Salmonella Rissen phage ϕ1 isolated from the ϕ1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RRϕ1+, which can harbour or not ϕ1.The study describes the W cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RRϕ1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth.Following this approach, we found that ϕ1, upon excision from RW and RRϕ1+ strains differ by 10 genes; of these, only two  display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes.The RSalmonella Rissen lifestyle.Phage ϕ1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of The online version of this article (10.1186/s12866-018-1360-z) contains supplementary material, which is available to authorized users. S. enterica ser. Typhimurium to express alternative forms of the O-antigen and escape phage attack [Escherichia (E.) coli phage Mu [Bacteria are under constant attack by bacteriophages (phages), the most abundant life forms in the biosphere . They hae attack , 9; Eschphage Mu  and othephage Mu  to alterphage Mu , a featuphage Mu . In concphage Mu .S. enterica ser. Typhimurium carrying the lysogenic phage P22 confers protection against infection by the phages L, MG178, or MG40 [The term superinfection exclusion (SE) describes the property of a preexisting prophage to inhibit a secondary infection by the same – or a very close – phage , 14. SE  or MG40 . The pro or MG40 , where t or MG40 , or prom or MG40 .S. Rissen phage ϕ1. This phage was excised from the ϕ1-sensitive S. Rissen strain RW and then used to select the ϕ1-resistant strain RRϕ1+, which can spontaneously lose ϕ1. The ϕ1 excised from RW cells with mitomycin behaves as an inducible temperate phage since lyses host cells and generates phage particles. Instead, the spontaneous excision of ϕ1 from RRϕ1+ cells does not generate phage particles, promotes biofilm production, loss of phage resistance, and the switch of the O-antigen from smooth to rough. To carry out the above tasks, phage ϕ1 uses all the resources described earlier: phase variation, SE, and SE inhibition.Here we describe the W, all display identical mutations at two phosphomannomutase genes.Finally, because of their rapid evolution and easy replication of experiments, bacteria are frequently used to investigate whether evolution is contingent or repeatable, an issue still debated . Here weS. Rissen bacteria (RW) yielded phage ϕ1  and release of ϕ1 particles was followed by host cell lysis. Instead, the spontaneous release of ϕ1 from RRϕ1+ cells (RSϕ1-) occurs without recovery of phage particles and is also associated with increased biofilm production  method with ϕ1, indicating that the ϕ1 prophage is resistant to the SE mechanism.Following incubation with mitomycin C, the wild type Rϕ1+ and RW colonies differ in curli production: RRϕ1+ colonies express the “ras” (red and smooth) phenotype, while RW colonies display the “pdar” (pink red and dry) phenotype (characterized by a reduced amount of curli) [Sϕ1- and RW are both ϕ1-sensitive, while that RRϕ1+ is ϕ1-resistant  aeruginosa [Moreover, Rf curli)   and therefore assigned to this family  and an average coverage of 13,200 x. The de-novo assembled phage ϕ1 genome is 51,738 bp long with a GC content of 48,4%. The genome contains 87 predicted coding sequences (CDSs): 30 affecting bacteriophage physiology, 12 encoding phage structures, 10 regulating DNA replication, and 3 encoding bacterial lysis. Genome sequence and general phage organization can be found in the annotation (available on GenBank accession: KY709687). The phylogenetic tree of phage ϕ1 genome was reconstructed by comparing its proteome with those of 37 fully sequenced phage genomes. Phage ϕ1 disclosed a robust orthology with 5 members of W and RRϕ1+ was generated 8,036,355  and 12,639,370 reads , respectively. To identify the insertion site of ϕ1, we assembled de novo the reads generated from RW and RRϕ1+ and compared them. This approach yielded 104,974 reads with an average length of 5305 bp for a total of 556 Mb and an average coverage of 116X. We could thus establish that in the RRϕ1+ strain phage ϕ1 is inserted at the end of the genome   all displayed the same differential gene expression already observed in the original strains RW and RR ϕ1 + .Comparative genomics showed that the two strains Rϕ1+ Fig. . This rebacteria –27, phagS. Rissen life style. In general, prophages aid bacteria with the production of virulence molecules [This study describes the properties of ϕ1, a prophage which modulates several phases of olecules , toxins olecules , antibioolecules , or  monocytogenes ϕ10403S, we speculate that ϕ1 or the RRϕ1+ host cells can disconnect phage excision from phage replication and release [We found that induction of ϕ1 excision with mitomycin in R release .The cases of bacterial genes controlled by prophage excision generally involve cryptic prophages , 31. PhaSalmonella, gain phage resistance by altering the genes of the LPS biosynthesis pathway [E. coli, nine different genes are involved in the LPS biosynthesis pathway, which potentially could lead to T7 phage resistance, but bacteria reach resistance by altering waaG, the gene associated with reduced biological cost [Many bacterial species, including  pathway . In E. ccal cost .W) and grown under the same conditions displayed the same regulatory alteration at two genes (phosphomannomutase1 and phosphomannomutase2)  acnes [Campylobacter (C.) jejuni [Vibrio (V.) cholerae [L. monocytogenes [Herpes (H.) influenzae [Staphylococcus (S.) aureus [S. Rissen (this study). Also, in most of these bacterial species (including S. Rissen), phase variation originates from HTs frameshift mutations  Fig. . Paralleytogenes , E. coli[E. coli  and Prop.) acnes . These r) jejuni , Vibrio cholerae , L. monoytogenes , 33, Herfluenzae , Staphyl) aureus , and S. ons Fig. a and b aThis study describes a phage which modulates several properties of its host. The results of this study may stimulate researchers to better understand benefits and negative outcomes associated with the therapeutic use of phages; how the stability of mutations is influenced by environmental stresses; how phages affect evolution and pathogenicity of bacteria. Finally, the study demonstrates that, at least in bacteria, natural selection uses repeatedly the same evolutionary path, when it requires a lower biological cost.S. Rissen strain RW  was isolated from a food matrix and characterized by Istituto Zooprofilattico Sperimentale Del Mezzogiorno . The S. Rissen strain RR was derived - in the course of this research - from the RW strain following selection for resistance to phage ϕ1 as described in this study. RR cells can spontaneously lose the prophage and thus occur with (RRϕ1+) or without ϕ1 (RSϕ1-) (the superscript S indicates that loss of ϕ1 causes loss of phage resistance). All the bacterial strains were analyzed for cellulose production and LPS phenotype and stored at − 20 °C in LB  supplemented with glycerol .The W cells (2 × 108 CFU/5 mL) in LB broth containing 1 μg/mL mitomycin C  for 1 h at 37 °C. Following centrifugation (5.7 × 103 g), the supernatant was stored at + 4 °C, and the pellet resuspended in 5 mL of LB broth and incubated again at 37 °C for 4 h and then centrifuged. The pellet was discarded, while the supernatants from the two centrifugations were pooled and filtered  [Phage ϕ1 excision was induced by incubating RGermany) .The titer of phage, expressed as plaque forming units (PFU), was evaluated by using the DLA technique as reported by Sambrook et al.  Phage ϕ1W bacteria in early exponential growth phase were mixed with warm soft agar. The mixture was poured on LB agar  plates and allowed to solidify. Phage ϕ1 was then spotted (10 μL/spot) and the plates were incubated overnight at 37 °C. The following morning, the colonies grown inside the lysis plaques were picked with a sterile loop and streaked on LB agar plate. This procedure was repeated 3 times. Phage-resistant bacteria were further tested for phage ϕ1 resistance by the spot test. Plaque absence after overnight incubation confirmed that bacteria were phage ϕ1-resistant (RRϕ1+). Phage ϕ1-resistant colonies were detected after about 24 h of selection.RSϕ1- bacteria (108 CFU in 500 μL LB) were incubated with ϕ1 isolated from RW bacteria (108 PFU/mL) for 72 h. The suspension was mixed with soft agar (4 mL) and then poured on a solid agar. Phage ϕ1was spotted on soft agar (10 μL/spot) and plates were incubated at + 37 °C and inspected daily for plaque formation.RCellulose production was detected by growing bacteria on LB agar supplemented with 200 μg/mL calcofluor . Plates were incubated at 37 °C for 2–4 days. Colonies were visualized under a 366-nm light source . Congo rRϕ1+, RSϕ1-, and RW strains grown in LB medium were diluted to a final concentration of 0.001 and seeded into a chamber slide at 37 °C for 36 h to assess biofilm thickness and cell viability. The biofilm cell viability was determined with the FilmTracer™ LIVE/DEAD® Biofilm Viability Kit  following the manufacturer’s instructions. Microscopic observations and image acquisitions were performed as described [Biofilms were formed on polystyrene Chamber Slides . For this purpose, overnight cultures of Rescribed .W or RR strains were expanded in LB broth starting from a single colony. Genomic DNA was then extracted by the phenol-chloroform method, purified with Agencourt AMPure XP beads , and quantified by the Qubit dsDNA BR Assay Kit . DNA size and purity were measured by the 2200 Tape Station Instrument (Agilent Genomics) and Nanodrop (Thermo Fisher), respectively. Illumina libraries were obtained from 1 μg of genomic DNA, and sequenced with the NextSeq500 instrument using the 150 nt paired-end protocol . Illumina reads were quality filtered, trimmed using Sickle and finally quality corrected with BayesHammer. Genomes were assembled de novo from Illumina reads using SPAdes 2.9.0 with multiple k-mer combinations: from 101 to 125 with 2 nt steps for the 202 RR genome, with 95, 97, 111, 113 for the RW genome and 101, 105, 109, 113, 117, 121,125 for the ϕ1 genome. The resultant contigs were scaffolded using SSPACE 3.0. Five μg high-molecular-weight genomic DNA (peak >60Kb) were used to prepare ~ 20 Kb-insert SMRT-bell libraries . The library templates were sequenced using the single molecule real time (SMRT) Sequencing technology on a PacBio RSII sequencer . PacBio subreads were extracted using Bash5tools (version 0.8.0), filtered and assembled de-novo with Falcon-Integrate and the settings suggested for bacterial genome. The assembled genome sequence was polished by Quiver v 0.9.2. and gene annotation performed using RAST web service (http://rast.nmpdr.org/) [Rϕ1+ bacteria against the phage genome assembly and soft-clipped bases were retrieved.The Rdr.org/) . The ϕ1 SNP calling was carried out using MUMmer 3.23 tool . Single-WSalmonella genome was used as reference. Each assembly was queried with each SNP context from the MUMmer output using BLAST + [The output was filtered by BUFF > 50 with the Show- SNPs flags ClIrx 25 and the SNP position was assembly by quality > 80. The R BLAST + , retainiPAGE was performed using the system of Laemmli  with sodW strain. The reference gene was the housekeeping InvA. The relative gene expression was carried out using the Delta Delta ct Method [Total RNA was extracted from individual bacterial