New chemical tools to ‘trap’ post translational modification (PTM)-mediated protein–protein interactions. e.g., phosphorylation, acetylation and methylation) of histone play important roles in regulating many fundamental cellular processes such as gene transcription, DNA replication and damage repair. While ‘writer’ and ‘eraser’ enzymes modify histones by catalyzing the addition and removal of histone PTMs, ‘reader’ proteins recognize these modified histones and ‘translate’ the PTMs by executing distinct cellular programs. Therefore, identification of the regulating enzymes and binding partners of histone PTMs is essential for understanding their regulatory mechanisms and cellular functions. Here we report the development of diazirine-based photoaffinity probes for identification of ‘readers’ and ‘erasers’ of histone PTMs. When compared with previously described benzophenone-based photoaffinity probes, the present probes demonstrate significantly improved photo-cross-linking rates, yields and specificities for capturing proteins that recognize a trimethylation mark on histone H3 lysine 4 (H3K4Me3). Furthermore, we show that the diazirine-based probes can also be used to identify enzymes that catalyse the removal of histone lysine acetylation and malonylation. This study provides new chemical tools for examining PTM-mediated protein–protein interactions and broadens the scope of our photo-cross-linking strategy from finding histone PTM ‘readers’ to identifying dynamic and transient interactions between PTMs and their ‘erasers’.Post translational modifications (PTMs,  These photoaffinity probes, in combination with stable isotope labelling amino acid in cell culture (SILAC)-based quantitative proteomics technology, have been used to profile the ‘readers’ of histone H3 trimethylation at lysine 4 (H3K4Me3) and lysine 9 (H3K9Me3) and phosphorylation at threonine 3 (H3T3-Phos).9 However, this chemical proteomics approach has suffered from low protein labelling efficiency and specificity. For example, in a model study, only less than 5% of total cellular inhibitor of growth protein 2 (ING2), a known histone H3K4Me3 ‘reader’, was labelled by an H3K4Me3 peptide-based photoaffinity probe (probe 1),7 and the quantitative proteomics analysis revealed that 98% of proteins captured by probe 1 were not specific binders of the histone H3K4Me3 mark.9 All of these problems are likely caused by the low efficiencies and specificities of the photo-cross-linking reactions mediated by the benzophenone moiety in probe 1.To link a particular histone PTM to its downstream biological functions requires comprehensive identification of the ‘readers’ of the PTM. However, it is challenging to find PTM-mediated protein–protein interactions, as PTMs are often present on a small fraction of proteins, with dynamic, or mediating transient and weak interactions. To address this difficulty, a chemical approach has been developed to ‘trap’ proteins that recognize histone PTMs.10 Besides, steric hindrance of bulky benzophenone is likely to disrupt the protein–probe interactions when it is incorporated too close to the binding sites. In addition to benzophenone, arylazide and diazirine are another two commonly used photoreactive groups. However, the application of arylazide as a photo-cross-linker is limited as it tends to photoisomerize to less reactive dehydroazepine, which leads to low cross-linking yields and specificities.11 In addition, arylazide also has a relatively large size that may cause steric hindrance. Diazirine, on the other hand, exhibits more advantages for photoaffinity labelling, including its small size, its short lifetime upon UV irradiation and high subsequent reactivity.16 Taking these aspects into consideration, here we use diazirine as a photoreactive group to develop new photoaffinity probes for the identification of histone PTM-mediated protein–protein interactions. We directly compare the benzophenone- and diazirine-based probes for their photo-cross-linking rates, yields and specificities in complex protein mixtures and for their steric effects on PTM-mediated protein–protein interactions. We show that the diazirine-based probes have significantly improved performances for capturing histone H3K4Me3 ‘readers’. We further demonstrate that our diazirine-based photoaffinity probes can also be used to identify enzymes that catalyse the removal of histone PTMs , including the lysine deacetylases and demalonylase.It is known that benzophenone requires a long period of irradiation and that its excitation is reversible, which may lead to non-specific labelling.3 mark as a model system. We designed probes 2 and 3 (3 peptide (residues 1–15) in which two diazirine-containing amino acids, photo-leucine (photo-Leu) and photo-methionine (photo-Met), respectively, were used to replace Ala 7, the residue that was replaced by benzoyl phenylalanine (Bpa) in the design of probe 1. To study the steric effects of the two photoreactive groups on the protein labelling efficiency, probes 4 and 5  as a starting material.19 To avoid these problems, we developed an efficient six-step route to synthesize Fmoc-protected photo-Leu (l-aspartic acid α-methyl ester (i) was transformed into the side-chain β-ketoester ii by reaction with monobenzyl malonate. Hydrogenolysis of ii using Pd/C and H2 followed by a simultaneous decarboxylation smoothly gave ketone iii,20 which was then treated with LiOH to hydrolyze the methyl ester, affording carboxylic acid iv. The diazirine moiety was then installed by reaction of iv with liquid ammonia followed by hydroxylamine-O-sulfonic acid to give the diaziridine intermediate, which was subsequently oxidized to give v.19 Deprotection of Boc and reprotection with Fmoc-OSu gave compound vi and Fmoc-protected photo-Leu (vii), respectively. The overall yield of vii was 20%. Fmoc-photo-Leu and Fmoc-photo-Met were then used to assemble the diazirine-based peptide probes through solid-phase peptide synthesis (SPPS) with a Fmoc-protection strategy.We first synthesized Fmoc-protected photo-Leu and photo-Met as building blocks for the assembly of the corresponding photoaffinity peptide probes. Fmoc-protected photo-Met was synthesized in five steps following a procedure described in the literature.hoto-Leu . Briefly1 and diazirine-containing probes 2 and 3 to covalently label a known H3K4Me3 ‘reader’, spindlin 1 (SPIN1),21in vitro. The recombinant GST-fused mouse SPIN1 was incubated with probes 1, 2 and 3, respectively, and irradiated with UV light for 1 h. The probe-labelled proteins were then conjugated with rhodamine azide (Rho-N3) via Cu(i)-catalysed azide–alkyne cycloadditions (“click chemistry”). The resulting reaction mixtures were resolved by SDS-PAGE and analysed by in-gel fluorescence scanning. The results showed dose-dependent labelling of SPIN1 by all probes, and the labelling was saturated with around 2 μM of each probe  with an amine-reactive rhodamine dye that has the same fluorophore as Rho-N3. After determining the degree of labelling, this rhodamine-labelled BSA (Rho-BSA) was used as a standard to generate a calibration curve for the quantitative analysis of the proteins labelled by the photoaffinity probes  within the first 20 minutes. In contrast, probe 1 labelled only 19% of SPIN1 after 90 minutes irradiation. Inspired by this result, we sought to test whether probes 2 and 3 could also mediate efficient labelling of other histone H3K4Me3 ‘readers’. To this end, we chose another two known H3K4Me3 binders, ING223 and MORC39 that recognize this histone mark with their PHD finger and ZW finger, respectively. Together with SPIN1 that binds H3K4Me3 with its double-tudor domain,24 these three chosen proteins represent ‘readers’ with three types of distinguished structural domains for H3K4Me3 recognition. Each protein was then labelled by probes 1–3 for 20 minutes, respectively, and the labelling yields were determined using the method described above. As shown in 1, whereas probes 2 and 3 labelled the protein with 33% and 20% yields, respectively, indicating the largely improved photo-cross-linking efficiency of these diazirine-based probes. Unlike SPIN1 and ING2, no significant difference in the labelling yields of MORC3 by probes 1–3 was observed  with 10-fold excesses of four other proteins that have not been related to H3K4Me3, including BSA, Sirt3, Sirt5 and MenE. Probes 1–3 were then each incubated with this protein mixture. After UV irradiation and click chemistry, the mixtures were resolved by SDS-PAGE and analysed by in-gel fluorescence scanning. As shown in 2 and 3 specifically labelled SPIN1 without interference from the other 4 proteins present in large excesses in the mixtures. However, although able to capture SPIN1, probe 1 showed much stronger labelling of Sirt3, which is not supposed to interact with the histone H3K4Me3 peptide.We next examined the specificity of the photo-cross-linking mediated by the benzophenone- and diazirine-containing probes. We mixed one specific H3K4Me1 is not caused by a specific recognition of the histone H3K4Me3 mark, we compared the behaviours of SPIN1 and Sirt3 in a series of photo-cross-linking experiments. Interestingly, similarly to SPIN1, Sirt3 was labelled only by probe 1 and not by probe C1, an unmodified H3 control probe, indicating that the labelling of Sirt3 is K4-methylation-dependent. However, unlike for SPIN1, the labelling of Sirt3 by probe 1 (2 μM) cannot be competed with by an H3K4Me3 peptide (30 μM)  . This re3 ‘readers’ in complex proteomes. SPIN1 was added to HeLa S3 cell lysates and subjected to photo-cross-linking reactions using probes 1 and 2. We found that while both the probes labelled SPIN1 in the cell lysates, probe 2 demonstrated a more robust labelling of SPIN1 with much higher ‘signal-to-noise’  ratio when compared with probe 1  via click chemistry, followed by streptavidin enrichment. The enriched proteins were then resolved by SDS-PAGE and subjected to a western blot analysis using an antibody against endogenous ING2. As shown in 1 and probe 2 can capture endogenous ING2, the