Cip1/CDKN1A (p21) is an important and universal Cdk-interacting protein. Recently, we have reported that p21 is involved in the regulation of the mitotic kinase Cdk1/cyclin B1 and critical for successful mitosis and cytokinesis. In the present work we show that S130 of p21 is phosphorylated by Cdk1/cyclin B1 during mitosis, which reduces p21′s stability and binding affinity to Cdk1/cyclin B1. Interfering with this phosphorylation results in extended mitotic duration and defective chromosome segregation, indicating that this regulation ensures proper mitotic progression. Given that p53, the major transcriptional activator of p21, is the most frequently mutated gene in human cancer and that deregulated Cdk1 associates with the development of different types of cancer, this work provides new insight into the understanding of how deregulated p21 contributes to chromosomal instability and oncogenesis.The multifunctional protein p21 Cip1/CDKN1A (p21) is an intrinsically unstructured protein [in vitro by non-receptor tyrosine kinases, which influences its Cdk/cyclin binding and stability [Cyclin-dependent kinases (Cdk), a family of proline-directed serine/threonine kinases, regulate the cell cycle . Cdk1 is protein , which b protein –7. It is protein , 8. p21  protein , 8. The  protein . It has  protein , whereas protein . Phospho protein , 12. In  protein . It is p protein , Cdk2/cy protein  and Cdk6 protein . Moreovetability .We have recently shown that p21 is involved in the regulation of the mitotic progression, whereby its loss prolongs mitotic duration resulting in defects in chromosome segregation and cytokinesis . Moreovest panel, lanes 2, 4, 6, 8 and 10), p21 is clearly present accompanied by an appearance of several additional slowly migrating bands indicative of being post-modified . To address if these slowly migrating bands are induced by phosphorylation events, we treated cells additionally with λ-phosphatase (λ-PPase) and the band/bands disappeared , suggesting that the slowly migrating band/bands of p21 are indeed induced by phosphorylation events. Moreover, when cells were treated with the proteasome inhibitor MG132, p21 was stabilized in prometaphase, both fast and slowly migrating bands . Cytoplasmic-nuclear separation of HeLa and MCF7 cells revealed that phosphorylated p21 is more abundant in the cytoplasm than in the nucleus of prometaphase cells . To follow the p21 phosphorylation status over the cell cycle, thymidine release kinetics was performed. When the cyclin B1 level peaked at 8 h post release, p21 was phosphorylated, which disappeared with the degradation of cyclin B1 , phosphorylated p21 almost disappeared in cells deficient of cyclin B1 , whereas phosphorylated p21 receded only slightly in cells treated with siRNA targeting Cdk2 , indicating that Cdk1 could be the main kinase for phosphorylating mitotic p21. The signals of phospho-histone H3 (p-HH3) were slightly reduced yet still abundant in cells knocked down of cyclin B1 or Cdk2, indicating that most of these cells with left cyclin B1 or Cdk2 were still able to enter mitosis . This was further supported by cell cycle analysis showing a reduction of 6-7% in the G2/M population in cyclin B1 or Cdk2 downregulated cells relative to control siRNA treated cells , further underscoring that phosphorylated p21 is associated with the activity of Cdk1. Moreover, the phosphorylation signal of p21 was abolished in mitotic HeLa cells released into medium containing RO-3306, a specific and efficient Cdk1 inhibitor [rd panel, lane 4). To halt RO-3306 treated cells in mitosis, mitotic cells were treated additionally with the proteasome inhibitor MG132. In this case, the phosphorylation signal of p21 was definitely decreased , whereas the levels of the mitotic markers Plk1 and cyclin B1 were hardly changed , suggesting that the reduction of the phosphorylation signal is ascribed to the reduced Cdk1 kinase activity. Of note, RO-3306 treated mitotic cells showed hardly the p-HH3 (S10) signal , which is in line with the previous observation [To define the mitotic kinase responsible for this phosphorylation, HeLa cells were treated with siRNA targeting cyclin B1, the regulatory subunit of Cdk1, or siRNA against Cdk2, and synchronized to prometaphase for Western blot analysis. Compared to control siRNA treated cells , the levels of precipitated wild type FLAG-p21 and its mutant FLAG-p21S130A  [in vitro [To reveal if the mutants affect the kinase activity of Cdk1, an n (MCAK) , in the in vitro , 25, a drd panel, lane 4), its protein stability was also affected by the pan-caspase inhibitor Z-VAD , whereas calpain cleavage inhibition impacted scarcely its protein turnover . Of note, p53 is increased after the inhibition of the caspase and the proteasome pathway, indicating that the accumulated p21 could result from both the stabilization