strains according to the Allprep Bacterial DNA/RNA/Protein Kit protocol (Quiagen) and then reverse-transcribed using the high-capacity cDNA reverse transcription kit (Applied Biosystem). Real-time PCR was carried out using the Step One Real-Time PCR Systems machine (Thermo-Fisher scientific). Reactions were carried out in a 20 μl of Master SYBR Green I mix . The amplification protocol included 10 min at 95 °C and 40 cycles, each consisting of 10 s at 95 °C for denaturation, 120 min at 57 °C for annealing, and 60 s at 60 °C for extension; the final step was at 4 °C. PCR reactions were carried out in triplicate. Expression values were normalized versus the Rt Method .Following the genome sequencing experiment, we designed the primers for the 5 kb region using primer 3 as primer design tool. We used the following overlapping eight primer pairs. Primers are designed to amplify regions within a size range of 400–600 bp.W or RRϕ1+ cells was carried out by exposing the cells at − 20 °C for 1 h and + 40 °C for 2 h. The cells were then tested for loss of phage resistance. The phylogenetic tree was constructed using the maximum likelihood method [The thermal shock of Rd method ; for datd method .Additional file 1:Figure S1. Gas chromatography-mass spectrometry (GC-MS) analysis of the (A) RW, (B) RRϕ1+, and (C) RSϕ1-strains. All of the strains display the presence of glucose, glucosamine, heptose, and KDO. Acquisition of phage resistance by RRϕ1+ strain is associated with loss of mannose. Peaks marked with X represent methyl esters of fatty acids. (PDF 673 kb)Additional file 2:Figure S2. Electrophoresis gel of PCR for detecting the presence of 5 kb region. Lines 1–5: RWϕ1+; lines 6–9: RWϕ1-; lines 10–14: RRϕ1+; lines 15–18: RSϕ1-; M = marker (100 kb). (PDF 35 kb)Additional file 3:Table S1. SNPs detection analysis of RW and RRɸ1+ strains. (PDF 107 kb)


The purpose of this research is to understand the effect of family participation in care planning assessments on the use of advance directives for newly admitted nursing home (NH) residents. This is a retrospective cohort study using data from 115 nursing homes involved in a pragmatic randomized clinical trial testing a video intervention for advance care planning . Data sources included the electronic health record and the Minimum Data Set (MDS). Competing risks regression analyses estimated the cumulative incidence of establishing an advance directive, after accounting for death, discharge from NH, and time censoring (12-month observation window). 18,978 residents admitted to eligible NHs without an advance directive (full code) between April 2, 2016 and August 31, 2017 were followed for one year. 11,905 (63%) died or were discharged without an advance directive; 4,155 (22%) remained in the NH without an advance directive; and 2,918 (15%) established an advance directive. Median time to establishing an advance directive was 75 days . After adjusting for competing risks and resident factors known to be associated with advance directive use, each care planning assessment involving a family member was associated with a 6% increase in the cumulative incidence advance directive use. Involving family members of NH residents in routine care planning assessments may reduce the amount of futile care received by NH residents. As we test interventions to improve advance care planning for NH residents, focus should be placed upstream on the first 90 days after admission.


Accreditation Council for Graduate Medical Education’s Clinical Learning Environment Review report suggests that residents in clinical learning environments underreport their near-misses, an indicator of patient safety. Furthermore, when the residents report these events, they receive little feedback from their attendings. Although, various studies emphasize the importance of feedback given to the residents, the association between feedback and patient safety has not been explored. This study was conducted in 28 emergency medicine residents in a level 1 trauma center. A recent study in the same cohort found that residents’ stress biomarker levels and patient acuity were positively related to their near-misses reports. The current study hypothesizes that residents that receive constructive feedback on their performance from their attendings would report more near-misses.Linear regression was used to determine whether ratings of attendings’ feedback predicted residents’ reports of near-misses. Feedback was positively related to residents’ near-misses reports even after controlling for patient acuity and stress biomarker levels. This may be due to the residents becoming more aware of what a near miss is or it may also be that constructive feedback encourages them to report more near-misses as they may view this as a learning opportunity. Residents in training in clinical learning environments are at increased risk of experiencing patient safety events , 2. ResiThe Clinical Learning Environment Review (CLER) program of the Accreditation Council for Graduate Medical Education (ACGME) conducts site visits every 2 years to assess teaching hospitals across the United States on six focus areas . CLER idPrior work from our group reported that residents’ biological stress measured by Tumor Necrosis Factor-alpha (TNF-α) was related to their reported near-misses in a large level 1 trauma center  stressinThe study was conducted in the emergency department (ED) of a level 1 trauma center in 2011. There was a total of 42 emergency medicine residents of which 34 (81%) consented. Complete data was secured from 28 (67%). The participants were between 26 and 35 years of age , primarily males (71%), and in their second (60.7%) or third year (39.3%) of residency. The residents’ program director was not involved in recruitment or data collection to avoid undue pressure on the residents to consent to participate. Rather, a senior Emergency Medicine physician who did not practice on a regular basis in the ED provided information about the study to the residents. The residents’ perceptions of the clinical environment were assessed after a rest period to control for the effect of shift-related fatigue. Over half of the residents reported having rested between 4 and 8 h prior to the start of the shift, about 38% rested over 8 h and about 7% had rested less than 4 h. The study design and reporting follow the Strengthening the Reporting of Observational Studies in Epidemiology guidelines [The residents’ rating of their attendings’ feedback was assessed 1 week before the shift in order to capture the residents’ overall assessment of the clinical learning environment and to ensure that these ratings were not affected by shift-related factors. The study used the Quality-Work-Competence (QWC) questionnaires to assess residents’ ratings of feedback from their attendings on their performance as well as residents’ chronic exhaustion level rather than just daily fluctuations . Blood samples (10 mL via venipuncture) were collected and were drawn on-site in the ED, placed on ice, and immediately transported to the laboratory. Samples were centrifuged, and plasma was stored at − 80 °C for later measurement of the biological stress biomarker TNF-α, using routine, commercially available human Enzyme Linked Immunosorbent Assay kits [Immediately after the shift ended (around 4 pm), residents were asked to report how many critically-ill and trauma patients, respectively, they had cared for during the shift using an 11-point scale (from 0 to 10 or greater). These two measures were summed to denote patient acuity. The residents also rated the number of near-misses that they had experienced during the shift . Bivariate analysis using Spearman’s rho was used to examine correlations between the independent factors: patient acuity, TNF-α, work-related exhaustion, feedback and the outcome near-misses reported by the residents. Linear regression analysis was used to predict residents’ reports of near-misses. In the first step, patient acuity, TNF-α and work-related exhaustion were entered as predictors of reported near-misses. In the final step, residents’ ratings of feedback from their attendings were added. Statistical significance was set at a two-sided p value of < 0.05.Bivariate associations and descriptive statistics are depicted in Table Table In partial support of our hypothesis, we found a positive association between how residents rated feedback in general from their attendings, and their reported near-misses. However, the association was only significant in the multivariate analysis, when patient acuity and pre-shift TNF-α were included in the model. When feedback was added to the model, the explained variance increased by 10%. A higher number of reported near-misses when residents give higher ratings to the feedback from their attendings may be due to residents becoming more aware when he or she commits a near-miss. Alternatively, when the attendings’ provide regular and constructive feedback on the residents’ performance, this may encourage them to report any near-misses encountered as they may view this as a learning opportunity. CLER findings suggest that a clear majority of residents do not report their near-misses due to a lack of knowledge of what a near-miss is . It alsoThe impact of patient acuity and TNF-α on residents’ reporting of near-misses in this same population has been previously reported . ResidenThere is a clear need to identify reliable measures of residents’ performance that allow an open and data-informed discussion between the residents and the attendings on near-misses and actual adverse medical events. Although this was a small study, the 28 participating residents reported 8 near-misses over a short time period, which is not an insignificant number. Near-misses are encountered more frequently compared to adverse patient outcomes and provide an opportunity to improve patient safety and prevent actual adverse events by having an open dialogue between the residents and the attendings since the patient was not hurt in this instance , 10. FurThe current study provides a self-report measure that can be used to quantitatively assess residents’ appraisals of their attendings’ feedback on the residents’ performance. The establishment of quantitative and psychometrically validated measures are critical to be able to define and achieve high-performance clinical learning environments as well as benchmarking and learning across academic healthcare centers. This study assessed the attendings’ feedback on residents’ performance in close proximity to the actual ED shift. This allowed us to study short-term temporal relationships between the residents’ rating of the feedback they receive from attendings and reported near-misses.This pilot study suggests that the management’s efforts to encourage reporting of near-misses by residents should focus on improving the malleable factor in clinical learning environments i.e. feedback given to the residents by their attendings. Future large-scale studies should explore the association between attendings’ feedback and residents’ near-misses in different clinical learning environments to validate the findings from this study.The sample size was small and included residents from only one emergency department. Furthermore, actual numbers of documented adverse medical events were not assessed.


