underlying molecular details. Phosphorylation of yeast securin by Cdk1 impedes its degradation. As small levels of securin are degraded by the APC/C, Cdc14 is liberated, which dephosphorylates securin, promoting its degradation. Thus, a positive feedback loop is generated that leads to switch-like anaphase onset .Major interactions between cell cycle phosphatases and the APC/C are outlined in Once all chromosomes are aligned and the SAC is switched off, APC/C-Cdc20 becomes active. In addition to securin destruction, Cyclin B also gets degraded which reduces the overall kinase activity and initiates phosphatase reactivation. Extensive phosphoregulation of both the APC/C and co-activators governs its activity throughout mitosis. Phosphorylation precipitates opposite outcomes in this regard: phosphorylation of the APC/C activates it while co-activator phosphorylation prevents its association with the E3 ligase. Co-activators bind to APC/C at multiple sites, employing evolutionarily conserved regions within their largely intrinsically unstructured N- and C-terminal tails. These tails flank the highly structured WD40 propeller domain, which only mediates substrate recruitment rather than APC/C association. Of chief importance are two short linear binding motifs within co-activators (the C box and IR tail) that cooperatively enable Cdc20 and Cdh1 association with the APC/C by binding to the TPR lobe (the substrate recognition sub-complex). The C box, named so due to its evolutionary conservation within Cdc20 family of proteins , typicalLoop300), both of which are conserved in vertebrates. Mutation of Apc3 or Apc1 sites of phosphorylation to alanine considerably reduced APC/C-Cdc20 activity, with simultaneous mutation of both rendering it almost inactive and unable to bind Cdc20 [Loop300. These results pointed to an inhibitory function for Apc1Loop300 that is relieved upon its phosphorylation. Cryo-EM studies revealed that this loop contained a stretch similar to the co-activator C box and physically occluded the C box binding site within Apc8 in the non-phosphorylated form of APC/C [Loop300 peels it away from this region by disrupting its interactions and allows Cdc20 binding. Consistent with this model, deletion of Apc1Loop300 in the context of the holocomplex permitted Cdc20-dependent activity even in the absence of phosphorylation [Loop300 could co-precipitate holo-APC/C while its phosphomimetic counterpart could not, further bolstering this conclusion [Although both co-activators possess these binding sites, it had long been known that Cdc20 can only bind APC/C hyperphosphorylated in early mitosis by Cdk1 and Plk kinases ,195,196.nd Cdc20 . It was nd Cdc20 , which hnd Cdc20 ,201. Thind Cdc20 ,203, whiof APC/C ,199. Phorylation ,199. Recnclusion . Thus, iLoop300 to bind the enzyme. Higher affinity of Cdh1 compared to Cdc20 for the E3 ligase, evidenced by increased interaction sites within its N-terminus with multiple APC/C subunits, and reduced flexibility of the IR tail within Apc3 in cryo-EM structures has been suggested as an explanation for their differential behavior [Phosphorylation only affects access of Cdc20 to the APC/C, with Cdh1 evidently outcompeting the autoinhibition of Apc1behavior ,204.The budding and fission yeast APC/C adopts a very similar architecture to its vertebrate counterpart (barring the absence of Apc7) ,206,207.A majority of APC/C subunits undergo phosphorylation ,199. WheLoop300 is protected from, or impervious to dephosphorylation in early anaphase to preserve Cdc20 function. APC/C is not homogenously distributed throughout the cell. For instance, it is localized to mitotic chromosomes by the Ska complex in a dephosphorylated yet active form [At some point following entry into anaphase, APC/C dephosphorylation occurs and increased electrophoretic mobility of Apc3 is readily observed by SDS-PAGE . Preciseive form . How thiAs mentioned previously, Cdc20 is an early activator of the APC/C, while Cdh1 takes the reins in late mitosis upon its dephosphorylation. The overall structure and organization of the co-activators is remarkably similar; nonetheless, important differences persist and define their characteristic behaviors. First, Cdc20 can only bind phosphorylated APC/C due to reasons outlined in the previous section. Secondly, both co-activators employ the use of D- and KEN boxes within their substrates; however, only vertebrate Cdc20 appears to bind the ABBA motif which contributes to its inhibition by BubR1 . BuddingIn contrast to APC/C, Cdk1 phosphorylation of both Cdc20 and Cdh1 is inhibitory and prevents their association with the E3 ligase ,213,214.As Cyclin B is degraded, the previously inhibited phosphatases PP1 and PP2A-B55 get reactivated (discussed later). Cdh1 is dephosphorylated, probably primarily by active PP2A-B55 in vertebrates , and repmK, a higher catk or both, instigating faster turnover. In summary, both Cdc20 and Cdh1 are dephosphorylated at different points in the cell cycle to drive APC/C activation.A recent study addressed how PP2A-B55 creates distinct temporal windows for co-activator dephosphorylation and activation: while Cdc20 is dephosphorylated in early anaphase, Cdh1 is only targeted later. Phosphothreonine residues within Cdc20 are preferred PP2A-B55 substrates compared to phosphoserines, a property likely shared by all members of the PPP family owing to the similarities of their catalytic cores. This allows faster activation of Cdc20 by preferential dephosphorylation of its residues that are mostly Thr. In contrast, p-Ser residues predominate Cdh1 N-terminus and are disfavored PP2A substrates. Their dephosphorylation occurs later in anaphase when other factors such as reduced substrate competition possibly make its dephosphorylation more amenable. Importance of late Cdh1 dephosphorylation is underscored by the aberrant mitotic phenotypes associated with a phosphothreonine variant of the co-activator that is dephosphorylated earlier . How PP2dK [C. elegans [Cdc20 dephosphorylation invokes a catch-22 scenario: on the one hand it requires PP2A-B55 