final ‘pull-down’ yield of probe 2 (∼8%) is four times higher than that of probe 1 (∼2%). This result further demonstrates the superiority of diazirine-based photoaffinity probes for identifying histone PTM readers in complex proteomes.We then compared the abilities of probe 3 ‘readers’, we sought to test whether this photoreactive group could be used to develop chemical probes for the identification of more dynamic or transient interactors, such as enzymes that remove histone PTMs . To develop this method, we focused on sirtuins, a family of enzymes known as nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylases.25 There are seven human sirtuins called Sirt1–Sirt7. While Sirt1–Sirt3 demonstrate robust deacetylase activities, Sirt4–Sirt7 have very weak or undetectable deacetylase activities. However, recent studies have revealed that these sirtuins with weak deacetylase activities can function as deacylases for the removal of different acyl groups from protein lysine residues.26 For example, it was recently demonstrated that Sirt5 preferentially hydrolyzes malonyl and succinyl lysine  and malonylation . These two probes are based on a histone H3 peptide (residues 1–15), where residue Lys 9 was acetylated and malonylated in probes 6 and 7, respectively. In addition, photo-Leu was incorporated at the Thr 6 position as the photoreactive moiety, and propargyl glycine was attached at the C-termini for bioorthogonal conjugations in these probes (Inspired by the high efficiency of the diazirine-based probes in the labelling of H3K4Mel lysine .27–29 Bae probes .6 and 7 can capture their corresponding ‘erasers’, we performed photo-cross-linking experiments using probes 6 and 7 to react with recombinant human Sirt3 and Sirt5, respectively. As shown in 6 and 7 successfully labelled Sirt3 and Sirt5, respectively, whereas an unmodified control probe C2 failed to label either of these two enzymes. To analyse the labelling specificity, Sirt1, Sirt3 and Sirt5 were photo-cross-linked with probes 6 and 7. Among the three sirtuins, only Sirt1 and Sirt3 were labelled by probe 6, while probe 7 specifically labelled Sirt5 (Fig. S46 and 7, respectively, in total cell lysates of human HeLa S3 cells (To examine whether probes 5 Fig. S4. This reS3 cells .6 and 7 could also be used to label endogenous Sirt3 and Sirt5, respectively, from whole cell lysates. Probes 6, 7 and C2 were each added to HeLa S3 lysates, followed by UV-radiation, click chemistry using biotin-N3, SDS-PAGE and western blot analysis using antibodies against Sirt3 and Sirt5. As shown in 6 and 7, respectively, whereas probe C2 failed to capture these enzymes. Taken together, these data suggest that the photoaffinity probes can be used to ‘trap’ ‘erasers’ of histone lysine acylations from complex proteomes.Finally, we examined whether probes We have developed diazirine-based photoaffinity probes and examined their ability to capture the ‘readers’ of histone lysine methylation as well as the ‘erasers’ of histone lysine acetylation and malonylation. When compared with previously reported benzophenone-based photoaffinity probes, these new probes demonstrated higher photo-cross-linking efficiencies and specificities in the tested systems. A key issue in the design of photoaffinity probes is to determine where a photoreactive group should be placed. Our study showed that to ensure robust labelling of histone PTM ‘readers’, diazirine, due to its small size, has more flexibility with respect to incorporation sites, while bulky benzophenone is likely to ‘bump’ against ‘readers’ if it is placed at a ‘wrong’ position. In addition, the small size of diazirine also enables it to be incorporated closer to PTM sites, which is expected to facilitate more efficient and specific labelling of proteins that recognize the PTMs. These advantages therefore make diazirine a better choice for the development of photoaffinity probes to identify histone PTM ‘readers’, particularly when structural information to guide the design of probes is lacking. Furthermore, we demonstrated that the diazirine-based probes can also be used to capture lysine deacetylases and demalonylase. This study has therefore broadened the scope of our photo-cross-linking strategy from finding histone PTM ‘readers’ to identifying dynamic and transient interactions between PTMs and their ‘erasers’. We anticipate to use this strategy in conjunction with SILAC technology to comprehensively profile ‘erasers’ of newly identified histone PTMs such as lysine crotonylation. These studies will be reported in due course.


Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome.IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells. Macroautophagy, here referred to as autophagy, is an ancient survival mechanism . SimilarAutophagy is also upregulated during infection with varicella-zoster virus (VZV), human herpesvirus 3, another herpesvirus of a more recent lineage in geologic time, perhaps 70 mya . VZV is 7–Among the herpesviruses, VZV is renowned for its extreme cell association with impaired egress of viral particles and low infectivity; the particle-to-PFU ratio is 40,000:1 –14. Upon–NC_001348) (VZV strains included the low-passage-number laboratory strain VZV-32 and the attenuated VZV Oka strain (vOka); both strains have been sequenced , 21. Eac_001348) . The vir_001348) . The tec_001348) .Antibody reagents included anti-VZV murine monoclonal antibodies (MAbs) 3B3 , 5C6 (anti-ORF62), and 251D9 (anti-ORF41), produced in our laboratory . HoechstOur laboratory reported several studies between 1979 and 1983 that detailed methodology for VZV virion purification and enumeration of the structural and nonstructural viral proteins –29. Init–2 infected MRC-5 cell monolayer and layered in a 1-ml aliquot onto a preformed KT-glycerol gradient. The gradient was prepared by mixing 7 ml of 50% (wt/vol) KT and 8 ml of 30% (vol/vol) glycerol in a dual-chambered gradient maker feeding into a 16- by 102-mm centrifuge tube . The gradient was sedimented for 16 h at 115,000 × g in a Beckman SW27 rotor at 4°C. After the first sedimentation, there was an obvious particular band (infectivity virus band) in the lower one-third of the gradient and a less apparent opalescent band (light particle band) in the upper one-third. Each band was removed and placed onto a second potassium tartrate-glycerol gradient for a second sedimentation under identical conditions. After the second sedimentation, the lower and upper bands were removed and diluted, and their contents were concentrated by a subsequent sedimentation for 1 h at 124,000 × g.For the above-mentioned reasons, cell-free virus required for the studies outlined below was harvested from a 150-cmMonolayers grown on glass coverslips were labeled with antibodies and fluorophores as described above, mounted onto glass slides, and then viewed with a Zeiss LSM710 confocal laser scanning microscope, using 10× and 20× dry objective lenses and 40× and 63× high-numerical-aperture oil immersion objective lenses , 34. TheConfocal images (z-stacks acquired by using the 63× objective) from each experiment described below in Results were analyzed by using the Cell Counter plug-in for ImageJ . At leasPelleted viral fractions from the KT-glycerol gradients were resuspended in 200 μl of phosphate-buffered saline (PBS) after a short (5-s) sonication burst. Next, 25 μl of this material was dropped onto carbon-coated transmission electron microscopy (TEM) grids (Formvar-coated nickel TEM grids that were carbon coated) and incubated at room temperature (RT) for 1 h, with occasional mixing by movement of the grids in the drop. Washing of TEM grids was done by briefly dipping the grids into water and then suspending the grids on drops of water (25 μl). After adsorption of viral material onto the grids, the grids were washed and then blocked in 5% nonfat milk with 5% normal goat serum, and the grids were incubated in drops of primary antibody (1:50 dilution) at RT for 4 h and then overnight at 4°C. After washing, the grids were incubated with secondary immunogold antibody  for 4 h at RT. After washing, the grids were suspended in freshly prepared silver enhancement solution (Aurion) for 30 min, washed with water, and then negatively stained with 2.5% uranium acetate drops for 2 min at RT. The uranyl acetate was wicked off with filter paper, and the grids were allowed to air dry overnight. Grids were viewed with a JEOL 1230 transmission electron microscope.Cells were scraped into minimal essential medium (MEM) containing 1% gelatin with a rubber policeman. In a series of centrifugation and medium replacement steps, the cells were fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in PBS for 30 min at RT, followed by increasing concentrations of warm gelatin  in PBS. The final gelatin solution was allowed to solidify at RT and then placed at 4°C overnight. The solid gelatinized cells were incubated in a 2 M solution of sucrose in PBS at RT overnight. The resulting solid sample was plunge frozen in liquid nitrogen, and thin sections were cut with a cryoultramicrotome and collected on grids. The resulting sections were then antibody labeled as described above, poststained with 2.5% uranium acetate and 2% lead citrate for 5 min, and then washed and dried before viewing with a JEOL 1230 transmission electron microscope.Our previous studies of autophagy documented the important observation that autophagic flux is not inhibited by VZV infection. In fact, the converse is true; namely, inhibition of flux inhibits virus spread and viral titers. Based on these observations, we postulated that components of the autophagy pathway were contributing to the VZV infectious cycle. Also, based on extensive previous imaging studies, we postulated that any intersection of the autophagy and endosomal pathways would be detectable in VZV-infected cells by probing the pathways for a major marker of the autophagy pathway, microtubule-associated protein 1 light chain 3B (MAP1LC3B or simply LC3B). Confocal laser scanning microscopy is an ideal technology for these