of the protein and increased transcription via the p53 signaling. These data support the common observation that the degradation of p21 is majorly dependent on the proteasome pathway.The protein degradation and stability of p21 is well studied and it is known that the degradation is often triggered by its site-specific phosphorylation and different binding partners , 26, 27.nd panel). Interestingly, compared to HCT116 cells with p21, cyclin B1 is more stable in HCT116 cells without p21 , which was also observed in HeLa cells treated with siRNA against p21 , suggesting that the p21 status may influence directly the stability of cyclin B1 or indirectly via interfering with mitotic progression. Intriguingly, while non-phosphorylated p21 retained till 90 min in cells released from nocodazole treatment and incubated with fresh medium containing CHX , the phosphorylated form was reduced at 45 min  and undetectable at 75 min , when cyclin B1, the regulatory subunit of Cdk1, started to be degraded . The data indicate that phosphorylation of p21 alters its turnover during mitosis.To study precisely the turnover of p21 during mitosis, HCT116 cells with and without p21 were synchronized to prometaphase and released into medium containing cycloheximide (CHX), a protein synthesis inhibitor, and harvested for Western blot analysis. p21 is a short-lived protein during mitosis , non-phosphorylatable FLAG-p21S130A was more stable , whereas phosphomimetic FLAG-p21S130D was less stable . Moreover, FLAG-p21S130D degraded faster than wild type FLAG-p21 and FLAG-p21S130A during the release course and it was almost disappeared at 2 h in HeLa cells . Further quantification analysis clearly indicates that FLAG-p21S130D is the most unstable form at 4 h  compared to nocodazole treated cells . To underscore these results, we transfected HCT116 p21−/− cells with wild type FLAG-p21 or its mutants and the CHX kinetics was carried out. In line with the observations from HeLa cells, FLAG-p21S130D was less stable compared to wild type FLAG-p21 and its non-phosphorylatable mutant FLAG-p21S130A . Notably, relative to wild type FLAG-p21 , both mutants were hardly affected by the treatment with the proteasome inhibitor MG132 , suggesting strongly that S130 is involved in the proteasome dependent degradation of mitotic p21. Taken together, the data underline the notion that phosphorylation of S130 by Cdk1 renders p21 unstable.To explore if the phosphorylation of p21 by Cdk1 impacts its stability, HeLa cells were transfected with wild type FLAG-p21 or its mutants, synchronized and released into CHX-containing medium Figure . Westernh Figure . InteresTo gain insight into the binding affinity of p21 and its mutants to Cdk1/cyclin B1, pulldown assays were performed using different GST-p21 recombinant proteins incubated with cellular extracts from mitotic HCT116 p21−/− cells. Compared to wild type p21 or the phospho-mimetic mutant p21S130D Figure , lane 4.To analyze if phosphorylation changes p21′s subcellular localization, which has been reported for T145, S153 or T57/S130 , HeLa ceTo examine the effect of the phosphorylation at S130 on mitotic progression, time-lapse imaging was performed in living HCT116 p21−/− cells stably expressing fluorescent histones (H2B-tdTomato in red) [nd panel, DNA staining), which were partially alleviated by wild type FLAG-p21 . While p21S130A was able to mitigate these failures in a marginal extent, p21S130D expressing cells exhibited defects comparable to p21-deficient cells , indicative of the importance of subsequent dephosphorylation of p21 during mitosis.We were then interested in the mitotic phenotype of cells overexpressing wild type FLAG-p21 or its mutants. Transfected cells Figure  were staIn the present work we show that S130 of p21 is mainly phosphorylated by Cdk1/cyclin B1 during mitosis, which attenuates its stability and reduces its binding to Cdk1/cyclin B1. This regulation is of importance for a proper mitotic progression evidenced by extended prometaphase and metaphase in cells expressing the non-phosphorylatable mutant FLAG-p21S130A or the phosphomimetic FLAG-p21S130D, respectively. Interfering with this phosphorylation leads further to defective segregation and delayed mitotic exit indicating that a precise control of p21 is important for a successful mitotic progression.in vitro as well as ex vivo. In support of our notion, mass spectrometry analysis revealed that S130 in p21 is indeed phosphorylated in mitotic HeLa cells [Cdk1 becomes activated for the mitotic entry, its activity increases at prophase, reaches its peak shortly after nuclear envelope breakdown, remains almost constant in metaphase and is immediately reduced at the onset of anaphase by degradation of its regulatory subunit cyclin B1 –37. It hLa cells , 40, altLa cells , Cdk6/cyLa cells  and ERK2La cells . These dLa