activity; on the other, it promotes B55 reactivation by degrading Cyclin B . It begsKd , it is l elegans . However elegans , althoug elegans .Mitotic exit witnesses degradation of at least a 100 protein substrates in a strict temporal order—a prerequisite for successful division. Perturbations in this order upset cell division. Coupled with the fact that protein synthesis exacts a considerable energetic cost, cells have evolved mechanisms to ensure specific, timely and efficient degradation of substrates by the APC/C. Computational modeling of APC/C-Cdc20-mediated substrate degradation in yeast concluded that substrate affinity, catalytic rate of ubiquitin transfer to achieve multi-ubiquitylation and substrate competition can together determine the order of substrate degradation . Co-actiThere is considerable plasticity among what constitutes a ‘degron’ motif and seemingly incompatible residues can be allowed in a particular sequence context. For instance, divergence from consensus can be tolerated if contacts at additional positions stabilize binding, a concept reviewed by Davey and Morgan . The degMultivalent substrate interactions, either with the WD40 domains by presence of multiple degron motifs, or with other APC/C subunits can elicit higher affinity—a strategy often employed by APC/C inhibitors. Higher order properties can also determine substrate binding and degradation. The example of Nek2A degradation illustrates this concept: the MR tail of Nek2A resembles co-activator and Apc10 IR tails and allows it to be independently bound and degraded by the APC/C during prometaphase, when the SAC is active. Although necessary, the MR tail is not sufficient for degradation; dimerization of Nek2A is also essential . It is lDegron modification is another major mechanism regulating substrate degradation. Acetylation of BubR1 during metaphase blocks its degradation during metaphase . AdditioPhosphostate of a protein is always in flux; it is a readout of the equilibrium kinase and phosphatase activities governing it. Any change in one of the parameters alters this equilibrium and is swiftly reflected in the phosphate occupancy of the protein. This is the principle behind rapid phosphatase activation as cells commence exit from mitosis. Cyclin B degradation by APC/C-Cdc20 decreases Cdk1 activity and creates a domino effect. The balance is shifted towards PP1 autoactivation by dephosphorylation of its C-terminal tail and I-1 ,109,110.Xenopus has only been prevented upon pan-phosphatase inhibition by toxins such as okadaic acid (OA)  [This leads us to an important discussion point: there likely is not one ‘mitotic exit phosphatase’ in higher organisms. In contrast, inactivation of Cdc14 in budding yeast elicits a complete block and arrests cells in telophase with elongated spindles due to its role in downregulating kinase activity ,228,229.nstance) ,231,232,Assembly of a central spindle, nuclear envelope reformation, chromosome decondensation and finally abscission into two daughter cells along the cytokinetic furrow are some of the events associated with mitotic exit. PP2A-B55 directly dephosphorylates p-Thr481 within Prc1, allowing its association with the central spindle where it bundles microtubules in an antiparallel array and defines the position of the cytokinetic furrow. PP2A-B55 also controls dephosphorylation of Aurora A regulator Tpx2 and its central spindle localization . PP2A-B5PP1 also plays a major role in regulating mitotic exit and cytokinesis ,235. ForXenopus egg extracts and human cells, high levels of non-degradable Cyclin B block cells in metaphase, intermediate levels allow progression into a pseudo-anaphase state with separated sister chromatids, while persistent low levels arrest cells in telophase [This is far from an exhaustive list of phosphatase regulation of mitosis; one can nonetheless appreciate that it is essential that these events occur in a specific order to avoid mitotic failure. For instance, premature recruitment of Prc1 to the central spindle upon Greatwall depletion induces early cytokinesis and causes a ‘cut’ phenotype in a majority of cells . Therefoelophase ,239. Theelophase . Howeverelophase . It is lHow are hundreds of proteins discriminated by mitotic phosphatases? Part of the answer lies in intrinsic preference for phosphothreonines exhibited by PP2A phosphatases. Accordingly, survey of proteins dephosphorylated very early during an induced mitotic exit in human cells revealed that p-Thr-Pro (p-TP) sites were significantly more dephosphorylated than p-Ser-Pro (p-SP) sites by OA sensitive phosphatases . A smallA majority of cellular phosphorylation events are catalyzed on serine residues (98.2%) . This coFinally, most proteins contain multiple phosphosites that could be potential phosphatase targets upon binding to substrate docking motifs such as LxxIxE. How the presence of these substrate affinity elements colludes with phosphosite preferences in the context of the whole protein is unknown.Our discussion thus far has been simplified by referring to each phosphatase as a homogeneous entity. This view, however, does the complexity of the system a disservice: each phosphatase has multiple isoforms. Do they exist to provide redundancy and buffer the system against inactivating mutations? Or do they perform distinct functions within the cell? A recent report examined the importance of multiple B56 isoforms, all of which contain an LxxIxE-binding pocket but exhibit distinct localizations within cells: while B56α binds Sgo2 at centromeres, B56γ prefers BubR1 binding via its LxxIxE motif. A small loop within B56α is thought to prevent docking interactions with BubR1 by repressing its binding to LxxIxE motifs, and enabling Sgo2 interactions instead . SpecifiAs the APC/C and phosphatases have pivotal roles in the cell cycle and beyond, they have been implicated in various diseases such as cancer. In case of the APC/C, heterozygous mutations within core subunits Apc3, Apc8 and Apc6 are found in human colon cancer cells . InvolveXenopus extracts [APC/C has been proposed as a potential