investigations because its resolution and optical sectioning capacities facilitate the visualization of structures with a diameter of 400 to 1,000 nm, such as an autophagosome .We had not observed colocalization in previous autophagy studies when we examined cultures labeled with individual antibody fluoroprobes to LC3B and VZV gE. We proposed that our high-affinity murine anti-VZV gE MAb 3B3 may have obscured binding by a lower-affinity polyclonal anti-LC3B antibody in previous experiments. For the present experiments, we added the anti-LC3B antibody before the addition of the high-affinity anti-gE antibody. Using this strategy, we observed some colocalization between LC3B and VZV gE in cytoplasmic vesicles . Because+) puncta per cell than LC3B+ puncta per cell . Near the outer cell membrane, the spots are oriented in linear rows called viral highways in a previous report .2) were harvested and then subjected to sonication. Two viral strains were included: the wild-type VZV-32 strain and the attenuated VZV Oka vaccine strain. An uninfected monolayer was subjected to the same experimental conditions. After removal of cellular debris from a sonicated monolayer by low-speed sedimentation, infectious virus was purified from the supernatant by an ultracentrifugation protocol: density-viscosity gradients made from potassium tartrate and glycerol. After a 16-h high-speed sedimentation, the virus-enriched band (V) (infectivity fraction) and the light particle band (L) (fraction with noninfectious enveloped particles lacking a capsid) were isolated from the first density gradient, and each band was then layered onto a separate second density gradient for a second high-speed sedimentation in an ultracentrifuge , 2726, 2ntrifuge . UninfecAs shown in In order to verify that the infectious virus fraction and the light particle fraction in the second sedimentations contained viral products, aliquots were solubilized and probed for the predominant VZV structural glycoprotein gE. VZV gE has been the standard marker for VZV virions since the earliest studies of VZV purification methods, because the protein is easily detectable by Western blotting with MAb 3B3, which binds to a defined linear epitope (amino acids 150 to 162) on the N terminus , 38.As shown in To further pursue our hypothesis about a convergence of the viral secondary envelopment-and-trafficking pathway with the autophagy pathway, we investigated whether the lipidated form of the LC3 protein (LC3-II) was detectable in either the purified virus fraction or the light particle fraction after the second sedimentation . When thFurthermore, we also probed for the Rab11 protein, based on the data from confocal microscopy  39, 40), 4039, 4Under the secondary envelopment model of herpesvirus assembly, viral capsids migrate from the nucleus after deenvelopment and enter vesicles in the cytoplasm that are populated by viral envelope and tegument proteins as well as cellular proteins . The cytSubsequently, we immunolabeled the viral fractions with LC3B and Rab11 antibodies. We were at a disadvantage because the binding affinities of the latter antibodies have not been characterized. Therefore, each of the following experiments was performed with at least two TEM grids per antibody. We also compared the sensitivities of secondary antibodies attached to ultrasmall gold beads (with silver enhancement) or 10-nm gold beads (without silver enhancement). Numerous images were taken, and representative examples are shown in In order to evaluate whether both Rab11 and LC3B can be found on the same viral particle, we carried out a cryo-EM experiment whereby infected cells were embedded in gelatin, frozen, and then sectioned. This TEM method is preferred for greater antibody binding with a less-well-defined ultrastructure. Three viral particles were seen on the surface of an infected cell . Of impoFor a negative-control experiment, we applied the antireovirus MAb (isotype IgG2a) along with secondary goat anti-mouse antibody (10-nm gold beads) to the grids and examined them by TEM. We enumerated a total of 112 viral particles; only 2 viral particles were observed with an adjacent gold bead. A representative micrograph is shown in Because of the above-described observations about (i) copurification of LC3B and Rab11 proteins with the sedimented virus band (containing gE) as well as (ii) colocalization of the same two proteins with gE, we pursued further investigations by TEM. First, we wanted to confirm that viral particles were not found in typical double-membraned autophagosomes within VZV-infected monolayers , 16. As Based on autophagy data in the HSV literature, we investigated whether an alternative source of LC3-II in VZV particles may be LC3A resident in the nuclear membrane. LC3A is localized primarily in the nuclear and perinuclear regions, while LC3B is found throughout the cytoplasm . All aboThis study provides insight into a previously unrecognized relationship between autophagy and the VZV infectious cycle. A proviral role for autophagy during herpesvirus infection was initially overlooked. The main explanation was the frequently cited pivotal report in 2007 that identified the HSV-1 neurovirulence protein ICP34.5 as an inhibitor of autophagy  The neurThe induction of autophagy by VZV infection is easily observed in cultured cells by confocal laser scanning microscopy , 50. Whetrans-Golgi network and/or recycling endosome  researchers recently reported finding LC3 in the EBV virion, but further characterization of other autophagy proteins was not performed . Little endosome , 54. Of endosome , 55. Forendosome , 57. VZVendosome , 58.+) that fuses with an autophagosome (LC3+) to form a single-walled vesicle containing both Rab11 and LC3-II; these vesicles in turn traffic to the outer cell membrane. Based on the data in this report, we therefore propose that there are at least 2 pathways by which virus can egress from the infected cell. The first pathway  containing only 1 or 2 particles remain intact (or mainly intact) throughout the purification process and therefore are detected by LC3/Rab11 immunoblotting in the virus band. The only other single-walled compartment with the LC3-II protein in its outer membrane is the autolysosome. Since the autolysosome transitions into a lysosome and does not traffic to the outer cell membrane, we eliminated the autolysosome as a candidate trafficking vesicle for viruses.In a recent commentary , amphisoIn a recent comprehensive review of secondary envelopment of alphaherpesviruses, the autophagy pathway was not recognized as a contributor . TherefoSome RNA viruses exploit endosomal and autophagy pathways for their replication and release from cells . Poliovi


Casson nanofluid, unsteady flow over an isothermal vertical plate with Newtonian heating (NH) is investigated. Sodium alginate (base fluid)is taken as counter example of Casson fluid. MHD and porosity effects are considered. Effects of thermal radiation along with heat generation are examined. Sodium alginate with Silver, Titanium oxide, Copper and Aluminum oxide are added as nano particles. Initial value problem with physical boundary condition is solved by using Laplace transform method. Exact results are obtained for temperature and velocity fields. Skin-friction and Nusselt number are calculated. The obtained results are analyzed graphically for emerging flow parameters and discussed. It is bring into being that temperature and velocity profile are decreasing with increasing nano particles volume fraction. Fluids are mainly of two type’s i.e Newtonian and non-Newtonian. Non-Newtonian fluids have numerous industrial applications3. Furthermore, its application with magnetohydrodynamic (MHD) flow in a porous medium can widely be seen in irrigation problem, biological system, petroleum, textile, polymer industries. More investigations have been published on numerous aspects of MHD non-Newtonian fluid passes over a porous medium7. The entropy analysis for nanofluid with different type of nano particles and water type base fluid for unsteady MHD flow was studied by8. The impact of magnetic field on free convection of nanofluid in a porous medium is presented by9. The effects of heat transfer on MHD nanofluid in a porous semi annulus has investigated by10 using numerical methods. Sheikholeslami et al.11 examined the influence of free convection in a semi annulus enclosure for ferrofluid flow in the presence of magnetic source with the consideration of thermal radiation. The observation of non-uniform magnetic field and variable magnetic field on forced convection heat is investigated by13. The observation of MHD on fluid flow with heat transfer is studded by16. Recently18 investigated the nanofluid transportation in a in the presence of magnetic source and porous cavity using CuO nano particles. The influence of external magnetic field for nanofluid as water is a base fluid of free convection flow is studied in19. Sheikholeslami and Ganji20 have investigated the effect of convective heat transfer for the nanofluid by semi analytical and numerical approaches. The same author has also investigated the influence of heat transfer for nanofluid between parallel plates in21. The influence of Lorentz forces and convection nanofluid flow is investigated by24. Dissimilar types of nano particles with water based fluid are studied by26. The influence of melting heat for nanofluid is studied by27. The transportation of nanofluid in porous media is investigated by28. The influence of magnetic field for nanofluid with entropy generation is analysed by31.The fluid is a particular kind of matter which have no fixed shape and deforms easily due to external pressure32. Carbon based nano materials, metal based nano materials, Dendrimers and composite. The terminology of nanofluid was first investigated by Choi33. He defined that the fluids occupying the sizes of particles less than 100 nm is called nanofluid. The categorieswith different attitude of nano particles are particle material, Base fluid, size and concentration, of the nanofluid. Suspend