As an emerging type of Internet of Things (IoT), multimedia IoT (MIoT) has been widely used in the domains of healthcare, smart buildings/homes, transportation and surveillance. In the mobile surveillance system for vehicle tracking, multiple mobile camera nodes capture and upload videos to a cloud server to track the target. Due to the random distribution and mobility of camera nodes, wireless networks are chosen for video transmission. However, the tracking precision can be decreased because of degradation of video quality caused by limited wireless transmission resources and transmission errors. In this paper, we propose a joint source and channel rate allocation scheme to optimize the performance of vehicle tracking in cloud servers. The proposed scheme considers the video content features that impact tracking precision for optimal rate allocation. To improve the reliability of data transmission and the real-time video communication, forward error correction is adopted in the application layer. Extensive experiments are conducted on videos from the Object Tracking Benchmark using the H.264/AVC standard and a kernelized correlation filter tracking scheme. The results show that the proposed scheme can allocate rates efficiently and provide high quality tracking service under the total transmission rate constraints. The Internet of Things (IoT) is a global infrastructure for the information society, enabling the advanced services by interconnecting physical and virtual things based on the existing and evolving interoperable information and communication technologies . The IoTA lot of existing research has considered video coding and transmission schemes to improve the human visual quality ,13,14,15To improve the service quality of video analytics in surveillance networks, plenty of researchers have worked on improving the performance of video analysis schemes in the computer vision field. Detection, recognition and tracking of human/vehicles are the main analysis tasks of smart surveillance systems ,18,19,20The main challenges for the schemes which chose to upload videos and analyze videos on a server, are the limited transmission resources and network state fluctuations. In ,27, videOnly a few researchers have studied the effect of video compression and transmission parameters on video analysis performance. In , a salieIn a realistic surveillance network for vehicle tracking, all surveillance videos are uploaded to server for video analysis purposes. When video sequences are uploaded via a wireless network, multiple camera nodes compete for the transmission resources which are limited by the bandwidth. Therefore, considering the effect of video content and video quality degradation caused by compression and transmission error, a joint source and channel rate allocation scheme is proposed in this paper to optimize performance of KCF tracking scheme in server. The main contributions of this paper can be summarized as follows:(1) Content-aware tracking precision prediction model. The features of the video content, which are not considered in existing prediction models, have a great impact on tracking precision. Based on the effect of video compression and video content on KCF tracking scheme, we take the bits per pixel, luminance values and complexity of the video content as key factors in the process of HOG extraction and object matching in KCF scheme. Then, based on the obtained relationship between these factors and the tracking precision, we establish a content-aware tracking precision prediction model by using curve-fitting method. (2) Tracking-precision-driven joint source and channel rate allocation scheme. Due to the limited wireless resources and the unreliability of wireless networks, it is important to design a joint source and channel rate allocation scheme to avoid degradation of tracking precision caused by video compression or transmission errors. We optimize the rate allocation for camera sensors by a tracking-precision-driven optimization problem. Based on our content-aware tracking precision prediction model, the optimization problem is formulated to maximize the product of tracking precision of each surveillance video in MIoT server under the constraint of the total transmission rate in the wireless network. To improve the reliability of data transmission and the real-time of the video communication, we adopt forward error correction (FEC) in the application layer. The rest of this paper is organized as follows: The proposed structure of the MIoT system for vehicle tracking, which consists of three main components, namely a mobile camera node, wireless network and MIoT server, is shown in Mobile camera node: This can be a mobile sensor device like a car recorder, smart phone, or an unmanned aerial vehicle, etc. In every group of picture (GOP) period, the camera node has two tasks: (1) The camera node calculates and uploads video content features of current uncoded GOP to a rate allocation module in the server; (2) the camera node encodes and uploads the GOP according to the rate allocation policy from the MIoT server. The H.264 encoder and Raptor  FEC encoWireless Network: A wireless wide area network (WWAN) is chosen for video transmission due to the random distribution and mobility of camera nodes. When uploading videos to the server via a WWAN, multiple camera nodes compete for transmission resources which are limited by the bandwidth. Due to the inherent transmission characteristics of wireless networks, packet loss occurs in the transmission network. In this paper, we assume that the packet loss rate of each camera node is perfectly known by the MIoT server.MIoT server: In the MIoT server, a joint source and channel rate allocation module optimizes the source and FEC rates for each camera node according to the content features and the current channel packet loss rates of GOPs in all camera nodes. Then the target source and redundancy rates of all camera nodes are sent back to each camera node for current GOP encoding. Besides, the MIoP server stores or performs vehicle tracking on received videos according to the actual demand. Since the tracking tasks usually are assigned with a delay in realistic scenarios, there is no real-time feedback about the tracking results in the MIoT server.K source packets of video stream, (N−K) redundant data packets are produced by the FEC encoder and are sent with source packets of the source block. As long as the receiver, which is a MIoT server in our structure, receives at least any K of the N packets, it can recover all the source packets [The FEC encoder shown in  packets .The KCF tracking scheme is proposed by Henriques et al. in 2015 . Since KAs shown in ppB) can be calculated as follows:r, Since HOG features can be affected by the artifacts created by video encoder at different compression rates , we emplL) to represent the effect of lighting conditions on HOG computation.When a camera is capturing video, the luminance value of pixels can be greatly affected by the lighting conditions at that time. Under the circumstance of weak lighting conditions, cameras are prone to lose detailed information of objects because of the lack of reflected light from objects and the interference of noise, and HOG features are affected consequently. We employ luminance level , in the covered field frames Based on the luminance levels of classified frames, the video scenarios can be detected and the video luminance level is obtained consequently. According to the previous description about scenarios, the video that has several consecutive covered field frames is regarded as a covered field scenario. Therefore, a video with more than five consecutive covered frames is considered a covered field video. The other videos are considered as open field videos. Due to the error propagation caused by the tracking scheme, different luminance level calculation methods are designed for different scenarios. In the tracking scheme, once a wrong target is determined in a frame due to the loss of HOG information caused by a low luminance level, the right target is hard to choose in all following frames even when the following frames have high luminance levels. Therefore, the open field frames are excluded when calculating the luminance level of video that has daytime covered field scenarios, as shown in Equation (5):f-th frame, N is the total frames in the video sequence. cN is the total covered field frames in the video sequence. Hence, f-th frame in video which can be obtained by Equation (7):f-th frame. In other words, The other videos, which have open field scenarios in daytime or at night have a different luminance level calculation method, that is:V) to represent the effect of video content complexity on tracking precision.In the KCF tracking scheme, the correlation results from kernelized correlation filter depend on the shapes of objects in the frame. In a video with complex content, the target object is surrounded by other background objects and the target object is hard to separate from the surroundings, which decreases the matching precision. We employ the video adjacent block difference  is the total number of foreground MBs in the f-th frame, and clstN(f) is the total cluster number of the f-th frame, which can be obtained based on the cluster results of foreground macroblocks (MBs) in the f-th frame. Here, the foreground MBs refers to the MBs which have more edges than other MBs in the frame. As the analysis in minG is the minimum of meanG is the mean magnitudes of intensity gradients in a frame. Then foreground MBs are detected based on the gradients magnitudes in the frame. Objects, such as vehicles, trees and pedestrians, always have more edges than background like road and sky. Then, we define the foreground set The adjacent block difference of a video sequence is defined as the maximum frame adjacent block difference just as follows:fmbN is defined as the total number of MBs in Then, clstN is then obtained based on clustering of foreground MBs. As shown in c) is the maximum cluster number when the c) + 1 indicates that the The total cluster number clstN is defined as the maximum c in the frame. In one cluster, magnitudes of gradients between adjacent MBs are prone to be small, which makes object matching difficult. Thus a smaller clstN indicates more complex content.Then, We select six videos which take vehicles as the target objects from the Online Object Tracking Benchmark (OOTB) . Among tppB to be our main tracking precision prediction parameter. According to the relationship between tracking precision and ppB shown in P is the tracking precision, and L and V of the video as follows:We use The finally obtained model coefficients are shown in k and n are the Mk represents the source packets of mobile camera node m. The element n represents the FEC packets of mobile camera node m, which contains the source packets and the protect packets. The proposed scheme allocates the appropriate source and FEC packets for GOP in each camera node, such that the product of the tracking precision of all GOPs is maximized. The joint source and channel rate allocation scheme can be formulated as follows:m. S is the packet size and m in application layer [In the MIoT system, multiple mobile camera nodes have to compete for the limited wireless transmission data rate. Due to the packet loss in the wireless network transmission, FEC needs to be used for source data protection. Therefore, we develop a joint source and channel rate allocation scheme for both video and FEC rate allocation with the objective to maximize the product of tracking precision of each uploaded GOP. Let M be the number of mobile camera nodes in system, and on layer :(17)f of the current raw GOP and sends these features to the server. The server jointly optimizes the source coding rate and FEC rate for all camera nodes based on these features and packet loss rates of all camera nodes. Then the optimal source coding rates and FEC rates are fed back to each camera node for current GOP coding. The encoded GOPs of six camera node are uploaded to the server. Next GOPs are also processed like this. Finally, we evaluate the tracking performance on the uploaded GOPs. In our simulation, we selected six video sequences from OOTB [We simulate a scenario where six mobile camera nodes capture different scenes and upload videos to a server via lossy wireless networks. The six mobile camera nodes have to compete for the limited transmission resource which in our simulation is represented by the total transmission bitrates. The channel condition of each camera node is represented by its packet loss rate. Each camera node extracts the two content features (rom OOTB  and visurom OOTB  data setm. We compare our proposed scheme with three schemes: the mean square error (MSE) driven rate allocation scheme, QoE driven rate allocation scheme and constant bitrate control (CBR) scheme. In CBR scheme, the total transmission rate is equally allocated to each camera node, and the packet correct rate of each node is set to be 0.995. For the MSE-driven and QoE-driven schemes, we adopt a classical rate-distortion model in  and a QoWe set a fixed total data rate of 800 kb/s for the proposed tracking-precision-driven scheme (TP-driven scheme), MSE-driven scheme, QoE-driven scheme and CBR scheme. The packet loss rates of all camera nodes are set to be 1%. Next, we conduct tracking on these video sequences by KCF scheme. The number of correct tracking frames in each GOP is illustrated in Here, we assume that the ground truth of the first frame in each video sequence is assigned by the server. From The performance of the proposed TP-driven scheme, MSE-driven scheme, QoE-driven scheme and CBR scheme is tested under different total rate constraints. We test the FEC rate allocation by setting different packet loss rates for each video. Since in the CBR scheme, the source data rates of all camera nodes are equally allocated to all videos, only the TP-driven, MSE-driven and QoE-driven schemes are tested. In the first case, the packet loss of each video is 1%. In our simulation, the GOP structure is IPPP. In fact, several B frames are allowed in the GOP, GOP structure like IBBPBBP can provide similar performance like IPPP. However, the GOP structures which have a lot of consecutive B frames (IBB…BP structure for example) are not suitable for our scheme. Due to the blurring and fast movement in the video, a lot of consecutive B frames can cause obvious video quality degradation. Consequently, tracking precision decreases. Therefore, I or P frames are needed to be inserted every several B frames in the GOP for high quality service.In this paper, we have proposed a joint source and channel coding algorithm for vehicle tracking in MIoT systems. Our algorithm aims to provide better tracking performance in MIoT servers, which is different with previous QoS/QoE-driven schemes which aim to improve the human visual experience. We first study the effect of video content and compression on the KCF tracking scheme and establish a content-aware tracking precision prediction model, which can estimate the tracking precision of videos with different content features and coding rates. In this model, video luminance level and adjacent block difference are extracted to represent the effect of lighting conditions and video content complexity on tracking precision, respectively. Then, based on the precision prediction model, the optimization problem is formulated to maximize the product of tracking precision of each surveillance video under the constraint of the total transmission rate in the wireless network. Considered the transmission error in wireless network, FEC is adopted to improve the reliability of data transmission. Compared with the traditional QoS/QoE-driven algorithm and CBR algorithm, our joint source and channel rate allocation algorithm more efficiently allocates the bit rates under the constraint total transmission rate and achieve better tracking performance.