cells . In thisWe showed previously that p21 deficiency causes segregation defects which could be rescued by inhibiting Cdk1 activity with the Cdk1 inhibitor RO-3306 or by adding back wild type p21  and chroTaken together, we show here that p21 is phosphorylated at S130 by Cdk1/cyclin B1 during mitosis influencing its stability/degradation and ensuring a successful mitotic progression. Given that p53, the major transcriptional activator of p21, is the most frequently mutated gene in human cancer  and that® HD in a ratio 1:3 .HCT116 p21+/+, HCT116 p21−/−, HeLa, MDA-MB-231, MCF7 and U2OS cells were cultured as instructed. Stable HeLa 776-6, expressing shRNA targeting cyclin B1, were generated as described , 19. To BamH I/Not I sites of the pGEX-5×3 vector , into the EcoR I/BamH I sites (5′-AATTGAATTCTATGTCAGAACCGGCTGGGGATGTC-3′ and 5′-GATAGGATCCTTAGGGCTTCCTCTTGGAGAAGATCAG-3′) of the 3xFlag-CMV 7.1 vector (Sigma), and into the BamH I/Hind III sites of the pBI-CMV3 vector (Clontech), described in Kreis et. al [Full-length human p21 cDNA (pOTB7-vector) was obtained from ImaGenes GmbH (Berlin). By use of primers (5′-GATAGGATCCAGATGTCAGAACCGGCTGGGGATGTC-3′ and 5′-GACAGCGGCCGCTTAGGGCTTCCTCTTGGAGAAGATCAG-3′), the cDNA was amplified by PCR and subcloned into the s et. al . Point ms et. al . Recombi® M2 . Mouse monoclonal antibody against calnexin was from BD Biosciences and against lamin B1 from MBL . To generate a polyclonal antibody against p21 phosphorylated on S130, rabbits were immunized using the peptide GEQAEGpSPGGPGD (p = phospho), and antibodies were affinity purified . Kinase assay in vitro was performed as described [ex vivo was performed with HeLa cells transfected with FLAG-p21 or FLAG-p21S130A. Cells were synchronized with nocodazole and immunoprecipitated with FLAG® M2 beads (Sigma). The beads were subjected with radioactive ATP  without adding additional kinase and incubated for 30 min at 37°C with or without inhibitors.Cells were lysed in RIPA buffer and Western blot analysis was performed, as described . Cytoplaescribed . Cdk1/cy®, Cambridge, UK), monoclonal mouse antibody against FITC-conjugated α-tubulin (Sigma-Aldrich) and human immune serum against centromere . Cy3 and Cy5-conjugated secondary antibodies were obtained from Jackson Immunoresearch . DNA was stained using DAPI . Slides were examined using an AxioObserver.Z1 microscope with a HCX PL APO CS 63.0×1.4 oil UV objective  and images were taken using a confocal laser scanning microscope .For indirect immunofluorescence staining, cells were seeded on Nunc™ Lab-Tek™ II CC2™ chamber slides from Thermo Fisher Scientific (Schwerte). Briefly, cells were fixed for 15 min with 4% PFA and permeabilized for 5 min with 0.1% Triton™ X-100 at room temperature. The following primary antibodies were used for staining: polyclonal rabbit antibody against pericentrin (abcamt-test (two tailed and paired or homoscedastic) was used to evaluate the significance of difference between the different conditions. Difference was considered as statistically significant when p < 0.05.Student's


Recently, smart mobile devices and wireless communication technologies such as WiFi, third generation (3G), and long-term evolution (LTE) have been rapidly deployed. Many smart mobile device users can access the Internet wirelessly, which has increased mobile traffic. In 2014, more than half of the mobile traffic around the world was devoted to satisfying the increased demand for the video streaming. In this paper, we propose a scalable video streaming relay scheme. Because many collisions degrade the scalability of video streaming, we first separate networks to prevent excessive contention between devices. In addition, the member device controls the video download rate in order to adapt to video playback. If the data are sufficiently buffered, the member device stops the download. If not, it requests additional video data. We implemented apps to evaluate the proposed scheme and conducted experiments with smart mobile devices. The results showed that our scheme improves the scalability of video streaming in a wireless local area network (WLAN). With the advent of smart mobile devices and the development of wireless communication technology, mobile traffic is increasing dramatically. In addition, with the introduction of cyber physical systems (CPSs) to control and monitor physical objects with a software algorithm, mobile traffic is likely to further increases in the future . Video tIn a wireless local area network (WLAN), many smart mobile devices with N-screen technology connect to an access point (AP) to share video content. However, as the number of devices increases, many devices suffer from frequent playback interruptions and buffering during video streaming. Because all devices share the bandwidth of the wireless channel, the throughput of each smart