these nano particles into any type of conventional fluid like oil, water, ethylene glycol to make nanofluids. The reason why nano size particles are preferred over micro size particles has been explained by34. Nano particles over micro particles, good improvement have seen in thermo physical properties. Nanofluids have various applications such as in air conditioning cooling, automotive, power plant cooling, improving diesel generator efficiency etc.35. Usually water, ethylene glycol are utilized as heat transfer base fluids. Different substances are used for the production of nanoparticles, which are generally divided into metallic i.e. copper36, metal-oxide i.e. CuO37, chalcogenides sulphides, selenides and telluride’s, mentioned38 and different particles, such like carbon nanotubes39. In literature the size of one particle is in between 20 nm40 and 100 nm41.Nanotechnology is that kind of technology which provides the materials with size less than 42. Examples of Casson fluids are honey, blood, soup, jelly, stuffs, slurries, artificial fibers etc. Cassonnanofluid flow with Newtonian heatingpresented by43. Sarojamma et al.44 investigated Casson nanofluid past over perpendicular cylinder in the occurrence of a transverse magnetic field with internal heat generation or absorption.Casson fluid model was first presented by Casson in 1959. Casson fluids in tubes was first studied by Okaet al.45 examined unsteady MHD Casson fluid withfree convection flow in a porous medium. Bhattacharyya et al.46 studied systematically magnetohydrodynamic Casson fluid flow over a stretching shrinking sheet with wall mass transfer. Arthur et al.47 studied Casson fluid flow in excess of a perpendicular porous surface, chemical reaction in the existence of magnetic field. Recently, Fetecau et al.48 has investigated fractional nanofluids for natural convection flow over an isothermal perpendicular plate with thermal radiation. Hussanan et al.49 investigates the unsteady heat transfer flow of a non-Newtonian Casson fluid over an oscillating perpendicular plate with Newtonian heating. Recently, Imran et al.50 analyzed the effect of Newtonian heating with slip condition on MHD flow of Casson fluid. MHD flow of Casson fluid with heat transfer and Newtonian heating is analyzed by Hussanan et al.51. The effect of Newtonian heating for nanofluid is recently investigated by52. But no work is done until now on heat transfer enhancement in Sodium alginate fluid with additional effects of NH, MHD, porosity, heat generation, and thermal radiation. Silver (Ag), Titanium oxide (TiO2), Copper (Cu) and Aluminum oxide (Al2O3) are nano particles suspended in base fluid. Problem is solved and interpreted graphically with some conclusions.Khalid Ag), Titanium oxide (TiO2), Copper (Cu) and Aluminum oxide (Al2O3) nano particles is considered. Heat transfer, thermal radiation and heat generation are taken. Unsteady flow is over an infinite vertical plate (ξ > 0) embedded in a saturated porous medium. MHD effect with uniform magnetic field B of strength B0 and small magnetic Reynolds number. Initially both the plate and fluid are at rest with constant temperature Θ∞. At time t = 0+ the plate originates oscillation in its plane ξ = 0 according to conditionSodium alginate with Silver ah factor of the deformation rate, π is represent the product of the factor of deformation rate with itself, πc is represent the critical value of this product based on the non-Newtonian model, μη is represent the plastic dynamic viscosity of the non- Newtonian fluid and Pλ is yield stress of fluid. Under these conditions alongside with the assumption that the viscous dissipation term in the energy equation is neglected, we get the following system56:k* is absorption coefficient and σ* is Stefan-Boltzmann constant. Where Q0 is the heat generation term, ρnf is the density of nanofluids, μnf is the dynamic viscosity, u is the fluid velocity in the γ is the Casson fluid parameter, ψ(0 < ψ < 1), K > 0, ψ is the porous medium and K is the permeability of porous medium, hs is a constant heat transfer coefficient, Θw is the constant plate temperature , g is the acceleration due to gravity, and βnf is the thermal expansion coefficient of the nanofluid.After some time, plate temperature is raised to Θρcp)nf, (ρβ)nf, μnf, ρnf, σnf, knf are given by24:ϕ the volume fraction of nano particles, ρf and ρs is represent the density of base fluid and particle respectively, and cp is specific heat on constant pressure. knf, kf, and ks are the thermal conductivities of the nanofluid, the base-fluid, and the solid particles, respectively. The expressions of Eq. .Taking , in Eq. , it modeSkin friction and Nusselt Numberγ, ϕ, Gr, M, K, Pr, Nr Figs γ on u which shows oscillatory behavior increasing first then decreasing is highlighted in Fig. In this section different parameters including ϕ on u and θ.φ is take in between 0 ≤ ϕ ≤ 0.04 due to sedimentation when the range goes above from 0.08. It is observed in both cases if the nano particles volume fraction ϕ is increased it leads to the decreasing of temperature and velocity profile.Figures Gr for Sodium alginate -based, Casson nanofluids on velocity profile. It is found that with increasing Gr, velocity increases. Because increasing effect in Gr, due to increase of buoyancy forces and decrease of viscous forces. Figure M = 0, 1, 2 on the velocity profile. u decreases due to increasing dragging force. M = 0, shows absence of MHD. Figure K effect of on u. Velocity decrease due to decreasing friction. Figure Nr. The effect is studied for TiO2 nano particle.Figure Al2O3 Sodium alginate -based Casson nanofluid and Ag-Sodium alginate -based nanofluid) on profile of velocity is studied in Fig. Al2O3 Sodium alginate -based Casson nanofluid and lower profile velocity for Ag-Sodium alginate -based nanofluid is observed.The impact of two different types of nano particles  on u. Velocity of Ag-Sodium alginate -based nanofluid is lower than copper Sodium alginate -based nanofluid. This shows that Cu nano particles have more thermal diffusivity compare to Ag which is physically true. Furthermore, the same comparison is study for Al2O3 and TiO2 models in Fig. Al2O3 nano particles have high thermal diffusivity as compare to Titanium oxide TiO2.Figure u decreases as γ increasesTemperature and velocity profile are decreasing with increasing nano particles volumeϕ.Fraction Al2O3 nanofluid has higher velocity from TiO2 nanofluid and Cu nanofluid has higher velocity from Ag nanofluid.K and MHD Μ show opposite behavior.The pours medium The following remarks are concluded from this work:


Little empirical evidence exists on the effectiveness of using Twitter as a two-way communication tool for public health practice, such as Twitter chats.We analyzed whether Twitter chats facilitate engagement in two-way communications between public health entities and their audience. We also describe how to measure two-way communications, incoming and outgoing mentions, between users in a protocol using free and publicly available tools .We used a mixed-methods approach, social network analysis, and content analysis. The study population comprised individuals and organizations participating or who were mentioned in the first #LiveFitNOLA chat, during a 75-min period on March 5, 2015, from 12:00 PM to 1:15 PM Central Time. We assessed audience engagement in two-way communications with two metrics: engagement ratio and return on engagement (ROE).The #LiveFitNOLA chat had 744 tweets and 66 participants with an average of 11 tweets per participant. The resulting network had 134 network members and 474 engagements. The engagement ratios and ROEs for the #LiveFitNOLA organizers were 1:1, 40% (13/32) (@TulanePRC) and 2:1, −40% (−25/63) (@FitNOLA). Content analysis showed information sharing  and health information  as the most salient theme and sub-theme, respectively.Our findings suggest Twitter chats facilitate audience engagement in two-way communications between public health entities and their audience. The #LiveFitNOLA organizers’ engagement ratios and ROEs indicated a moderate level of engagement with their audience. The practical significance of the engagement ratio and ROE depends on the audience, context, scope, scale, and goal of a Twitter chat or other organized hashtag-based communications on Twitter. Approximately a quarter 23%) of American adult age 18+) Internet users and a fifth (19%) of the entire American adult population use Twitter [8+ Intern3% of AmePublic health practitioners use Twitter for disease surveillance -7, inforCommercial Twitter engagement measuring tools exist; many include metrics such as the number of “likes” and the click-through rate in their engagement metrics. However, they do not focus on the incoming and outgoing communications that occur when one user mentions another user in a tweet. In addition, they require a purchasing fee or a recurring membership fee.Our primary aim was to analyze whether Twitter chats facilitate engagement in two-way communications between public health entities and their audience. We also describe how to measure this two-way communication, incoming and outgoing mentions, between users in a protocol using free and publicly available tools  organized by Tulane Prevention Research Center (@TulanePRC), a university-based research and education center, and the City of New Orleans Health Department’s Fit NOLA Initiative (@FitNOLA). Twitter chats are interactive, organized, and curated communications on Twitter. They focus on a specific topic and take place at predesignated times.With a mixed-method approach, social network analysis (SNA), and content analysis, we analyzed (1) whether Twitter chats facilitated audience engagement among public health entities and their audience and (2) whether functional themes were present in the engagements between Twitter chat participants.Twitter defines engagement as the number of times a user interacted with a tweet, including clicks, retweets, replies, follows, likes, links, cards, hashtags, embedded media, username, profile