target in cancer therapy, as its inhibition leads to an arrest in mitosis followed by cell death . Two smaextracts . TAME blextracts . Apicin extracts —a strateextracts . CombinaPP2A has been heavily implicated in several diseases such as cancer, Alzheimer’s disease, diabetes, etc. ,267. TraCell cycle phosphatases have been implicated in numerous signaling pathways, regulating myriad functions. A systems-level analysis of the consequences of phosphatase perturbation in various contexts would be a beneficial complement to various studies.Mitosis is a tour de force achieved by phosphatases and the APC/C working in concert with mitotic kinases. They integrate a variety of information to ensure timely and accurate execution of cell division. Inhibitors, regulatory modifications and opposing activities modulate their activities to fine-tune passage through cell cycle transitions. The past few years have unfurled layers of regulation by critical molecular pathways that control their activities.A larger theme emerging in phosphatase and APC/C function is the ubiquity of regulation by intrinsically unstructured loop domains. These act as regulatory hubs to occasion myriad effects: they underlie efficient catalysis by increasing substrate residence time or local concentrations of regulators with respect to the enzyme. The example of PP1 shows that one enzyme can possess multiple pockets that bind their own short linear motifs. Thus, it is likely that many other such interactions within other phosphatase and the APC/C await discovery.Equilibrium protein modification relies on many factors. Some of them are intrinsic to the substrate, such as the nature of the phosphosite presented to phosphatases, or indeed the accessibility of the substrate lysines by the APC/C. These can be further potentiated by the strength of degrons, or presence of docking motif interactions. However, many factors extrinsic to the substrates are likely to exert equally important effects on protein modifications. Higher local concentrations of enzymes, achieved by tethering them to cellular structures for instance, would aid the formation of substrate-enzyme complex and catalysis. Competition provided by more efficient substrates could also delay enzyme action on less preferred ones, as would the presence of opposing enzyme activities. Hence, unraveling how these factors work together would help us better understand how two cells are successfully produced from one.


Emerging evidence suggests a link between allergic disease and cardiovascular disease. The idea that there could be a connection between ‘allergies’ and heart disease is not readily apparent. Nonetheless, over the past 20 years research in mice and humans has linked hematopoietic cells and soluble factors that mediate allergic disease with coronary artery disease (CAD) [se (CAD) ,2. Specise (CAD)  and elevse (CAD)  have beese (CAD) . It was Sensitization to α-Gal, an oligosaccharide allergen in mammalian meat, relates to tick bites. Galactose-α-1,3-galactose  is a ‘blood-group-like’ antigen that is present as both glycoproteins and glycolipids in mammalian muscle and secretions  and is the causal epitope in a syndrome of IgE-mediated delayed anaphylaxis [Amblyomma americanum (the lone star tick) is a dominant ‘vector’ for inducing IgE to α-Gal, a finding that is supported by the strong overlap between cases of α-Gal syndrome and the established distribution of the lone star tick [bona fide α-Gal syndrome implies that there are likely many subjects who make IgE to α-Gal but nonetheless continue to consume mammalian products because they do not experience any allergic symptoms.phylaxis . Becausephylaxis . There atar tick ,7. A criEvidence supporting a link between IgE to α-Gal and CAD. The hypothesized connection between IgE to α-Gal and CAD was experimentally approached by a collaboration between cardiologists and allergists with a shared interest in understanding how immune regulation shapes health and disease. Adult subjects (n=118) from central Virginia who had been deemed to be at cardiovascular risk and had undergone heart catheterizations including advanced imaging with intravascular ultrasound (IVUS) formed the basis of the investigation [ects n=11 from cenBuilding the case: α-Gal has a glycolipid form which could be the key to understanding delayed onset of allergic reactions and a link with CAD. The recent report, which suggests the possibility that IgE to α-Gal represents a tick-related risk factor for CAD, is provocative but nonetheless only represents an association at the current time. However, there are additional elements that support the possibility of a mechanistic link. One is the realization that the prevalence of CAD, like α-Gal sensitization, also has marked regional variation and in the United States is most common in the southeast. In fact, there is significant overlap in the areas of the country where CAD and α-Gal syndrome are most common. Additionally, unique among major allergens, α-Gal can be present in a glycolipid form. There are many reasons to think that the glycolipid form of the allergen is the key to explain the characteristic delay of 3-6 hours that occurs with ingestion of mammalian meat, kinetics which are much slower than the onset of symptoms that result from parenteral exposure to α-Gal-containing allergen . Of course this ‘glycolipid hypothesis’ suggests another important tie with CAD given that it is well established that serum levels of low-density-lipoprotein (LDL) cholesterol are a risk factor for CAD and LDL deposits are found within lesions of atherosclerotic plaques. A working model to explain the connection between α-Gal and CAD posits that subjects who consume mammalian products containing α-Gal, and also make IgE to α-Gal, have chronic inflammation in the walls of coronary arteries that relates to this interaction between an antigen in food and a ‘unique’ immune response that recognizes that antigen. Further epidemiological and mechanistic studies will be imperative to further understand the relationship between IgE to α-Gal and CAD.