Introduction: Operative surgical videos are a popular educational resource, not commonly a part of a peer-reviewed article. We wanted to evaluate the impact of either reading a peer-reviewed manuscript or watching an operative video on a surgeon's confidence in performing a complex case.Methods: Pediatric surgeons and fellows were asked to complete an initial questionnaire to assess their confidence (formulated as a score) in the diagnosis and operative repair of anal stenosis and rectal atresia.Results: Of 101 pediatric surgeons and fellows, 52 (51%) were randomized into a “manuscript” group and 49 (49%) into a “video” group. The mean confidence before the intervention was the same in the two groups (6.4 vs. 6.6). Attending surgeons started with more confidence than trainees . In the manuscript group, the average confidence increased to 7.7 (p = 0.005), and in the video group the average confidence increased to 7.9 (p = 0.001) globally. Trainees in the video group significantly improved their confidence to a score of 6.6 (p = 0.035), as did attending surgeons to 8.5 (p = 0.01). In the manuscript group, only attendings significantly improved their confidence by 1.5–8.3 (p < 0.001), whereas trainees did not with a difference of 1.3 (p = 0.194). When considering experience level, physicians who reported never having performed this surgery improved only by reading the manuscript (3.9–6.2) (p = 0.004), not by watching the video (5.4–6.6) (p = 0.106). Surgeons with experience doing this operation (>5 times) did not improve their confidence by reading the manuscript (p = 0.10), nor by watching the video (p = 0.112).Conclusion: Reviewing either a detailed manuscript or operative video on the surgical management of rectal atresia and anal stenosis demonstrated a significant increase in self-reported confidence. Trainees benefitted the most from operative videos, whereas experienced surgeons did not improve their confidence by reading the manuscript nor watching the video. Traditionally, surgeons and residents have learned new operative techniques by reading about surgical techniques and trying to improve their skills in the operating room. Recently, newly emerged media, such as video, are already being used in educating medical students and seem to have value as an addition to traditional educational measures , 2. In rAny pediatric surgeon or pediatric surgery resident could participate in this study. The study was approved and excempted from a full review by the Institutional Review Board of Nationwide Childrens Hospital due to the voluntary, consented and anonymous participation of our study population. We contacted surgeons and residents by email and asked them to anonymously complete a questionnaire assessing their confidence in diagnosing and operating on patients with anal stenosis or rectal atresia. The participants were then randomized into two groups: one group watched the video, and the other group read the manuscript. Thereafter, they completed the same questionnaire as before the intervention, to assess any difference in confidence.To assess the confidence of physicians in performing the surgery, we developed an 11-item questionnaire (see addendum A). Participants were asked about their experience in these operations and whether they were a trainee or an attending surgeon, enabling us to perform a subgroup analysis on these groups. For the overall analysis, we looked at the individual questions, but averaged the confidences of the questions into a total score as well.The manuscript describing the operative technique was published earlier in a peer-reviewed journal . FurtherU-tests were performed to compare the total pre- and post-confidence between the video and the manuscript groups. The same test was used to see if there was any improvement in either group for the subgroup analysis and to look at the individual questions. For the overall comparison, a p < 0.05 was considered significant. When comparing the individual questions, after applying a Bonferroni correction, a p < 0.00833 was considered significant.Mann-Whitney One hundred one surgeons completed the pre- and post-intervention questionnaires. Of those, 52 (51%) were randomized to the manuscript group and 49 (49%) to the video group. Of the surgeons completing both surveys, 66 (65%) were attending pediatric surgeons, 19 (65%) were pediatric surgery residents (19%), and 16 (16%) were pediatric surgery fellows. Thirty-two (32%) participants reported never having performed either surgery, whereas 46 (46%) participants had performed it between one to five times, and 23 (23%) surgeons had performed the surgery more than five times.p = 0.962). Attending surgeons reported an overall confidence of 7.4 in the video group and 6.8 in the manuscript group, whereas these numbers were 5.2 and 5.5 for trainees. Physicians who had not previously performed the surgery noted a pre-intervention confidence of 5.4 in the video group and 3.9 in the manuscript group, whereas these were 6.4 and 6.7 for surgeons having performed this surgery one to five times. Finally, surgeons that had performed this particular technique at least five times reported a confidence of 8.7 in the video group and 8.5 in the manuscript group before receiving the intervention.The baseline overall confidence was 6.6 in the video group and 6.4 in the manuscript group (p = 0.005) in the video group and to 7.9 (p = 0.001) in the manuscript group. When comparing the two groups, there was no significant difference between the total scores (p = 0.790) or any of the separate questions. However, when looking at the individual questions in the video and manuscript groups separately, the manuscript group improved significantly on five of six questions, whereas the video group improved only on two questions  to 6.6 . The confidence of attending surgeons who watched the educational video increased from 7.4 (n = 43) to 8.5 .We also analyzed the influence of experience on the increase of confidence (see Table p = 0.001). This was not the case for trainees: as a result of reading the article, there was no statistically significant increase in confidence .Attending surgeons who read the manuscript significantly increased their scores, as well  in the video group and from 3.9 to 6.2 (p < 0.001) in the manuscript group. The same conclusion can be drawn when looking at surgeons who had performed this surgery between one and five times: when watching the video, their confidence rose from 6.4 to 7.8 (p = 0.006), whereas it increased from 6.7 to 8.2 (p = 0.001) when reading the article.We also took into account the number of times a surgeon had done this particular operation. Surgeons having never performed this operation significantly improved their confidence in both groups: from 5.4 to 6.6 (p = 0.042), but did not improve by reading the manuscript .Experienced surgeons (>5 surgeries performed) significantly increased their confidence only by watching the video . These finding are in the range of what one might expect of the confidence of a group of pediatric surgeons in a specific surgical technique.As expected, both groups significantly improved their confidence in performing this particular operation. When looking at the overall confidence, the video group and the manuscript group improved similarly: from 6.6 to 7.7 and 6.4 to 7.9, respectively. However, when looking at the six questions separately, the surgeons who read the manuscript improved their confidence significantly on five of six questions, whereas surgeons who watched the video improved only on two questions. One could therefore argue that the manuscript seems better at improving the broader confidence of every aspect of diagnosing and treating rectal atresia and stenosis.Attending surgeons benefited from both the video and the manuscript, whereas trainees benefitted only by watching the video. It is conceivable that with more general surgery knowledge , it is easier to learn from an article. On the other hand, a trainee with fewer skills benefits from the visual instruction that a video offers. An earlier study found that faculty can name more laparoscopic facts when shown a video than can residents shown the same video, but the study did not measure actual proficiency in surgery or confidence to perform the operation .Interestingly, when categorizing participants according to the number of times they have performed the operation, surgeons with the most experience do not significantly improve their scores when reading the manuscript vs. watching the video. Participants with no experience in this particular surgery at all are better off reading the manuscript.Watching a video and reading a manuscript about a specific surgical technique similarly improve a surgeon's confidence in performing the operative technique. However, reading a manuscript improves almost every aspect of the surgery, whereas watching a video does not. Surgeons with no experience seem to win greater confidence in performing a procedure after watching a video, but an actual performance on the procedure itself, and its outcomes can't be concluded from this data.CR and AD had the idea, planned and performed the study. VL edited the video and manuscript used for the study. JC did the statistical analysis on the paper. RW and ML edited and corrected the manuscript.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.