photo, or tweet expansion . Similar@TulanePRC and @FitNOLA organize, host, and curate a monthly Twitter chat using the hashtag #LiveFitNOLA. The chat is focused on health and wellness in New Orleans. It is used to inform and engage participants in open discussions about the New Orleans’ culture of health. The target audiences for the #LiveFitNOLA chat are organizations and individuals in New Orleans interested in health and wellness. As hosts, @TulanePRC and @FitNOLA coordinated the chat plans and preparations, created visual promotions and tweets, and invited Twitter followers and community partners to participate.Health & Fitness Magazine , a Louisiana-based and operated magazine, participated as a guest host and determined the chat topic; approved all questions in advance; and invited their Twitter followers and community partners to participate. In addition, the Centers for Disease Control and Prevention’s National Center for Chronic Disease Prevention and Health Promotion (@CDCChronic) played a supporting role by participating as a subject matter expert. @CDCChronic provided evidence-based information on healthy living but did not intend to initiate or seek discussions with chat participants. The chat organizers selected the chat’s date and time (lunch hour on a weekday in New Orleans) after consulting other national organizations that host recurring, public health-related Twitter chats. Posting tweets during lunch hour toward the end of the week is a recommended best practice for social media scheduling, including Twitter chats [The first #LiveFitNOLA Twitter chat occurred on March 5, 2015, between 12:00 PM and 1:00 PM Central Time (CT). er chats -38.We collected the #LiveFitNOLA Twitter chat transcript from the Symplur Healthcare Hashtag Project . SymplurFor this study, we collected the #LiveFitNOLA chat transcript during a 75-min period on March 5, 2015, from 12:00 PM to 1:15 PM CT. This time frame included the hour-long #LiveFitNOLA chat and 15 min of residual conversations. Thus, our study population was composed of the individuals and organizations that participated and were mentioned in the #LiveFitNOLA chat during the previously defined time frame. We identified the chat participants’ geographic location based on self-disclosed location listed in user profiles. We categorized the #LiveFitNOLA chat participants’ locations as New Orleans, other US locations, and not available.All information included in our analysis is secondary data obtained from publicly available Twitter data. The CDC Human Research Protection Office (HRPO) designated the research activity conducted in this case study as exempt—HRPO Exemption Determination for Protocol #6803 “#LiveFitNOLA: A dissemination and translation case study.” The Tulane Institutional Review Board determined this research activity does not constitute human subjects research—ID #15-857090U “#LiveFitNOLA: Monthly Twitter Chats – A Community Engagement Project.”We conducted an SNA to measure and visualize engagement in two-way communications between #LiveFitNOLA chat organizers and other chat participants. The SNA allowed us to create an engagement ratio of outgoing to incoming mentions among Twitter chat participants, and a return on engagement measure (ROE). The engagement ratio and ROE provide a simple way for public health practitioners to measure audience engagement on Twitter.An SNA is the study of relationships between a connection’s source and its target within a specifically defined and bounded network ,40. We dIn our study, SNA focused on simple network components and metrics, including node, edge, degree, in degree, out degree, and a number of communities. A node  is an individual or an organization in a network; it is represented by a circle in a network map ,40. An eThe engagement ratio compares a network member’s total number of outgoing mentions (out degree) with their total number of incoming mentions (The ROE is based on the concept of return on investment (ROI), an investment performance measure. Like ROI does for monetary investments, the ROE measures the engagement gain or loss generated relative to the amount of engagement invested. In other words, the engagement gain or loss calculated as incoming mentions related to the number of outgoing mentions invested .To estimate the level of incoming and outgoing communications between public health entities and their audience, we focused our analysis on the #LiveFitNOLA Twitter chat organizers (@TulanePRC and @FitNOLA), the chat guest host , and the supporting organization (@CDCChronic). We identified these usernames on the network map . Not allThe step-by-step protocol for creating the SNA dataset is described in The content analysis assessed the primary function of tweets posted during the #LiveFitNOLA Twitter chat. We adapted a preexisting coding scheme originally developed by Lovejoy and Saxton to characterize tweets posted by public health entities . They idWe applied the adjusted (I-P-A) coding scheme to a dataset of original tweets, which excluded all retweets to avoid biasing the distribution of themes and sub-themes. Of the 744 tweets in the full #LiveFitNOLA Twitter chat transcript, 491 (66%) were original tweets and 253 (34%) were retweets. Two analysts independently coded all original tweets for the most applicable theme and subtheme. They collaboratively reconciled coding disagreements resulting in percentages of agreement of 89% for themes and 74% for subthemes. We calculated inter-coder reliability for theme and subtheme using Cohen Kappa and Scott Pi. For theme, Cohen Kappa and Scott Pi were both .78. For subtheme, Cohen Kappa and Scott Pi were both .66.The #LiveFitNOLA Twitter chat had 744 tweets and 66 participants, with an average of 11 tweets per participant, during the 75-min study period. More than half of the #LiveFitNOLA chat participants were individual users . Twenty-six participants (39%) were organizational users, and 5 (8%) were uncategorized users. As expected, the majority  of the #LiveFitNOLA chat participants were from New Orleans. Fifteen (23%) were from other US locations, and 12 (18%) were from unidentified locations .The resulting #LiveFitNOLA network had 135 network members and 474 edges connections.The network members included all 66 #LiveFitNOLA participants and 69 other Twitter users mentioned during the chat. Independent of direction, the overall number of connections  per network member ranged from 1 to 101, with an average of 8 (SD=16) connections per network member. On average, there were 4 (SD=9) outgoing mentions per network number, and 4 (SD=7) incoming mentions per network member .The network had 5 distinct communities. More than half of the network’s connections formed 2 distinct communities, centered on the 2 #LiveFitNOLA chat organizers, @TulanePRC (33.3%) and @FitNOLA (31.8%) . Among aThe trends in our findings indicate public health entities can use Twitter chats as a two-way communication audience engagement tool with their audience. To our knowledge, this is the first study that empirically examined Twitter audience engagement based on the incoming and outgoing communications between public health entities and their audience. To respond for the need to further assess the effectiveness of public health entities in two-way communications on Twitter, we developed metrics and a protocol to assess the incoming and outgoing communications between public health entities and their audience during a Twitter chat.The #LiveFitNOLA chat organizers targeted New Orleans residents to engage in open discussions about the culture of health in the New Orleans area. A hashtag trends on Twitter when it is algorithmically determined to be one of the most popular hashtags or topics at a particular time . Trends @TulanePRC was the main driver of this chat. Its engagement ratio revealed it received one incoming mention from a chat participant for each outgoing mention to another network member. Furthermore, @TulanePRC had a 40% ROE, which means it generated a 40% gain of incoming engagement related to the outgoing engagement it invested. @FitNOLA experienced a 40% engagement loss related to its invested engagement. By reading the chat transcript, we learned @FitNOLA retweeted most of @TulanePRC’s original tweets, which doubled its outgoing mentions compared with @TulanePRC’s outgoing mentions. @FitNOLA’s engagement level might be improved in the future if both #LiveFitNOLA organizers meet and coordinate to send original tweets rather than retweeting each other.The guest host, @HealthFitMag, largely played a supporting role. It repeated most of the information shared by both #LiveFitNOLA organizers and did not provide original content. In future chats, the chat organizers should encourage guest hosts to share original content and actively respond to the chat participants. Interestingly, while playing a supporting role, @CDCChronic had the biggest return on its engagement at 138% and a 1:2 engagement ratio. @CDCChronic received twice as many incoming mentions as its outgoing mentions. Popular and influential Twitter accounts like @CDCChronic attract incoming communications from other users participating in hashtag and time-bound Twitter activities. As popularity begets attention, this might introduce false positives or false negatives to the engagement ratio and ROE. In this instance, #LiveFitNOLA chat participants sought to engage directly in two-way communications with @CDCChronic. @CDCChronic did not respond to the participants because its role was to disseminate evidence-based information on healthy living during the chat, not initiate or seek discussions like the Twitter chat hosts (@TulanePRC and @FitNOLA). The negative or positive popularity of certain users participating in hashtag-based Twitter interactions might influence how others will react to them; and in some cases, this is something beyond the control of the said user. For public health entities seeking to increase audience engagement in two-way communications, we recommend they address the roles of each account at the beginning