Faced with the global health threat of increasing resistance to antibiotics, researchers are exploring interventions that target bacterial virulence factors. Quorum sensing is a particularly attractive target because several bacterial virulence factors are controlled by this mechanism. Furthermore, attacking the quorum-sensing signaling network is less likely to select for resistant strains than using conventional antibiotics. Strategies that focus on the inhibition of quorum-sensing signal production are especially attractive because the enzymes involved are expressed in bacterial cells but are not present in their mammalian counterparts. We review here various approaches that are being taken to interfere with quorum-sensing signal production via the inhibition of autoinducer-2 synthesis, PQS synthesis, peptide autoinducer synthesis, and N-acyl-homoserine lactone synthesis. We expect these approaches will lead to the discovery of new quorum-sensing inhibitors that can help to stem the tide of antibiotic resistance. The increase in bacterial resistance to antimicrobial compounds and the spread of drug-resistant pathogens have become serious threats to human health. Currently, most antimicrobial compounds target essential bacterial physiological processes, thereby exerting a strong selective pressure on bacteria and facilitating the emergence and dissemination of resistant strains , diffusible signal factor (DSF), γ-butyrolactone, 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) among others . These AI molecules comprise a diversity of molecular species such as autoinducer-2 (AI-2), acylated homoserine lactones (acyl-HSLs), oligopeptides, the Interference with quorum-sensing systems has been envisioned as a suitable strategy to address the multi-drug resistance problem  enzyme, which synthesizes AI-2]  respond to AI-2 external supply, and AI-2 mediates the interaction between polymicrobial biofilm members , growth was not impaired in nutrient-rich conditions but it was affected in zebrafish embryos  whereas the growth rate and swarming motility were lower than the wild-type strain ethylthio-DADMe-Imm-A, all of which showed dissociation constants in the pM range  was expressed and characterized kinetically, showing a preference for 5′-deoxyadenosine as the substrate in comparison with MTA and SAH. For Rv0091, DADMe-Imm-A inhibitors consisting of derivatized analogs with long alkyl groups at the C5′ position exerted potent inhibitory activity . The authors suggested that Rv0091 plays a role in 5′-deoxyadenosine recycling but is not essential for the growth of M. tuberculosis or M. smegmatis , anti-malarial, and antineoplastic agents. Some of them are in clinical trials or have been approved for use in humans. Immucillin-based inhibitors are chemically stable, specific, and it is possible to chemically modify them to gain bioavailability without severely affecting their inhibitory activity  is a potential target for the development of new therapeutic agents because it is present in numerous bacterial species but not in mammals  turns it into the ΔrbsAΔlsrAΔluxS mutant, which was also as virulent as the wild-type strain  followed by a β-elimination step to yielding L-homocysteine and the enol form of 4,5 dihydroxy-2,3-pentanedione (DPD) -L-homocysteine] -sulfur bond or the hydrogen atom at C4 position in the SRH substrate. In the inhibitor S-homoribosyl-L-cysteine, the carbon (C5)-sulfur bond was replaced by a C5-C6 carbon-carbon bond, which interferes with the cleavage of the carbon-sulfur bond  homocysteine] that exerted a time-dependent inhibition of LuxS have been identified. They act as slow-binding inhibitors of improved potency, similarly to the halogenated S--L-homocysteine and S--L-homocysteine analogs  genome-editing technology now permits the expression of genes to be modulated with a high specificity and reduced off-target effect. This technology has been proposed as a promising method for fighting against antimicrobial resistance  was down-regulated  employed by pathogens. However, if this approach will be effective in vivo remains to be seen. Nevertheless, the feasibility of CRISPR-Cas9 genome-editing technology for attenuating the virulence of pathogens in vivo has been demonstrated. In this respect, the use of CRISPR-Cas9 phagemid vectors attenuated the virulence of enterohaemorrhagic E. coli in a Galleria mellonella infection model and S. aureus in a mouse skin colonization model , Zuberi et al.  that were metabolically actives but with impaired biofilm formation capacity  approach, has been applied successfully for i et al.  obtainedE. coli the CyaR small RNA regulated luxS gene expression negatively via post-transcriptional binding to the luxS mRNA 5′end (including the ribosome binding site), consequently, CyaR small RNA expression reduced AI-2 production  that constitutively expressed the luxS antisense RNA, showed lower AI-2 production, reduced biofilm formation and reduced expression of esrA and orf26 genes in comparison to the control E. tarda TX1/pJRA strain. Furthermore, in E. tarda TX1/pJR18-infected Japanese flounder group the accumulated mortality, the bacteria recovered from blood and kidney, and orf 26 and esrA expression were lower than the E. tarda TX1/pJRA-infected group -quinolone, which is commonly known as Pseudomonas quinolone signal (PQS) and acts as a quorum-sensing signal molecule . Subsequently, two condensation reactions take place. First, anthraniloyl-coenzyme A condenses with malonyl-CoA to form (2-aminobenzoyl) acetate with the participation of the proteins PqsD and PqsE. Second, the (2-aminobenzoyl) acetate condenses with octanoate through the PqsB and PqsC catalytic activity, producing 2-heptyl-4(1H)-quinolone (HHQ). Finally, HHQ is hydroxylated by PqsH FAD-dependent monooxygenase to form PQS , hydrogen cyanide (hcnABC), chitinase (chiC), lectins (lecA and lecB), and elastase (lasB). Interestingly, these compounds are also effective in vivo, as they limited the virulence of P. aeruginosa in mice, delayed mortality in the treated animals, reduced the production of HHQ, and prevented systemic dissemination of the bacteria , showing the Cys-His-Asn catalytic triad typical of FabH-like enzymes methanol derivatives, improved in vitro PqsD inhibition does not necessarily mean improved inhibitory activity in cellulo , Allegretta et al.  and PqsD. This compound acted as a dual inhibitor that affected the production of the virulence factors, pyocyanin and pyoverdine, but without affecting bacterial growth. Additionally, this compound reduced biofilm formation by P. aeruginosa and boosted the anti-bacterial activity of ciprofloxacin under biofilm conditions. Importantly, the dual inhibitor increased, in a dose-dependent manner and without cytotoxic effects, the survival rate of G. mellonella larvae challenged with lethal doses of P. aeruginosa  system and E. faecalis fsr quorum-sensing system are the most extensively characterized peptide-based quorum sensing systems . Transcription of the agr operon produces the RNA II and RNA III transcripts. Specifically, RNA II translation produces the proteins AgrA, AgrB, AgrC, and AgrD, which are the structural components of the agr-system, while RNAIII is involved in the post-transcriptional control of virulence factors expression and encodes δ-hemolysin  and NIF] were developed that consisted of peptides that mimic the N-terminal cleavage site of AgrD. The inhibitor NIF showed improved stability and stronger inhibition of quorum sensing in comparison to inhibitor P+1 , in a dose-dependent manner -1,3-thiazole-4-carboxylic acid, 9H-xanthene-9-carboxylic acid, 4-phenoxyphenol, savirin, ω-hydroxyemodin, biaryl hydroxyketones, and norlichexanthone appear to act by blocking the binding of AgrA to the agr operon promoters via direct interaction with the AgrA C-terminal DNA binding domain  inhibited in vitro all the S. aureus agr-system types (I–IV). Consequently, ω-hydroxyemodin treatment reduced the RNAIII, psmα and hla transcription without bactericidal and cytotoxic-associated effects. In addition, ω-hydroxyemodin inhibited the S. epidermidis agr-system and attenuated the virulence of S. aureus in a mouse skin and soft tissue infection model, apparently via disruption of the arg-system, facilitating the bacterial clearance by the host immune system  treated with savirin down-regulated psmα transcript levels, and α-hemolysin activity was reduced in several MRSA and MSSA clinical isolates. Interestingly, resistance or tolerance to savirin were not observed, and the S. epidermidis agr system was not significantly disturbed  strain treated with F19 down-regulated AtlE, psmα and RNAIII transcript levels. F-19 protected monocyte and macrophage cells from the lysis caused by several Gram-positive pathogens. Importantly, in an MRSA wound infection model, compound F-19 potentiated β-lactam and fluoroquinolone antibiotic activity, whereas in an MRSA bacteremia/sepsis model, F-19 alone and in combination with cephalothin protected the animals from a lethal infection with MRSA . The antisense oligonucleotide treatment affected the agrA expression as well as the expression of several virulence factors, including psmα, psmβ, hla, and pvl. The CA-MRSA USA300 LAC strain virulence in a mouse subcutaneous infection model was attenuated by antisense oligonucleotide treatment   did not show a potent inhibitory activity toward S. aureus AgrC (group III-IV) in comparison with the parental peptide (truncated AIP-II), but displayed an inhibitory activity similar to the parental peptide toward S. aureus group I, which is one of the main etiologic agents in human infections  comprises more than 30 different molecules that share a common structural scaffold, consisting of an acyl chain linked to a homoserine lactone ring. The acyl chains vary in length (4-18 carbons), oxidation state, and degree of saturation  is synthesized by the acyl-HSL synthase TofI. Chung et al. (pyridin-2-yl)methyl-2-phenyl-1H-indol-1-ol] acted as a noncompetitive inhibitor of BmaI1 synthase, and some analogs of this compound showed inhibitory activity. One interesting finding in this study was that one of the five inhibitory compounds selected was the cephalosporin antibiotic cefatrizine, suggesting that cephalosporin antibiotics may inhibit acyl-HSL synthases -3-Benzyl-6-piperazine-2,5-dione] acted as a non-competitive inhibitor toward both C8-ACP and SAM substrates. This finding was also supported by molecular docking analysis, which showed several high-affinity contact sites for the inhibitor on the CepI structure, but none of these sites was the SAM- and acyl substrate-binding site. Besides, some of these diketopiperazine derivative compounds did not exert an antimicrobial effect on B. cenocepacia J2315 but