Research in fall prevention programs has increased in recent years in response to the aging demographics of the United States. To date, limited research and outreach programs have focused on ethnic minorities due to increased cost, language barriers, and cultural differences. Digital media platforms may be a cost-effective avenue to initiate fall prevention programs for minority populations.The objective of this study was to determine whether Facebook advertisements are a practical recruitment method for health education to the Chinese-speaking population.This was an observational, cross-sectional study. We uploaded a video on fall education on YouTube and initiated an advertisement campaign on Facebook that was linked to the video. The target population was older adults aged >45 years who used Facebook and were presented with the advertisement (N=1039). We recorded metrics such as the number of unique individuals reached, the number of views of the advertisement, the number of clicks, user gender and age, and traffic sources to the advertisement. Data were analyzed with descriptive statistics.Our Facebook advertisement had 1087 views (1039 unique viewers). There were 121 link clicks with a click-through rate of 11.13% (121/1087). The cost per link click was approximately US $0.06. Among the viewers, 91.41% (936/1024) were females and 8.59% (88/1024) were males. In the 45-54 age group, the ad reached 50 people, with 1 link click (2.00%). In the 55-64 age group, the ad reached 572 people, with 57 link clicks (9.97%). In the ≥65 age group, the ad reached 417 people, with 63 link clicks (15.11%).Facebook was able to directly target the advertisement to the desired older ethnic population at a low cost. Engagement was highest among females and among those aged ≥65 years. Hence, our results suggest that Facebook can serve as an alternative platform for dissemination of health information to geriatric patients in addition to print-based and face-to-face communication. Falls are highly prevalent among older adults in the United States. Every year, 2.8 million older adults are admitted to emergency departments for fall injuries, and >800,000 patients are hospitalized from fall-related injuries such as hip fracture or head injuries . Falls cIn response to the aging demographics in the United States, new guidelines and outreach programs are being implemented and reformed. For example, the Centers for Disease Control and Prevention created the Stopping Elderly Accidents, Deaths, and Injuries initiative, a multiapproach program that aims to reduce fall injuries by improving screening and intervention rates at clinical settings. However, few programs were able to accommodate ethnic minorities with different culture and language backgrounds.Minorities constitute the fastest growing segment in the rapid rise of older adults in the United States. Between 2001 and 2010, the older Asian American population increased by 145% . PreviouElectronic health and Web-based outreach programs have shown substantial success in promoting awareness -14. PopuWe uploaded a recording of a 37-minute-long video of a medical education talk show at the radio station KMRB AM1430 in Los Angeles on YouTube in May 2016 . The shoThe population sampled in this study included all viewers who saw the Facebook advertisement on Web during the 48-hour advertising campaign. According to the current literature, falls and related injuries are most prevalent in adults aged >65 years . TherefoData on the advertisement campaign were obtained via Facebook analytics. Parameters included the number of individuals reached (defined as unique advertisement viewers), the number of impressions , the number of engagements , the click-through rate (clicks/impressions), and the cost per click. In addition, demographic information including gender, age, and traffic sources was recorded. Data obtained via Facebook analytics were further confirmed using data directly obtained from the video link via YouTube analytics during the 48-hour campaign. Data were analyzed using descriptive statistics.Overall, the Facebook advertisement had 1087 impressions, of which 95.58% (1039/1087) views were by unique individuals (reach). Of 125 engagements recorded by Facebook analytics, 121 were link clicks, 3 were post reactions, and 1 was a post share. The click-through rate (clicks/impressions) was 11.13% (121/1087). The total cost of the advertisement for 48 hours was US $6.82. The cost per 1000 impressions was US $6.27, and the cost of the ad per link click was US $0.06.In the 45-54 years age group, the ad reached 4.81% (50/1039) viewers, with 1 (2.00%) link click. In the 55-64 years age group, the ad reached 55.05% (572/1039) people, with 57 (9.96%) link clicks. In the ≥65 years age group, the ad reached 40.13% (417/1039) people, with 63 (15.11%) link clicks. The costs per link click were US $0.23, US $0.07, and US $0.04 for each group, respectively.In the 45-54 years age group, 38 females and 12 males were reached; in the 55-64 years age group, 523 females and 43 males were reached; and in the ≥65 years age group, 375 females and 33 males were reached. There were 15 impressions with unknown gender. Only 1 female and no males clicked the advertisement in the 45-54 years age group; 52 females and 5 males clicked the advertisement in the 55-64 years age group; and 59 females and 3 males clicked in the ≥65 years age group.With regards to device usage, the advertisement reached 99.62% (1035/1039) mobile device users, resulting in all 121 of the link clicks recorded. Only 4 users were reached through desktop devices, and there were no link clicks via desktop devices.During the first year when the same educational video was uploaded on YouTube, YouTube data showed 588 views with an average view duration of 27% (9.82/37.1 minutes) of the total video length. Mobile phones accounted for 52.2% (307/588) of the views.In comparison, during the 48-hour advertisement run, the video had 75 views on YouTube, of which 66 viewers arrived from external websites. The total watch time was 440 minutes, and the average view duration was 16% (5.87/37.1 minutes) of the total video length. Data on audience retention revealed that 13% viewers (10/75 of viewers during this time period) finished all 37.1 minutes of the video. Mobile phones accounted for 85% (64/75) views.This study demonstrates that Facebook was able to attract viewers to Web-based health education resources at low cost, as well as correctly target the advertisement at the desired older ethnic population. Engagement and cost-effectiveness were highest among those aged ≥55 years. This ultimately suggests that older Asian individuals may benefit from targeted preventive health and educational interventions delivered through Facebook.One advantage of using Facebook is its ability to directly target a certain demographic group; in our study, we were able to target our advertisement on fall education toward Chinese-American older adults with an above-average click-through rate (11.13% vs 0.90%) and a lower cost per click (US $0.06 vs US $1.72) compared with the Health and Medical Industry average . On compA few other noteworthy patterns were also observed in this study. First, the advertisement disproportionately reached a very large number of female viewers compared with male viewers. Similar results have been noted in several previous studies on Facebook advertisements, which could be attributed to higher rates of Facebook usage by females across all ages -20. In aComparison of the data received from Facebook and the data gleaned from YouTube analytics during the 48-hour run of the advertisement revealed that the Facebook advertisement was able to increase traffic to the YouTube video. During the first year after uploading the video on YouTube, there were 588 views. During the 48 hours analyzed, there were 75 new views of the video, indicating that the advertisement was successful in directing traffic. Interestingly, the average view duration dropped from 9.82 minutes during the first year to 5.87 minutes during the 48 hours. We speculate that this may be due to the fact that many Facebook users were not actively seeking out information on fall prevention when they came across the advertisement and, thus, may have a shorter engagement time than those who were actively searching for such information. However, the average view duration recorded from this advertisement coincides with the reported median engagement time of 6 minutes for Web-based educational videos, suggesting that the video was able to capture viewers’ attention .Limitations of this study include the inability of researchers to assess knowledge before and after viewing of the video. As such, the efficacy of the video in providing information on falls remains unknown. Additionally, because of the anonymity of internet users, as stated previously, there is no way of knowing the motivation of viewers for watching the video or their actual socioeconomic status. Furthermore, the study video was conducted in Cantonese; therefore, the audience reached was likely a selected group of Chinese-American individuals who are fluent in that language. Moreover, the advertisement only ran for 2 days, which qualifies as a short campaign. It remains unknown whether a longer campaign would have better or worse impact on the overall views of the video. Finally, the engagement time for the video was short. In the future, the video should be shortened with more visual aids and focused content to improve viewers’ engagement.Future studies should explore how to optimize advertisement descriptions to maximize the click-through rate. Owing to our result showing the high prevalence of female users in the older ethnic Facebook user population, we suggest that Facebook may provide an attractive platform for future distribution of information regarding women’s health. In addition, future studies will likely have to address the increasing prevalence of mobile devices in exploring Web-based health care resources, as demonstrated by this study.In this study, we were successful in engaging users; however, more research is needed in determining how to receive feedback regarding what this population may need in terms of health education information. Most viewers were older individuals belonging to an ethnic population; however, the very anonymity that attracts internet and social media users also makes it difficult for researchers to determine why this patient population is watching health education videos and to figure out the type of help they need. The question of how to bridge this gap between remote-based tools, internet anonymity, and clinical care is the next problem to solve in the internet and social media health information delivery research.