of the chat. We also recommend taking into consideration the popularity or influence of certain accounts when interpreting the result of the engagement ratio and ROE.Tweet sentiments might also influence the engagement ratio and ROE. For the scope of this case study, we did not conduct any sentiment analyses on tweet contents. We do recommend such analyses whenever possible to complement the engagement ratio and ROE results. An example of the importance of sentiment analyses would be the disclosure of a negative action, which would likely get a high ROE but might have highly negative reputational impact on the user. Mathematically, the ROE would be high but its impact on relationship building could be detrimental. Stylistic differences might influence the engagement ratio and ROE, and introduce false positives or false negatives because bombastic or offensive tweets might incite strong negative or positive reactions from other users. To this end, when interpreting the findings of the engagement ratio and ROE, we recommend skimming through the transcript to see if a user with a surprisingly low or surprisingly high engagement ratio and ROE have a very different tweeting style to other users.The depth and breadth of incoming and outgoing communications between users might influence the interpretation of the engagement ratio. Depth represents how many times the same source mentions a specific target user (incoming) and how many times a specific source user mentions the same target user (outgoing). Breath measures mention volume; how many different users mention a specific target user (incoming), and how many different users mention a specific source user (outgoing). For example, of @TulanePRC’s 32 outgoing communications, 31 were sent to distinct different users and one was to itself, which indicates a broad-scale engagement with other users. In contrast, if a particular user had mentioned the same user 31 times, a single-source engagement would occur. If a single-source and a broad-scale engagement have the same engagement ratio and ROE, the engagement quality depends on the context. If the goal was to engage with more people such as in a Twitter chat, then a broad-scale engagement is better. If the goal was to engage in a one-on-one conversation, as might be the case for customer service interactions on Twitter, then a single-source engagement would be better.Bots might interfere with the activities around a particular hashtag, and affect the engagement ratio and ROE with false positives. Tweets sent by bots need to be removed from the transcript before analyzing the incoming and outgoing engagement between users around a particular hashtag. The #LiveFitNOLA chat described here did not include any bot. However, the #LiveFitNOLA hashtag was hijacked by a bot in later periods of its use. We recommended the chat organizers to remove the bot’s activities from any analyses they might conduct within the period of the bot’s activities. In conclusion, user popularity, tweet sentiment and stylistic difference, and bots might influence the engagement ratio and ROE. Their practical significance depends on the audience, context, scope, scale, and goal of a Twitter chat or other organized hashtag-based communications on Twitter.More advanced analyses could be conducted on networks formed on Twitter around hashtags. For example, in our case study, granular community-based analysis could be used to assess individual communities, cross-community, and within-community interactions. We did not conduct such analyses and did not include them in our protocol because they are beyond the practical purpose of our study. In addition, they require advanced knowledge of SNA beyond what is needed to obtain the engagement ratio and ROE. As previously mentioned, our intended audience are public health professionals able to use point and click apps and with basic quantitative skills.In line with previous studies, results of the content analysis revealed the majority of tweets shared health information or showed positive affect or interpersonal closeness. The distribution of themes, however, deviated from prior research using the I-C-A framework ,42. SpecA limitation for this case study is @FitNOLA and @HealthFitMag retweeted most of @TulanePRC’s tweets. Hosts retweeting hosts create an echo chamber and do not add new content, which might hinder host engagements with other users. Based on our findings, the hosts’ audience engagement might be improved by ensuring Twitter chats are organized and led by two or more organizations that do not retweet each other and individually tweet original contents. Each host should share carefully planned and curated original contents and refrain from sharing (retweeting) the same information as other hosts.Another limitation is our use of self-disclosed geographical locations in user profiles. This is not an ideal way to identify geographic origins of tweets. We chose this approach because using the Global Positioning System (GPS) tags in tweets or Internet Protocol (IP) addresses would require more advanced analytical skills than the ones needed for the protocol we used in our study. In addition, users might not always enable their GPS-based location in their tweets, and IP addresses might not be accurate location indicators.Further studies are needed to establish quantifiable parameters on what is a low, medium, or high engagement ratio and ROE such as through longitudinal analyses of a recurring Twitter chat. In addition, such a study could provide more nuanced information about a public health entity’s Twitter audience for their hashtag-based communications such as their constituents’ health topic of interests or the type of guest host who will engage more with their audience.The protocol described in Based on the lessons learned from this case study, to build further evidence on the effective use of Twitter as an audience engagement in two-way communication tool for public health entities our future research includes: (1) conducting longitudinal ROE assessments with recurring Twitter chats and (2) determining audience engagement effectiveness based on the hashtag-based communication’s purposes by examining larger public health campaigns.


Here, we report the case of a patient who developed protamine shock during a transcatheter aortic valve implant (TAVI) procedure, which was diagnosed by intraoperative transesophageal echocardiography (TEE). A 77-year-old man with symptomatic severe aortic stenosis and reduced left ventricular (LV) function underwent TAVI under general anesthesia. During the procedure, a transcatheter heart valve (THV) was deployed via the transfemoral approach, without any other major complications. The entire device system was then removed, and protamine sulfate was administered intravenously in 2 min.Two minutes after the protamine administration, severe hypotension occurred. TEE did not reveal THV malfunction or any other major complications. However, comparison of the TEE image obtained before protamine administration and that obtained 2 min after protamine administration showed right ventricular (RV) dilatation, RV free wall motion abnormality, and LV volume reduction, without any electrocardiographic changes. We diagnosed this as protamine shock and bolus infusions of phenylephrine and norepinephrine were administered, and chest compressions were initiated immediately. After 1 min, hypotension as well as the right and left ventricular size and dysfunction immediately reverted to baseline. The severe systemic hypotension resolved as well. Thereafter, he recovered from anesthesia without other complications.This case showed the clinical features of protamine shock with acute pulmonary hypertension. The TEE images, in this case, should be a reminder for all doctors who perform intraoperative TEE for patient monitoring when they perform procedures to treat structural heart diseases.The online version of this article (doi:10.1186/s40981-016-0053-6) contains supplementary material, which is available to authorized users. Protamine is the mainstay drug for heparin neutralization during cardiac surgery, structural heart disease (SHD) intervention, or coronary intervention . TranscaA 77-year-old man with symptomatic severe AS and reduced left ventricular (LV) function underwent TAVI under general anesthesia. During the procedure, dobutamine (2–5 μg/kg/min) and nicorandil (3 mg/h) were continuously infused to maintain hemodynamic stability. A transcatheter heart valve (THV) (SAPIEN XT 26 mm) was deployed via the transfemoral approach with an electrocardiographic change in the left bundle branch block (LBBB), without any other major complications Fig. . ThereafTwo minutes after the protamine administration, severe hypotension occurred 30/10 mmHg). TEE did not reveal THV malfunction or any other major complications; however, comparison of the TEE image obtained before the protamine administration This case showed the clinical features of protamine shock by acute pulmonary hypertension (APH), a rare cause of hypotension during the TAVI procedure. The transfemoral approach TAVI procedure has several well-known causes of systemic hypotension such as access site bleeding, cardiac tamponade, aortic root rupture, coronary obstruction, aortic regurgitation, mitral regurgitation, and THV malfunction . TEE imaProtamine remains the mainstay drug for heparin neutralization during coronary intervention or cardiac surgery, and it occasionally causes transient hypotension known as “protamine shock” in clinical settings . ProtamiThere are several suggested potential risk factors for protamine shock, including a history of vasectomy, previous protamine exposure, vertebrate fish allergy, and preexisting pulmonary hypertension , 9. It iHere, we reported the case of a patient who developed protamine shock with APH during a TAVI procedure in which TEE was useful for making the diagnosis. The images obtained, in this case, should remind all doctors to perform intraoperative TEE monitoring during TAVI and SHD procedures whenever protamine is used.Additional file 1: Video 1.TEE moving image after THV deployment. Normal RV size and RV free wall motion in the mid-esophageal four-chamber TEE view after THV deployment. RV, right ventricle; TEE, transesophageal echocardiography; THV, transcatheter heart valve. (AVI 3462 kb)Additional file 3: Video 3.TEE moving image after 1 min of chest compressions. Recovered size and wall motion of RV and LV to baseline in the mid-esophageal four-chamber TEE view after 1 min of chest compressions. (AVI 2758 kb)