did interfere with the production of virulence factors such as proteases and siderophores. Furthermore, they perturbed biofilm formation, protected Caenorhabditis elegans nematodes against infection with B. cenocepacia J2315, and had low toxicity for HeLa cells -3-benzyl-6-piperazine-2,5-dione displayed a protein expression pattern quite similar to B. cenocepacia ΔcepI mutant (B. cenocepacia J2315 with deleted cepI gene). Interestingly, both the inhibitor-treated strain and the ΔcepI mutant overexpressed the giant cable pilus protein CblA, which has been associated with B. cenocepacia virulence in vivo. In addition, using site-directed mutagenesis and enzymatic activity inhibition approaches it was observed that the pocket around the Ser 41 residue on the CepI structure appears to be the inhibitor binding site  and ExpI (acyl-HSL synthase), the mechanism of action of these quorum-quenching agents was suggested to involve direct interaction with ExpI/ExpR proteins and the consequent inhibition of acyl-HSL production  in vitro. This inhibition reduced the production of butyryl-HSL because FabI supplies the butyryl-ACP necessary for RhlI synthase-mediated butyryl-HSL synthesis  (Chuanchuen et al., Even though most of the strategies for inhibiting signal production have targeted the acyl-HSL synthases, it is possible that other enzymes linked to the signal biosynthesis pathway could also be targeted. In this respect, it has been reported that triclosan inhibited the Sun Tzu on The Art of War” postulated that “In the practical art of war, the best thing of all is to take the enemy's country whole and intact; to shatter and destroy it is not so good. So, too, it is better to recapture an army entire than to destroy it, to capture a regiment, a detachment or a company entire than to destroy them” (Giles, The book “” Giles, . In this” Giles, .in vivo application of such molecules, could lead to the next generation of antimicrobial agents.In this Review, we have summarized the main targets for quorum sensing signal biosynthesis inhibition. The control of bacterial behavior by small molecules has been viewed as a promising strategy for the control of biofilms; and despite the differences among species, quorum sensing plays a crucial role in the infectious process. Although therapies that affect quorum sensing are less likely to select for resistance in comparison with traditional antibiotics, some cases reported in the literature show that bacteria can become resistant to quorum-sensing inhibitors (Defoirdt et al., OF, OS, CFN, and OLF contributed conception and design of the review. OF, PR, ÁP, WP, and OS wrote the manuscript. PR and ÁP made the figures. OLF and CFN revised the manuscript. All authors read and approved the submitted version.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.


EMSY, which is known to be amplified in breast and ovarian cancers, encodes a protein reported to bind and inactivate BRCA2. Thus, EMSY overexpression may mimic BRCA2 mutation, resulting in HR deficiency. However, to our knowledge, the phenotypic consequences of EMSY overexpression in HGSOC patients has not been explored.Approximately half of high‐grade serous ovarian carcinomas (HGSOCs) demonstrate homologous recombination repair (HR) pathway defects, resulting in a distinct clinical phenotype comprising hypersensitivity to platinum, superior clinical outcome, and greater sensitivity to poly(adenosine diphosphate‐ribose) polymerase (PARP) inhibitors. EMSY expression on clinical outcome and sensitivity to platinum‐based chemotherapy using available data from transcriptomically characterized HGSOC cohorts.Here we investigate the impact of EMSY expression was associated with better clinical outcome in a cohort of 265 patients with HGSOC from Edinburgh . Superior outcome also was demonstrated in the Medical Research Council ICON7 clinical trial and multiple publicly available data sets. Patients within the Edinburgh cohort who had high EMSY expression were found to demonstrate greater rates of complete response to multiple platinum‐containing chemotherapy regimens  and corresponding prolonged time to disease progression .High EMSY expression appear to experience prolonged survival and greater platinum sensitivity, reminiscent of BRCA‐mutant cases. These data are consistent with the notion that EMSY overexpression may render HGSOCs HR deficient.Patients with HGSOCs demonstrating high  EMSY appear to experience prolonged survival and hypersensitivity to multiple lines of platinum‐based chemotherapy. These data support the notion that overexpression of the gene product of EMSY, which is reported to bind and inactivate BRCA2, may mimic the phenotype conferred by BRCA mutation.Patients with high‐grade serous ovarian carcinomas demonstrating high expression levels of  Ovarian cancer is the most lethal gynecological malignancy, accounting for >14,000 deaths per year in the United States alone.BRCA1 and BRCA2 (BRCA) is routinely used to identify molecular subgroups that are clinically actionable. BRCA‐mutant patients experience prolonged survival, enhanced sensitivity to platinum even with multiple exposures, and a greater sensitivity to poly(adenosine diphosphate‐ribose) polymerase (PARP) inhibition by virtue of HR deficiency.Over the last decade, a wealth of molecular data have been produced in an effort to better characterize HGSOC and to identify