Paenibacillus polymyxa WLY78 can strongly inhibit Fusarium oxysporum f. sp. Cucumerium, which causes Fusarium wilt disease. In this study, we screened the genome of WLY78 and found eight potential antibiotic biosynthesis gene clusters. Mutation analysis showed that among the eight clusters, the fusaricidin synthesis (fus) gene cluster is involved in inhibiting the Fusarium genus, Verticillium albo-atrum, Monilia persoon, Alternaria mali, Botrytis cinereal, and Aspergillus niger. Further mutation analysis revealed that with the exception of fusTE, the seven genes fusG, fusF, fusE, fusD, fusC, fusB, and fusA within the fus cluster were all involved in inhibiting fungi. This is the first time that demonstrated that fusTE was not essential. We first report the inhibitory mode of fusaricidin to inhibit spore germination and disrupt hyphal membranes. A biocontrol assay demonstrated that fusaricidin played a major role in controlling Fusarium wilt disease. Additionally, qRT-PCR demonstrated that fusaricidin could induce systemic resistance via salicylic acid (SA) signal against Fusarium wilt of cucumber. WLY78 is the first reported strain to both produce fusaricidin and fix nitrogen. Therefore, our results demonstrate that WLY78 will have great potential as a biocontrol agent in agriculture.Cucumber is an important vegetable crop in China. Fusarium wilt is a soil-borne disease that can significantly reduce cucumber yields.  Fusarium oxysporum f. sp. cucumerium is the causal agent of the Fusarium wilt of cucumber, a soil-borne disease that can significantly reduce cucumber yields [Cucumber is an important vegetable crop in China. r yields . To suppr yields . TherefoPaenibacillus polymyxa strains offer a biological solution to suppress plant diseases due to their ability to produce some compounds that inhibit plant pathogens and are listed as commercial biocontrol agents by the United States Environment Protection Agency [P. polymyxa strains have been used as biocontrol agents in controlling crown rot disease, hairy root disease, grapevine aerial disease, Phytophthora blight of pepper, Fusarium wilt of watermelon, and blackleg disease of canola [n Agency ,4. P. pof canola ,7,8,9,10Bacillus and Paenibacillus genera, can produce antimicrobial lipopeptides [Several mechanisms have been attributed to biocontrol agents for inhibiting plant diseases: the synthesis of antibiotics, root-colonization, and the induction of plant resistance . A wide peptides ,13,14. Tpeptides . Bacteripeptides . P. polymyxa, which contain a cyclic polypeptide (CP) consisting of six amino acids and a guanidinylated β-hydroxy fatty acid (GHPD) [P. polymyxa strains. P. polymyxa strain L1129 produced a peptide antibiotic complex, named LI-F, from which five kinds of fusaricidins LI-F03, F04, F05, F07, and F08 were isolated [P. polymyxa KT-8 and from P. polymyxa SQR-21 [Fusarium genus in vitro. The possible antibacterial mechanism of fusaricidin is that the compound interacts with the cytoplasmic membranes, while the inhibitory mode of action of fusaricidin to inhibit fungi remains unclear [Fusarium wilt of cucumber in vivo has not been rigorously proven up to now.Fusaricidins are a class of cyclic lipopeptide antibiotics produced by d (GHPD) . Severalisolated . Four kia SQR-21 . All kin unclear . AdditioPseudomonas spp., was reported to induce systemic resistance in rice [Bacillus spp. were reported to induce ISR via the salicylic acid (SA) pathway, rather than the ethylene pathway [PR genes [PR genes coding for proteins with antimicrobial activities in plant, providing plants with long-term resistance against subsequent pathogen infections [P. polymyxa WLY78 could colonize the rhizosphere of wheat, maize, and cucumber [P. polymyxa WLY78.In addition, orfamide, a lipopeptide antibiotic produced by  in rice . The ind in rice . Interes pathway ,24,25. IPR genes , while tPR genes . SAR canfections ,29. Prevcucumber . HoweverP. polymyxa WLY78, a nitrogen-fixer, showed strong antifungal activity against F. oxysporum f. sp. cucumerium in vitro [P. polymyxa WLY78 to inhibit plant pathogens, eight potential antibiotic biosynthesis gene clusters located on the genome of P. polymyxa WLY78 were predicted to be candidates. Mutation analysis revealed that the fusaricidin synthesis (fus) gene cluster was involved in inhibiting not only the Fusarium genus, but also Verticillium albo-atrum, Monilia persoon, Alternaria mali, Botrytis cinereal, and Aspergillus niger. The previously reported fus gene cluster consists of eight genes . Further mutation analysis revealed that with the exception of the fusTE gene, the seven genes fusA, fusB, fusC, fusD, fusE, fusF, and fusG within the fus gene cluster were all involved in antifungal activity. This is the first time that the fusTE gene has been demonstrated to not be essential for fusaricidin production. Moreover, we first report that the inhibitory mode of the action of fusaricidin inhibits spore germination and disrupts the hyphal tips of fungi by causing cytoplasm leakage. Importantly, our study also revealed that fusaricidin elicits the SA-mediated system resistance for the first time. P. polymyxa WLY78 is also the first reported strain that could both produce seven forms of fusaricidins and fix nitrogen. Taken together, our results show that P. polymyxa WLY78 has great potential as a biocontrol agent in agriculture. in vitro ,32. In tPaenibacillus species  and two Bacillus species (B. amyloliquefaciens LJ02 and B. subtilis 168). We found that P. polymyxa WLY78 showed an excellent antifungal activity against F. oxysporum f. sp. cucumerium  gene cluster of P. polymyxa WLY78 showed 95% similarity with that of P. polymyxa PKB1 (GenBank: EF451155.3). The paenicidin B biosynthesis (pab) gene cluster of P. polymyxa WLY78 showed 100% similarity with that of P. terrae NRRL B-30644 (GenBank: KF111343.1). The polymyxin biosynthesis (pmx) gene cluster of P. polymyxa WLY78 showed 100% similarity with that of P. polymyxa PKB1 (GenBank: JN660148.1). The paenibacterin biosynthesis (pbt) gene cluster of P. polymyxa WLY78 showed 60% similarity with that of Paenibacillus sp. OSY-SE (GenBank: JX899679.1). The tridecaptin biosynthesis (tri) gene cluster of P. polymyxa WLY78 showed 100% similarity with that of P. terrae NRRL B-30644 (GenBank: KF111342.1). The bacillibactin biosynthesis (dhb) gene cluster of P. polymyxa WLY78 showed 53% similarity with that of B. subtilis subsp. subtilis str. 168 (GenBank: AL009126.3). The paeninodin biosynthesis (pade) gene cluster of P. polymyxa WLY78 showed 40% similarity with that of P. dendritiformis C454 (GenBank: AHKH01000064.1). The paenibacillin biosynthesis (paen) gene cluster of P. polymyxa WLY78 showed 100% similarity with that of P. polymyxa OSY-DF (GenBank: JQ728481.1). For more details, please refer to P. polymyxa WLY78 might produce corresponding antibiotics including fusaricidin, paenicidin, polymyxin, tridecaptin, and paenibacillin. Furthermore, eight gene clusters in software A. DNA hoF. oxysporum f. sp. cucumerium, the core structural genes of each of the eight clusters  were inactivated via homologous recombination, yielding eight mutants including −fusA, −pabB, −pmxA1, −pbtC, −triE, −dhbE, −padeC, and −paenC, and these mutants were identified by PCR  showed the same antifungal activities as P. polymyxa WLY78. In addition, the −fusA mutant lost its antifungal activity against F. asiaticum, F. moniliforme, V. albo-atrum, F. graminearum, M. persoon, A. mali, B. cinereal, and A. niger  arranged within a 31.8 kb region in the following order: fusG, fusF, fusE, fusD, fusC, fusB, fusA, and fusTE. Of the eight genes, fusA is the largest gene with a length of 23.73 kb, which is responsible for synthesizing the cyclic polypeptide (CP) moiety of fusaricidin. The products of fusG, fusF, fusE, fusD, fusc, and fusB are responsible for synthesizing the lipid moiety of fusaricidin  is independently transcribed by its own promoter , B (897 Da), LI-F05b (911 Da), LI-F07a (931 Da), C (947 Da), D (961 Da), and E (975 Da)  side chain, and the ion of m/z 628.16 represents the cyclic polypeptide (CP) of fusaricidin A (883.54). Furthermore, the N-terminal stepwise cleavage of CP was Thr (503.21), Val (404.06), and Val (305.11), while the C-terminal stepwise cleavage of CP was Ala (539.24) and Asn (425.08). Therefore, the fracture fragments for the ion of m/z 883 were exactly consistent with the molecular structure and fragmentation pattern of fusaricidin A. Using a similar method, the other six molecular ions of m/z 897, 911, 931, 947, 961, and 975 were confirmed as fusaricidin B, LI-F05b, LI-F07a, C, D, and E, respectively (P. polymyxa WLY78 were determined to show nitrogenase activities (P. polymyxa WLY78 produces seven forms of fusaricidin and fixes nitrogen.Liquid chromatography-mass spectrum (LC-MS) analysis showed that (975 Da) A. These ectively C–H. In ativities . All of P. polymyxa WLY78 and the −fusA mutant were individually added to the suspensions of F. oxysporum f. sp. cucumerium spores and hyphae. After incubation for different times at 28 °C, the spores and hyphae were observed under an optical microscope. The hyphal tips treated with the methanol extracts from P. polymyxa WLY78 were expanded and ruptured, while those treated with the −fusA mutant extracts grew well  exhibited serious disease symptom  of F. oxysporum f. sp. cucumerium, while the quantities of F. oxysporum f. sp. cucumerium in the rhizosphere soil treated with P. polymyxa WLY78 and the −fusA mutant were decreased to 0.6 × 103 CFU/g of soil and 2.33 × 103 CFU/g of soil, respectively , PR1 (for pathogenesis-related protein 1), PR2 , PR3 (for chitinase), and Etr1 (for ethylene receptor) in cucumber leaves at days 1, 2, and 3 post inoculation were comparatively analyzed by qRT-PCR. As shown in NPR1 was enhanced in P. polymyxa WLY78 treatment at day 1, which was significantly higher than that of the −fusA treatment and control. Then, the expression levels decreased at day 3 and showed no significant changes in response to WLY78 when compared with −fusA treatment. Similarly, the expression levels of the PR1 gene in P. polymyxa WLY78 treatment at day 1 was 20-fold higher than that of the −fusA treatment and then decreased to the same levels as the −fusA treatment did at day 3  that might be involved in synthesizing potential antibiotics for its wide-spectrum inhibitory character  from WLY78 showed high similarities (>60%) with those known antibiotic biosynthetic clusters. Although the five highly potential antibiotic biosynthetic gene clusters showed a more or less inhibitory effect against fungi or bacteria, in fact, according to the literature, the fus gene cluster is involved in synthesizing fusaricidin, which is able to inhibit pathogenic fungi including A. niger, Leptosphaeria maculans, and Penicillium expansum [pab gene cluster is involved in synthesizing paenicidin, which has been reported to inhibit Campylobacter jejuni [pmx and tri gene clusters are involved in synthesizing polymyxin and tridecaptin, respectively, which are strongly against Gram-negative bacteria [paen gene cluster is responsible for synthesizing paenibacillin, which can effectively inhibit Gram-positive bacteria [pbt, dhb, and pade) showed low similarities (<60%) with the corresponding reported antibiotic biosynthetic gene clusters, indicating their functions are incomplete. Therefore, the pbt, dhb, and pade gene cluster may fail to synthesize paenibacterin, bacillibactin, and paeninodin, respectively, as previously reported to