Aldehyde dehydrogenase 1 (ALDH1) and Nestin immunohistochemistry were used as a measure of stem-like properties, showing significantly decreased Nestin expression (p < 0.0001) in the invasive margin. Array PCR of the core, rim, and invasive regions showed significantly increased fibroblast growth factor (FGF) and ALDH1 expression in the invasive zone, with elevated hypoxia inducing factor 1-alpha (HIF1α) in the rim region, adjacent to the hypoxic core. The influence of varying microenvironments in the intra-tumour regions is a major key to understanding intra-tumour heterogeneity. This study confirms the distinct molecular composition of the heterogeneous invasive margin and cautions against purported therapy strategies that target candidate glioblastoma stem-like genes that are predominantly expressed in the tumour core. Full characterization of tumour cells in the invasive margin is critical, as these cells may more closely resemble the residual cell population responsible for tumour recurrence. Their unique nature should be considered when developing targeted agents for residual glioblastoma multiforme (GBM).Glioblastoma, a WHO grade IV astrocytoma, is a highly aggressive and heterogeneous tumour that infiltrates deeply into surrounding brain parenchyma, making complete surgical resection impossible. Despite chemo-radiotherapy, the residual cell population within brain parenchyma post-surgery causes inevitable recurrence. Previously, the tumour core has been the focus of research and the basis for targeted therapeutic regimes, which have failed to improve survival in clinical trials. Here, we focus on the invasive margin as defined by the region with 5-aminolevulinic acid (5ALA) (GliolanTM) fluorescence at surgery beyond the T1 enhancing region on magnetic resonance imaging (MRI). This area is hypothesized to constitute unique microenvironmental pressures, and consequently be molecularly distinct to tumour core and enhancing rim regions. We conducted hematoxylin and eosin (H&E), array real time polymerase chain reaction (PCR), and immunohistochemistry staining on various intra-tumour regions of glioblastoma to determine molecular heterogeneity between regions. We analyzed 73 tumour samples from 21 patients and compared cellular density, cell proliferation, and the degree of vascularity. There is a statistically significant difference between the core, invasive margin and other regions for cell density ( Brain tumour mortality rates have increased in the last 32 years by 15% for men and 10% for women, making it an urgent target for novel therapies in the future. Astrocytomas are the most frequent type of primary brain tumour, with glioblastoma multiforme (GBM (Grade IV astrocytomas)) being the most malignant and most common type of brain tumour in adults. Even with aggressive treatment, median survival for patients is around 15 months from diagnosis [6-methylguanine-DNA-methyltransferase (MGMT), facilitating resistance to the drug and recurrence of the GBM [In 2014, around 10,981 people were diagnosed with a new brain, central nervous system (CNS), or intracranial tumour, making it the ninth most common cancer in the UK , isocitrate dehydrogenase 2 (IDH2), or tumour protein 53 (TP53); transcriptional signature; copy-number variation; amplification or mutation of epidermal growth factor receptor (EGFR), and hypermethylation of promoter regions .RNA sequencing and histological analysis, conducted by Gill et al. , revealeA possible explanation for this intra-tumour heterogeneity is thought to be the clonal evolution of the tumour cells. Studies show that GBM tumors have genetic alterations that are common amongst different regions and some alterations that are area specific. The common mutations are considered to have an evolutionary advantage when compared to other mutations, and were proposed as “driver mutations” occurring early in pathogenesis . DivergeCancer stem-like cells (CSCs) are self-renewing, multipotent cells that can give rise to progenitor cells, and eventually, differentiated cancer cells that have the ability to generate tumors when xenografted into immunocompromised animals . CSCs maIt is hypothesized that CSCs may be concentrated in close proximity to neo-angiogenic vessels in the growing and invading tumour, and, which may provide a supportive microenvironment . These nMany previous studies have focused on samples taken from the tumour core, consisting of “pure” tumour. However, the molecular studies discussed have shown different regions of tumour and have different profiles of molecular abnormality and differing expression profiles for known tumour markers. Although evaluation of the core, pure tumour area gives information regarding the central tumour, this is the region that is most likely to be resected when surgery is visually guided under conventional light alone. This study seeks to evaluate tumour characteristics, including levels of markers of stem-like cells in the periphery of GBM, comparing the area beyond the T1 enhancing tumour  with conventional core tumour samples. It is hypothesized that tumour cells in this area will share more characteristics with the residual tumour cells left behind after surgery that cause recurrence. By understanding and targeting cells, in particular treatment resistant CSCs that are the true source of recurrence (rather than the bulk tumour core), more effective therapeutic strategies may be developed.IDH1 gene, with 11 patients having wild type IDH1 GBM. One patient had hypermethylated MGMT promoter, with 10 hypomethylated and three of unknown status. Twelve patients completed 60Gy radiotherapy and adjuvant temozolomide after surgery, with two patients only receiving radiotherapy. IDH status was significantly associated with extended survival (p = 0.04 logrank test)  fluorescence positive areas beyond tumour enhancement as per the image guidance surgical navigation system. There were eight female and six male subjects and median age was 55 years old (26–73). Overall survival in the cohort was a median of 14.9 months (3.8–36.2 months), consistent with other published series, with a 12-month survival of 61%. Three patients had a tumour with the R132H mutation in the nk test) .p = 0.031). Core samples had a median cell density of 144 cells per HPF (range 66–335), rim samples a median of 152 cells per HPF (56–561), and invasive samples median of 76 cells per HPF (39–287). On a pairwise post-hoc t-test, there were significant differences between core and invasive regions (p = 0.018), and between rim and invasive regions (p = 0.045), but not between core and rim regions (p = 0.849)  across all of the tumour regions (range 39–561 cells per HPF). Using the non-parametric median test, there was a significant difference in cell density between core, rim, and invasive regions of tumour (= 0.849) . There wp = 0.089 Kruskal-Wallis) , but not between rim and invasive or rim and core samples .Using Ki67 immunohistochemistry, we assessed the fraction of proliferating cells in each tumour region across the various samples. Median Ki67 positive fraction was 39% for core samples (range 4–61%), 26% for rim samples (range 2–45%), and 22% for invasive samples (range 4–39%). This apparent trend towards a decrease in tumour proliferation in the periphery did not quite achieve statistical significance  . On post samples . The meap = 0.025 Kruskal Wallis), with significant differences between core and invasive and between core and rim regions on pairwise t-test (Tumour vascularity was assessed using CD31 (PECAM-1) immunohistochemistry, identifying the number of vascular structures per HPF. Core tumour regions showed a median of nine vessels pre HPF (range 0–28), rim regions a median of seven vessels per HPF (range 0–25), and invasive areas a median of 5 (range 0–12). There was statistically significant variation between areas (e t-test .p = 0.202). Nestin appeared to be showing a different pattern with reduced levels of expression (none in many samples) in the invasive areas but with elevated levels in tumour core and rim areas but again this did not achieve significance (p = 0.212) . ALDH1 immunohistochemistry (IHC) showed a trend toward increased expression in the fluorescent invasive region beyond the enhancing rim but this was not significant on Kruskal-Wallis (= 0.212) .ALDH1A (in keeping with the IHC result), ABCG2, and FGF1 were consistently and significantly over-expressed  in the invasive margin of GBM when compared to rim and core tumour regions from where samples are conventionally taken. The gap junction protein gene, GJB1, was also consistently over-expressed in the invasive region  demonstrated distinct stem cell expression profiles within different spatial regions of GBM, in line with the results that were observed by immunohistochemistry. The specific stem cell markers e region . Other sFGF1, GJB1, ABCG2, and ALDH1A1) could have a key role in GBM malignant infiltration through normal brain parenchyma, and may ultimately be functionally relevant to the inevitable GBM recurrence and treatment resistance despite adjuvant radio- and chemotherapy after surgical resection.Immunohistochemistry and array reverse transcriptase PCR were used in this study, aiming to assess phenotypic and genotypic cellular diversity within distinct GBM intra-tumour regions and to give an insight into the invasive margin region, which may be the most promising region for developing potential therapeutic targets to prevent the currently inevitable tumour recurrence. Our results support the hypothesis that GBMs display