molecular subtypes of disease with biology that may be exploited therapeutically.EMSY gene, also known as C11orf30, encodes a nuclear protein that has been identified to bind and inactivate BRCA2 and is reportedly amplified in approximately 6% to 18% of HGSOC cases and 7% to 13% of sporadic breast cancer cases.EMSY disrupts the BRCA2/RAD51 pathway after DNA damage and may override the HR players RPA and PALB2, which bind BRCA2 in the same region as EMSY.EMSY amplification may mimic those demonstrating mutational inactivation of BRCA2. Similar to BRCA mutation, EMSY amplification has been associated with poor prognosis in patients with breast cancer and is most common in the HGS histological subtype of OC.The EMSY expression and clinical outcome in patients with HGSOC has been made to date. In the current study, we sought to perform in silico analysis of available transcriptomic data to investigate whether patients with HGSOCs demonstrating high expression of EMSY experience differential clinical outcome or sensitivity to platinum‐based chemotherapy.However, to our knowledge, no association between The Edinburgh cohort comprised 265 HGSOC patients who were treated within the Edinburgh Cancer Centre between 1984 and 2006 and identified as part of a previous study of HGSOC.EMSY gene expression were extracted and per‐sample EMSY expression was calculated as the mean expression between probe sets  criteria, with the exception of the need for confirmatory scans. CA 125 tumor marker responses were reported according to Gynecological Cancer InterGroup (GCIG) guidelines.P values. Comparisons of categorical variables were performed using the chi‐square or Fisher's exact tests as appropriate. Differences in time to disease progression from receipt of platinum were evaluated using the Mann‐Whitney U test. Adjustments for multiple testing were made using the Bonferroni correction when specified.Statistical analyses were performed using R . Survival analyses were conducted using Cox proportional hazards models for progression‐free survival (PFS) and overall survival (OS). Survival differences were visualized using the Kaplan‐Meier method. Multivariable survival analyses accounted for the success of primary surgical debulking, FIGO stage at the time of diagnosis, and patient age at the time of diagnosis, with the exception of the Mateescu et al data set, in which data regarding surgical debulking and patient age were not available. Within the MRC ICON7 data set, chemotherapy regimen (bevacizumab treatment vs placebo) was also accounted for in multivariable analyses where patients from both treatment arms were analyzed together. Survival differences are presented as univariable or multivariable hazard ratios (uniHR or multiHR) alongside their corresponding 95% CIs and EMSY expression. 14% of HGSOC patients with the highest levels of EMSY expression (high‐EMSY) demonstrated prolonged OS compared with the remainder of the cohort (low‐EMSY)     Fig. .EMSY expression, the gene expression cutoff value from the Edinburgh data set was applied directly to the MRC ICON7 cohort characterized on the same gene expression platform.EMSY group within this cohort demonstrated prolonged OS when accounting for FIGO stage at diagnosis, residual disease after primary debulking surgery, trial arm (bevacizumab‐treated patients vs control arm), and age at diagnosis    Fig. , but didEMSY population demonstrated that patients in the high‐EMSY group within the bevacizumab treatment arm (14 patients) had inferior survival compared with those patients who received chemotherapy alone (10 patients)    Fig. . Within EMSY expression and superior clinical outcome, publicly available gene expression data sets were accessed using the curatedOvarianData package.EMSY expression within each data set was proposed. The validity of this approach was assessed using the MRC ICON7 control cohort arm and, consistent with results using the absolute threshold derived from the Edinburgh cohort, analysis of the 14% of HGSOCs with the highest EMSY expression demonstrated prolonged OS       Fig.  and Mate8]) Fig.  studies.6]) Fig. .26 The c8]) Fig. . NotablyP = .029])    Fig. .EMSY group    Fig. . High EMEMSY group with sampling of the primary adnexal mass  were found to demonstrate significantly superior OS     and second  platinum exposure. At the time of fourth platinum exposure, patients in the high‐EMSY group demonstrated a significantly greater objective CA 125 response rate   , which was significant when considering only progression by radiology  and CA 125 tumor marker  specifically.The median time to first  disease progression after second platinum exposure was 127 days in the high‐EMSY group remained alive without disease recurrence within the context of high‐risk disease at 2 years , 3 years , 5 years , and 10 years  from diagnosis  HGSOC with gross macroscopic residual disease after debulking surgery have particularly poor prognosis (“high‐risk” patients).P = .041), 18 months , and 2 years    and 18 months  from diagnosis .Through in silico analysis of local and publicly available transcriptomic data, we identify a subgroup of HGSOCs defined by high levels of BRCA‐mutant HGSOC, high‐EMSY HGSOCs appear to demonstrate prolonged survival across multiple independent data sets, and demonstrate a greater benefit from platinum‐based chemotherapy than their low‐EMSY counterparts. Patients