inhibit fungi [P. polymyxa WLY78, we found that the fus gene cluster might play a crucial role in inhibiting various fungi  within the fus gene cluster were essential for antifungal activity, but fusTE was not  were organized as an operon, and fusTE was independently transcribed  [fusG, fusF, fusE, fusD, fusc, and fusB are crucial for synthesizing the lipid moiety of fusaricidin  ,46. Sincaricidin , our finaricidin D. Our reP. polymyxa strains. For example, P. polymyxa E681 produced a mixture of two kinds of fusaricidins, LI-F05b (911 Da) and LI-F08b (925 Da) [P. polymyxa strain E681 to produce five kinds of fusaricidins by using LC-MS [P. polymyxa M1 [Paenibacillus sp. strain Lu16774 [P. polymyxa SQR-21 could also produce four kinds of fusaricidins, A (883 Da), B (897 Da), C (947 Da), and D (961 Da) [P. polymyxa WLY78 produced seven forms of fusaricidins and all of these variants were characterized efficiently by LC-MS-MS fragment ion analysis  . Interesng LC-MS . Mass spymyxa M1 . Additio Lu16774 . Li et a(961 Da) . Using aanalysis A–H. Our for mice . These cP. polymyxa WLY78 suppressed the Fusarium wilt of cucumber more effectively than the fusaricidin-deficient mutant  medium for the cloning of plasmids. P. polymyxa strains were cultivated at 30 °C in LB medium, and Katznelson & Lochhead (KL) broth for the production of fusaricidin [P. polymyxa strains were grown in nitrogen-limited medium under anaerobic condition [F. oxysporum f. sp. cucumerium, F. asiaticum, F. moniliforme, F. graminearum, Verticillium albo-atrum, Monilia persoon, Alternaria mali, Botrytis cinereal, and Aspergillus niger) were cultivated at 28 °C in potato dextrose agar (PDA) medium.The source of strains and plasmids are listed in aricidin . For theondition . When neP. polymyxa WLY78 (GenBank: ALJV00000000) were predicted by antiSMASH (https://antismash.secondarymetabolites.org/#!/start). There are eight gene clusters that might be involved in the synthesis of the potential antibiotics in this bacterium and each gene cluster contained one to four core genes with lengths of 4–23 kb. The gene clusters that might be involved in the synthesis of antifungal substances in the genome of P. polymyxa WLY78. Then, the two homologous arms were assembled into the suicide plasmid pRN5101 digested by BamHI , yielding a recombinant plasmid, before the plasmid was transformed into P. polymyxa WLY78 as described [fus gene cluster was disrupted by deleting a 3.7 kb region of the fusA (core gene) by homologous recombination. Briefly, a 5’ homologous arm (1 kb) flanking the upstream of the coding region of fusA was amplified using primers fusAUf and fusAUr, and a 3’ homologous arm (1 kb) flanking the downstream of the coding region of fusA was amplified using primers fusADf and fusADr. The two homologous arms were assembled into the suicide plasmid pRN5101 digested by BamHI (New England Biolabs Inc.), yielding a recombinant plasmid pRN5101-TFfusA, and then the plasmid was transformed into P. polymyxa WLY78. The single-crossover transformants were selected for erythromycin resistance. Subsequently, the double-crossover transformants were selected from the initial erythromycin resistance transformants after several rounds of non-selective growth at 39 °C. The double-crossover mutants were identified by PCR and then their antifungal activities against F. oxysporum f. sp. cucumerium were examined. The primers for PCR are listed in fusA AYC81015, pabB MN087474, pmxA1 MN087480, pbtC MN087479, triE MN087478, dhbE MN087477, padeC MN087476, and paenC MN087475).To investigate whether the eight gene clusters were involved in antibiotic production, each gene cluster was disrupted by deleting a partial (2–4 kb) coding region of a core gene via homologous recombination. Two homologous arms (each ~1 kb in length), flanking the deleted coding region of a core gene, were amplified from the genomic DNA of escribed . For exafus gene cluster was deposited in GenBank (accession number MH368541.1). There were eight genes  within the fus gene cluster predicted by using antiSMASH. Each gene within the fus gene cluster was mutated by deleting its coding region via homologous recombination as described above. The primers for PCR are listed in The nucleotide sequence of the P. polymyxa WLY78 and its mutants were grown in LB broth, and the bacterial cells were collected after 12 h of cultivation at 30 °C. The bacterial cells were suspended in sterilized water with a concentration of 107 colony-forming unit (CFU)/mL. To compare the antifungal activities of P. polymyxa WLY78 with that of the mutants, F. oxysporum f. sp. cucumerium was inoculated onto the center of the plate containing the PDA medium. Then, individual 0.5 μL of cell suspensions from P. polymyxa WLY78 and from mutant strains were inoculated onto the two sides of F. oxysporum f. sp. cucumerium at the same distance of 2.5 cm. All plates were cultured at 28 °C for four days. Then, the inhibition effect of fungal growth was recorded. fus cluster were organized in a single operon, several primers designed to span across intergenic regions were used to conduct the RT-PCR reaction. Total RNA of the P. polymyxa WLY78 cells collected from the LB broth was extracted by RNAiso Plus and converted into cDNA by a Reverse Transcription Reagent Kit . The primers for RT-PCR are listed in To verify if the eight genes within the P. polymyxa WLY78 and the fus mutants were grown in 100 mL of KL broth at 30 °C for 72 h with shaking at 200 rpm. The cells were collected by centrifugation at 12,000 rpm, 4 °C for 5 min, and extracted with 5 mL of methanol for 12 h. Then, the crude extracts were centrifuged at 12,000 rpm for 5 min to remove the cells and condensed by a vacuum freeze dryer, until ~2 mL of methanol remained. The crude extracts were fractioned by HPLC (Shimadzu LC-20AP) using the following method. A total of 1.5 mL of the methanol extract was injected into a C18 reversed-phase column (250 mm × 20 mm) with 90% acetonitrile (v/v) in 0.1% trifluoroacetic acid solution at a flow rate of 20 mL/min and detected by UV at 210 nm. The purified fractions were collected, air dried, and dissolved in 1 mL of methanol for antifungal activity assay. Then, 250 μL extracts of the purified fractions were dropped into the sterile iron rings (0.7 cm in diameter) on the PDA medium that had been mixed with 1 mL of the F. oxysporum f. sp. cucumerium spores (106 CFU/mL). After cultivation at 28 °C for four days, the size of the inhibition zone was recorded.To extract the purified fusaricidin, the cells of The active HPLC fractions were analyzed by LC-MS (Agilent 6520) under positive mode with an electrospray ionization/collision induced dissociation source. The following conditions were applied: gas temperature 300 °C, drying gas 5 L/min, nebulizer 30 psig, capillary voltage at 3.5 kV, spray voltage was 4 kV.P. polymyxa WLY78 and the −fusA mutant was determined by using acetylene reduction assays as described [2H4/mg protein/h.The nitrogenase activity of escribed . The nitF. oxysporum f. sp. cucumerium, 10 μL of the methanol extracts from P. polymyxa WLY78 and the −fusA mutant and 10 μL of the fusaricidin extracts were respectively added into 0.5 mL suspensions of the mycelia or spores of F. oxysporum f. sp. cucumerium. A total of 10 μL of methanol was used as the controls. For preparation of the spore suspension (106 CFU/mL), the spores were brushed in sterile PDA liquid broth from the disk. For preparation of the mycelia suspension, 1 mL of the spore suspension (106 CFU/mL) above was inoculated into 5 mL of PDA liquid broth and cultured for six hours at 28 °C with 200 rpm. The growth status of the fungal mycelia and fungal spores in the suspensions was observed under microscope after incubation at 28 °C with 200 rpm.To investigate the inhibitory mode of fusaricidin against 6 CFU/mL) was treated with 10 μL of fusaricidin extracts at 28 °C. A total of 10 μL of methanol was used as a control. After treatment for six hours, the supernatant was obtained by centrifugation at 5000 rpm for 2 min. Then, the OD260 and OD280 values were detected to evaluate the leakage of nucleic acid and proteins from F. oxysporum f. sp. cucumerium.A total of 1 mL of spore suspension (10Cucumis sativus Linn. variety Zhongnong 16) seeds were surface-sterilized as described previously [F. oxysporum f. sp. cucumerium spore suspensions (104 CFU/mL) in 15-cm pots. For detecting defense-related gene expression and SA accumulation, the seedlings were planted in 250-mL Erlenmeyer flasks containing 20 mL of Murashige & Skoog solid medium [Cucumber  of P. polymyxa WLY78 and the fusA− mutant (104 CFU/mL) were respectively inoculated into the rhizosphere of seedlings, with 100 mL of water being used for the control group. Each group contained fifteen seedlings. Then, the disease severity was investigated at day 21 post inoculation using the following grades (g): 0, plants with no yellowing or wilting symptoms; 1, plants with <25% of leaves with yellow spots; 2, plants with 25~50% of leaves with yellow spots; 3, plants with 50~75% of leaves with yellow spots; 4, plants with 75~100% of leaves with yellow spots; and 5, plants with 100% of leaves with yellow spots [g is the grade value; gN is the number of plants of the corresponding grade; h is the highest grade; and tN is the total number of plants in each group.To confirm the correlation between fusaricidin production and the biological control efficacy, we compared the biological control efficacy of ow spots . The disThen, the biological control efficacy (BE) was determined by the following formula:F. oxysporum f. sp. cucumerium in the cucumber rhizosphere soil were detected as described previously [P. polymyxa WLY78 treatment, the −fusA treatment, and water (control) treatment was respectively sampled at day 21 post inoculation. The sampled soil was suspended in 50 mL of 0.9% NaCl solution and then the suspension was spread on fungi selective medium. The colonies were counted after incubation for three days at 25 °C. The quantities of eviously . BrieflyNPR1, PR1, PR2, PR3, and Etr1) in cucumber leaves between P. polymyxa WLY78 treatment and the fusA− treatment by the following method. At the trefoil stage, the cell suspensions  of P. polymyxa WLY78 and the fusA− mutant were respectively inoculated into the rhizosphere medium of seedlings. A sample of 3 mL of water was inoculated as a control. The first leaves were sampled at days 1, 2, and 3 after treatment. The total RNA of sampled leaves was extracted by an RNAiso Plus Kit and converted into cDNA by a Reverse Transcription Reagent Kit (TaKaRa). The qRT-PCR program was conducted as described previously [Ct method [To confirm whether the plant resistance in cucumber seedlings was induced by fusaricidin, we compared the relative expression level of plant resistance genes  was mixed into the water phase at 80 °C for 1 h, and then extracted twice with 0.5 mL of ethyl acetate. Both of the two ethyl acetate phases were dried, and the dried material was re-suspended in 0.2 mL of methanol to obtain free SA and conjugated SA, respectively. The suspension was centrifuged at 12,000 rpm for 2 min and filtered with a 0.22 μm filter. SA was detected at 300 nm by a HPLC (Shimadzu LC-20AT) equipped with a UV detector. A total of 20 μL of sample was injected into a C-18 reverse-phase column (4.6 × 150 mm) with 80% methanol (v/v) in 0.1% acetic acid solution at a flow rate of 0.8 mL/min. At the trefoil stage, the purified fusaricidin obtained above were dissolved in 3 mL of water and was then inoculated into the rhizosphere of cucumber seedlings, with 3 mL of water used for the control group. The roots (0.5 g) of the cucumber seedlings were collected at days 0.5, 1, 2, 3, 5, 7, 10 after treatment. The SA was extracted as described with minor modifications . The samp ˂ 0.01) of data was analyzed by one-way ANOVA with Duncan’s multiple-range test using SPSS version 22 statistical software .All of the experiments were repeated three times with a similar result. The significant difference (