both inter- and intra-tumour heterogeneity specifically in terms of human stem cell gene expression, proliferative capacity, and microenvironment. In general, these results are consistent with previous work that was performed by genome-wide studies to identify tumour heterogeneity ,17. In pFGF1 with or without VEGF (stimulated by decreased oxygen supply through HIF1α or HIF2α) in the invasive region, could potentiate the malignant vasculature that is needed to provide the appropriate niche for malignant invasion throughout the disease course.Fibroblast growth factor 1 (FGF1) ligand is secreted independently from the extracellular matrix, and it is thought to enhance the formation of branching vasculature networks . FurtherGJB1 that is observed at the invasive margin relative to other intra-tumour regions could also contribute to neo-angiogenesis, as this gene is known to mediate the process of endothelial cell intercommunication [GJB1 remains complex within tumors in different tissues [GJB1 in the invasive margin could be due to the activation of mitogen-activated protein kinase (MAPK)-dependent downstream signaling of epidermal growth factor (EGF) and FGF1 pathways. Tyrosine kinase receptor activation by EGF and FGF1 ligands has been proposed to mobilize Ca2+ from its intracellular stores in the endoplasmic reticulum [GJB1 [2+ waves that result in escaping apoptosis, enhancing cell migration, and promoting angiogenesis [2+ gradients via GJB1 channels, which are already upregulated in response to high MAPK activity (expression of EGF and FGF1 ligands) in this region. These intracellular Ca2+ waves may enhance the invasive cell survival and promote their migration through normal tissue via remodeling of their actin cytoskeleton and focal adhesion [Furthermore, the high expression of nication . The reg tissues . Our fineticulum . This inum [GJB1 . In cancogenesis . Thus, wadhesion . Angiogeadhesion  it may bALDH1A1 and ABCG2 expression as compared to other tumour regions. These genes have been shown to be involved in radio- and chemo-resistance, and their expression in the invasive zone could contribute to treatment resistance post-surgically [Interestingly, the expression of 84 stem cell genes within different GBM regions reveals that the invasive region has the highest rgically . Dean etrgically  suggesteALDH1A1 and ABCG2 genes are strongly associated with the CSC phenotype [Stem-like cells also express ABC transporter channels, which have the ability to efflux different chemotherapeutic agents. henotype ,32. TargFGF1, GJB1, ABCG2, and ALDH1A1 stem-related genes in the invasive margin as compared to other regions of the tumors may indicate a distinct spectrum of precursor clones in the invasive margin of GBM. The stresses of prolonged hypoxia in the tumour core can stimulate the expansion of stem cell subpopulations in addition to the potentiation of reprograming non-stem glioma cells to a stem-like phenotype [Targeting of stem-related populations in GBM remains a significant challenge because of their cellular localization, identification, characterization, and behavioral variability. The results of our study also suggest that varying stem cell populations are located in the invasive margin and tumour mass regions, presumably in response to different micro-environmental conditions. Additionally, overexpressed henotype . This plFGF1 overexpression that enhances malignant angiogenesis, which in turn provides the appropriate niche that is needed for malignant cell migration and attraction towards the normal tissue. Upregulation of GJB1 may enhance Ca2+ mediated GBM cell migration by promoting endothelial intercellular communication. Similarly, ABCG2 and ALDH1A1 overexpression in the invasive region could be linked to stem-like subpopulations. Thus, targeting FGF1 and GJB1 could lead to the suppression of GBM invasion, whereas deactivation of ABCG2 and ALDH1A1 pathways could possibly impair GBM recurrence at the invasive margin by inhibiting the enrichment for stem-like subpopulations following chemo-radiation. Further confirmatory studies and broader molecular analysis are needed for a better understanding and efficient targeting of these molecular pathways.In conclusion, our findings revealed considerable inter- and intra-tumour heterogeneity within distinct GBM regions. Although we observe different CSC gene expression, our invasive margin is a molecular snapshot of before the selection pressure conferred by chemo- and radiotherapy has occurred. We may not be able to exclude the possibility that CSC marker expression starts to resemble levels in the tumour core post-treatment, when self-renewal must be very active to help to repopulate the tumour. Despite the high phenotypic and genotypic cellular diversity, we have identified the potentially targeted molecular pathways in the invasive region that can be linked to GBM invasiveness and recurrence. GBM invasive behavior may be attributed to the observed This project has consent and ethical approval from the Local Regional Ethics Committee in Nottingham . Patient information is from databases within the Children’s Brain Tumour Research Centre (CBTRC), and the samples were obtained during surgeries performed by Dr Stuart Smith and colleagues in the Queen’s Medical Centre (QMC). All patients provided consent for the use of tumour samples for research and were obtained by the operating neurosurgeon. The samples were paraffin-embedded and sectioned by the Queen’s Medical Centre Histopathology Department. Samples were collected from tumour core, rim, and peripheral/invasive regions as per Tonsil, pancreas, placenta, and IDH-mutant low grade glioma slides were used as controls to ensure that antibodies was specific and bound efficiently with low background staining. Tissue Microarrays (TMAs) were created within the CBTRC, and samples were separated in the TMA by tumour region and by patients.Immunohistochemistry slides were kept at 50–60 °C overnight to fix the tissue onto the slide and to prevent tissue loss during the procedure. Next, for the removal of paraffin wax, the slide rack was placed in Xylene for 15 min, followed by absolute ethanol for 10 min, 95% ethanol for 10 min, and washed in running tap water until it clears. Samples were placed in a steamer with sodium citrate buffer (pH 6) for 40 min at 90 °C. Slides were washed in a Coplin jar with phosphate buffered solution (PBS) for 2 min, and then placed in a humidified box. 200 μL of peroxidase blocking solution was applied to cover the specimen for 5 min and then washed with PBS for 5 min. Slides were dried, then primary antibody applied diluted in antibody diluent  (1:50 dilution for Ki-67 (DAKO) and CD31 (DAKO) and 1:300 dilution for ALDH1  and incubated for 1 h at room temperature. Sections were washed with PBS for 5 min and the secondary antibody (DAKO) was applied to cover the specimen and incubated at 37 °C for 30 min. Sections were washed again with PBS for 5 min and DAB solution made up (DAB and DAB buffer in a 1:50 dilution). The substrate-chromogen solution (DAB) was applied to cover the specimen, incubated for 5 min, and rinsed gently with distilled water. To counterstain, the slides were agitated briefly in filtered Harris hematoxylin for 10 s and washed in running water. Slides were placed in Lithium carbonate to blue for 10 s and were washed in running water before dehydrating the tissue in alcohols: 95% ethanol for 2 min and 30 s, absolute ethanol and xylene for 1 min each. Slides were mounted with DePeX medium onto a suitably sized coverslip.The Olympus BX41 light microscope was used to view the staining and to also capture the photographs of the regions. Three representative photos were taken per slide (per region). The intensity and proportion of positively stained cells was calculated by averaging counts of three representative images of every region. All of the counting was done blindly. Areas with large amounts of necrosis and burst blood vessels were not counted, as these areas were not deemed viable and would display inaccurate, non-specific staining.SPSS Statistics and GraphPad Prism V7.02 was used to perform two-way ANOVAs with Tukey’s multiple comparisons tests on data for each antibody and for cellular density to look for a significant statistical difference between the mean values for each region. Comparisons were made between the core and rim, rim and invasive margin, and core and invasive margin. Protein expression and cellular density was also compared between invasive and non-invasive regions.mirVANATM miRNA Isolation Kit  according to the manufacturer’s instructions. RNA was extracted from each specimen in triplicate and all of the RNA samples were assessed for purity and quantity by using Nanodrop spectrophotometry. cDNA was synthesized by using 1μg RNA from each sample according to the manufacturer’s instructions for the cDNA synthesis RT2 First Strand Kit . Gene expression profiling was performed using Human Stem Cell PCR Array  to assess 84 human stem-related genes based on published literature and selected by the manufacturer [® Green ROXTM qPCR; Qiagen) and high-quality nuclease free water, and then loaded equally (25 μL) into each well of the stem cell array. The arrays were run in a MX3005P RT-PCR machine. The cycle conditions for array RT PCR were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min.RNA was extracted using facturer . The arrp-Values were considered to be significant where less than 0.05.After confirming the equal fluorescence detection threshold for all of the arrays in the Array-PCR machine, the obtained array data was analyzed using the accompanying Excel based statistical software that was provided by the same company , and the significance of the fold changes was further confirmed and explored by SPSS software. Gene expression data was presented using volcano plots, in which the log fold of gene expression changes is directly plotted against the statistical significance. The fold-change threshold of gene expression was considered valid only if it was more or less than (1.5).