in the high‐EMSY group within the Edinburgh cohort demonstrated a >3‐fold radiological complete response rate to second platinum exposure compared with patients in the low‐EMSY group.Similar to EMSY expression may be abrogated upon addition of the antiangiogenic agent bevacizumab to first‐line therapy, although the power of these analyses was severely limited. Clearly, overinterpretation of these data must be avoided in light of the low numbers of patients in the high‐EMSY group between the two treatment arms, and the presented analyses do not fulfil the REporting recommendations for tumor MARKer prognostic studies (REMARK) criteria for biomarker studies.Intriguingly, within the MRC ICON7 cohort, we demonstrated that the benefit conferred by high EMSY expression have a greater chance of remaining free of disease recurrence (12.5% vs <1% at 10 years from diagnosis within the Edinburgh cohort), suggesting that patients in the high‐EMSY group could represent a group with favorable long‐term clinical outcome even in the face of otherwise poor prognostic markers.Advanced stage of disease at the time of diagnosis and suboptimal surgical resection both are associated with markedly inferior survival in patients with HGSOC.EMSY expression was associated with prolonged survival, this phenomenon was not apparent in patients in whom expression data were generated using samples that were not taken from the primary adnexal mass. There are several plausible explanations for this observation. First, differences in the tumor microenvironment at different anatomic sites may well have impacted EMSY expression. Second, the contamination of tumor samples with different nonmalignant cell types has the potential to alter bulk EMSY expression. Third, intratumor heterogeneity as a result of tumor evolution may have resulted in differential tumor cell EMSY expression between the primary tumor mass and more distant disease sites. In any respect, differential gene expression between sampling sites has clear implications for the identification of pertinent molecular events in both the clinical and research setting. In particular, the frequency of extra‐adnexal sampling may account for weakened trends between EMSY expression and clinical outcome in some data sets. Transcriptomic characterization of the EdinburghEMSY group.Within the data set from the study by Tothill et al,EMSY expression who appear to demonstrate improved clinical outcome and greater sensitivity to platinum‐based chemotherapy, consistent with the notion that EMSY overexpression renders HGSOCs HR deficient. Given the role of EMSY in disrupting the BRCA2/RAD51 HR pathway demonstrated by previous studiesBRCA‐like clinical phenotype described here in patients with high‐EMSY HGSOC, investigation of the sensitivity of EMSY‐overexpressing tumors to PARP inhibition has the potential to improve our understanding of which patients benefit the most from these agents. The correlation of EMSY expression with existing HR deficiency signatures and scores should also be explored. Moreover, the impact of differential EMSY expression in other disease settings, most notably breast and prostate cancer, should now be investigated.These data demonstrate the power of in silico analysis of preexisting data sets to identify novel, clinically meaningful subtypes of disease. Collectively, the current study findings allude to a subgroup of HGSOC patients defined by high levels of Funded by a Medical Research Council PhD Studentship and Medical Research Council‐funded Research Fellowship awarded to Robert L. Hollis, and by charitable donation from The Nicola Murray Foundation.Laura Knight, Andrena McCavigan, D. Paul Harkin, and Richard Kennedy are employees of Almac Diagnostics, a precision medicine company focused on the discovery, validation, and commercialization of novel diagnostic and companion diagnostic tests. Gordon C. Jayson has received grants from Roche and AstraZeneca for work performed outside of the current study. Charlie Gourley has received honoraria from AstraZeneca, Tesaro, Cor2Ed, Medscape, and Sierra Oncology; has acted as a paid consultant for AstraZeneca, Clovis, NuCana, Tesaro, Roche, Foundation One, and Cor2Ed; and is named on 1 patent issued (patent PCT/US2012/040805) and 4 pending patents  related to gene expression signatures predicting treatment response in ovarian cancer and his institution receives research funding from AstraZeneca, Aprea AB, NuCana, and Tesaro for work performed outside of the current study. The other authors made no disclosures.Robert L. Hollis: Conceptualization, data curation, formal analysis, investigation, visualization, writing–original draft, and writing–review and editing. Michael Churchman: Conceptualization and writing–review and editing. Caroline O. Michie: Investigation. Tzyvia Rye: Data curation. Laura Knight and Andrena McCavigan: Methodology. Timothy Perren, Gordon C. Jayson, Richard S. Kaplan, Amit Oza, D. Paul Harkin, and Richard Kennedy: Resources and writing–review and editing. Alistair Williams and W. Glenn McCluggage: Investigation and writing–review and editing. C. Simon Herrington: Investigation, supervision, conceptualization, and writing–review and editing. Charlie Gourley: Conceptualization, supervision, funding acquisition, writing–original draft, and writing–review and editing. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.