PHAP1 (Putative HLA‐DR‐associated protein 1), also termed acidic leucine‐rich nuclear phosphoprotein 32A (ANP32A), Phosphoprotein 32 (pp32) or protein phosphatase 2A inhibitor (I1PP2A), is a multifunctional protein aberrantly expressed in multiple types of human cancers. However, its expression pattern and clinical relevance in human glioma remain unknown. In this study, Western blotting and immunohistochemistry analysis demonstrated PHAP1 protein was highly expressed in glioma patients, especially in those with high‐grade disease. Publicly available data also revealed high levels of PHAP1 were associated with poor prognosis in glioma patients. The functional studies showed that knock‐down of PHAP1 suppressed the proliferation of glioma cells, while overexpression of PHAP1 facilitated it. The iTRAQ proteomic analysis suggested that stathmin might be a potential downstream target of PHAP1. Consistently, PHAP1 knock‐down significantly decreased the expression of stathmin, while overexpression of PHAP1 increased it. Also, the upstream negative regulator, p27, expression levels increased upon PHAP1 knock‐down and decreased when PHAP1 was overexpressed. As a result, the phosphorylated Akt (S473), an upstream regulator of p27, expression levels decreased upon silencing of PHAP1, but elevated after PHAP1 overexpression. Importantly, we demonstrate the p27 down‐regulation, stathmin up‐regulation and cell proliferation acceleration induced by PHAP1 overexpression were dependent on Akt activation. In conclusion, the above results suggest that PHAP1 expression is elevated in glioma patients, which may accelerate the proliferation of glioma cells by regulating the Akt/p27/stathmin pathway. Additionally, we determine PHAP1 plays a significant role in glioma cell proliferation. Finally, we explore the effects of PHAP1 on Akt/p27 regulated stathmin signalling.22.1PHAP1 and stathmin antibodies were bought from Abcam . Antibodies against Akt, p‐Akt (S473), p27 and β‐actin were purchased from Cell Signaling Technology .2.2Thirty specimens of human glioma tissues  and twelve specimens of non‐tumour brain tissues  were collected at the Affiliated Hospital of Xuzhou Medical University . All glioma specimens have been confirmed by the pathological diagnosis and were classified in line with the criteria of WHO. Written informed consent was acquired from each patient, and the study was permitted by the Research Ethics Committee of Xuzhou Medical University.2.32 at 37°C.Glioma cell lines C6, U251, U118, A172, U87 and human embryonic kidney cell line 293T were bought from Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences. These cells were all supplemented with Dulbecco's modified Eagle's medium (DMEM)  containing 10% foetal bovine serum  and cultured in a humidified incubator with 5% CO2.4To knock‐down PHAP1, three sets of shRNA duplexes were designed and synthesized as the followings:shPHAP1‐F1:5′‐GATCGGACGCCCTCTGATGTGAATTCAAGAGATTCACATCAGAGGGCGTCCTTTTTTG‐3′shPHAP1‐R1:5′‐AATTCAAAAAAGGACGCCCTCTGATGTGAATCTCTTGAATTACATCAGAGGGCGTCC‐3′shPHAP1‐F2:5′‐GATCGCAAGACTCAGTGGTGTATTTCAAGAGAATACACCACTGAGTCTTGCTTTTTTG‐3′shPHAP1‐R2:5′‐AATTCAAAAAAGCAAGACTCAGTGGTGTATTCTCTTGAAATACACCACTGAGTCTTGC‐3′shPHAP1‐F3:5′‐GATCGACTCTGATGTTACTCTTGTTCAAGAGACAAGAGTAACATCAGAGTCTTTTTTG‐3′shPHAP1‐R3:5′‐AATTCAAAAAAGACTCTGATGTTACTCTTGTCTCTTGAACAAGAGTAACATCAGAGTC‐3′Control‐F:5′‐GATCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG‐3′Control‐R5′‐AATTCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAA‐3′BamH I and EcoR I cloning sites. To overexpress PHAP1 in glioma cells, the PHAP1 cDNA was cloned into the pWPXLd‐puro plasmid by using BamH I and Mlu I enzyme sites. Cell transfection was carried out by PolyJet  according to the manufacturer's instructions. The lentiviruses were produced by cotransfecting the core plasmid and the packaging plasmids in 293T cells.The PHAP1 shRNAs and control shRNA oligomers were annealed and then cloned into the pLV‐shRNA plasmid using the 2.5The stable cell lines were developed as we previously described.2.64HCO3 pH 7.4 and alkylated with 10 mmol/L iodoacetamide for 1 hour at 37°C. Then each sample was diluted 10‐fold with 25 mmol/L NH4HCO3 and digested with trypsin at a ratio of 1:100 (trypsin/substrate) for 6 hours at 37°C. A 25 μg aliquot of digested peptides for each sample was subjected to eight‐plex iTRAQ labelling according to the manufacturer's instructions. Peptides from each iTRAQ experiment were subjected to capillary liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) using a Q Exactive Hybrid Quadrupole‐Orbitrap Mass Spectrometer . The quantitative analysis was conducted by calculating the ratios between experimental group and control group. To make the data more credible, the iTRAQ experiment was repeated at three times. The changes were considered significant if the increased or decreased fold change >1.5 and the P‐value <.05. The original mass spectrum data were searched by database using Mascot 2.2 and Proteome Discoverer 1.4 .Quantitative iTRAQ‐based proteomic analysis was performed by CapitalBio Technology Co. Ltd . Total protein was extracted from U251‐Control, U251‐shPHAP1#3, U87‐Control and U87‐shPHAP1#3 cells. 100 μg of each protein was denatured in 8 mol/L urea in 50 mmol/L NH2.73 cells/well. Twenty hours after culture, the cells were applied to 50 μmol/L of 5‐ethynyl‐20‐deoxyuridine  and incubated for 2 hours at 37°C. The cells were washed with PBS and fixed with 4% paraformaldehyde for 20 minutes, and then permeabilized with 0.5% Triton X‐100 for another 20 minutes. Afterwards, the cells were washed five times with PBS and incubated with 100 μL of 1 × Apollo® reaction cocktail for 30 minutes at room temperature. Finally, the nuclei of the cells were dyed with 100 μL of Hoechst 33342 (5 μg/mL) for 20 minutes and visualized with a fluorescent microscopy .The cells stably knocking down or overexpressing PHAP1 were cultivated in 96‐well plates at 4 × 102.8The plate colony formation assay was carried as we previously described.2.9Two thousand cells in 100 μL of medium were cultured in several 96‐well plates. At the designated time‐point, 10 μL of CCK‐8 reagent was applied into the medium. After reaction for 4 hours at 37°C, the absorbance at 450 nm was determined by a SynergyMx Multi‐Mode Microplate Reader . The cell viability was calculated according to the absorbance.2.10At the designated time‐point, the cells were harvested and subjected for total protein extraction. To analyse the protein expression by Western blotting, equal amounts of proteins were isolated on a 12% SDS‐PAGE and then transferred to 0.45 μm pore size PVDF membrane . After blocking with 3% bovine serum albumin (BSA), the membrane was incubated with the primary antibodies , p27 and β‐actin) at 4°C overnight. On the following day, the membranes were probed with a horseradish peroxidase (HRP)‐labelled goat anti‐rabbit/mouse IgG. Bound antibodies were detected by the ECL Plus Western Blotting Substrate  and then visualized with a ChemiDoc™ Imaging System . Band density was quantified by Image J Software . The relative amount of each protein was determined by normalizing the densitometry value of interest to that of the loading control.2.11t test. P values <.05 were considered statistically significant (*P < .05).The results shown were representative of experiments that were repeated at least three times. All quantitative data were presented as mean ± SEM. Statistical analysis was performed with the SPSS Version 13.0 . Differences in multiple groups were compared by a one‐way analysis of variance (ANOVA) followed by post hoc test. Differences between two groups were determined by Student's 33.1To study the role of PHAP1 in the development of human gliomas, the total protein was isolated from 30 cases of human glioma tissue  and 12 cases of non‐tumour brain tissue samples for Western blotting analysis. As shown in Figure 3.2To explore the roles of PHAP1 in glioma cell proliferation, we used specific shRNAs to down‐regulate PHAP1 expression. To knock‐down PHAP1, three shRNA targets  were examined for their efficacy in suppressing PHAP1 expression, and a non‐targeting shRNA served as a negative control. ShPHAP1#3 was the ideal candidate for silencing PHAP1 expression Figure A,B. To dAdditionally, we overexpressed GFP‐PHAP1 in U251 and U87 cells to further determine the function of PHAP1 in the proliferation of glioma cells. The expression efficiency of PHAP1 was confirmed by GFP images Figure A,B and W3.3The above results have indicated that PHAP1 is important for the proliferation of glioma cells. However, the underlined mechanisms are not yet elucidated. Thus, we performed an iTRAQ proteomic analysis to compare the protein expression between control and shPHAP1#3 in both U251 and U87 cells. A total of 5400 peptides were detected in three independent biological replicates among four groups. Then, we analysed the identified peptides. As a result, 57 and 71 changed proteins were identified in U251 and U87 cells, respectively (Figure 3.4Previous reports show stathmin is negatively controlled by the cell cycle inhibitor p27.4PHAP1 is a multi‐functional phosphoprotein that is involved in multiple cellular processes, including cell differentiation, gene transcription, apoptosis and cell cycle transition.Depression of specific tumour suppressor genes or activation of specific oncogenes is a key event for the development and malignant progression of glioblastoma.Stathmin regulates dynamic instability, the growth and shrinkage of microtubules by modulating microtubule plus end catastrophes and sequestering alpha‐beta tubulin dimers.In conclusion, we demonstrate how PHAP1 is linked to malignant proliferation of human glioma through the Akt/p27/stathmin pathway. Our work elucidates new findings that warrant further exploration of the PHAP1‐mediated signalling pathway, as well as, evaluation of the prognostic potential of PHAP1 expression in glioma patients. However, deeper investigations are still needed to elaborate how PHAP1 to regulate the activation of Akt in human glioma in the future study.The authors declare that they have no conflict of interest. Click here for additional data file. Click here for additional data file. Click here for additional data file.