device decreases as the number of smart devices increases –7.The multicast approach has been proposed for efficient video streaming because it constructs a hierarchical network topology to reduce duplicate transmissions –10. DespIn this study, we focused on the scalable video streaming technique for wireless networks. We exploited the relay node in order to extend the streaming service area and separate networks to reduce collisions among devices in a WLAN. As shown in In the progressive download scheme, a whole video file is transmitted from the source device to the member device via the relay device until the download is completed. Therefore, the progressive download scheme causes many collisions between devices during the download, and video playback interruptions occur frequently. To observe the performance degradation due to contention between member devices, we measured the transmission control protocol (TCP) throughput in WiFi Direct networks. In the experiment, seven smart devices transmitted packets to a relay device, and Iperf was used to generate packets . As showFor wireless networks, several streaming relay schemes have been proposed to share multimedia content to multiple smart mobile devices –18. Lin In this paper, we propose a scalable video streaming relay scheme to decrease the contention between devices and maintain the video resolution. In the progressive download scheme, much of the video data that are not playing are stored in a player buffer, which results in excessive congestion in the WLAN. In our proposed scheme, a member device decides whether or not to continue the download. First, a member device that wants to use a video streaming service requests a portion of video  from a source device. The source device transmits the video data to a relay device, and the relay device forwards it to the member device. If the size of the buffered data is larger than the threshold, the member device stops the download. Otherwise, the member device requests and downloads an additional chunk file. After the chunk is downloaded, because the member device is excluded from contention, the contention overhead decreases, and the efficiency of the video streaming relay scheme is improved.The rest of this paper is organized as follows. First, we present related works. We then present the proposed mechanism of the streaming relay scheme and architecture. We next discuss experiments performed to evaluate our scheme. Finally, we present our conclusions.Many papers have proposed method to support a scalable and seamless video streaming service. “Scalable” refers to the number of smart devices that rarely experience playback interruption. “Seamless” means that the video is playing without a blank screen or playback interruption. Multicast has been widely adopted to realize efficient streaming service , 9. HoweIn overlay multicast networks, an intermediate node with low system capability can become a bottleneck. This can cause a user to experience low-quality video streaming. To mitigate the computational burden of a bottleneck node, Lin et al. proposed one-to-many streaming splicing (OMSS) which utilizes a payload-sharing mechanism to improve the efficiency of memory copying and the worker-pool processing model for a multiprocessor . HoweverHwang et al. proposed the enhanced adaptive fast replica (EAFR) based digital living network alliance (DLNA) proxy to distribute video content in home networks . When thIn cellular networks, the network utilization varies over time. Because traffic jams occur during busy hours, a user experiences low throughput rather than the expected throughput. Keller et al. proposed MicroCast, which exploits both a cellular interface and wireless (or Bluetooth) interface to mitigate this problem . Each smBethanabhotla et al. proposed a scheduling policy to deal with the increased load of video on demand (VoD) in wireless networks . In wireAn optimal streaming strategy has been proposed to dynamically adjust the streaming rate over the Hypertext Transfer Protocol (HTTP) in wireless networks by utilizing a finite markov decision process (MDP) , 20. HowStockhammer proposed dynamic adaptive streaming over HTTP (DASH) to support videos with different levels of quality . A clienAs the demand for video streaming increases, a new Internet architecture called CCN has been proposed to efficiently transmit video files to end users , 23. In Do et al. exploited both cellular and wireless networks for scalable video streaming . They imAs the popularity of IoT increases, a scalable security scheme has become necessary. Butun et al. proposed the cloud-centric multi-level authentication (CMULA) scheme to support the scalability of IoT . A hieraFor seamless service, routing protocols have been proposed to prolong the lifetime of wireless sensor networks or support the security of mobile ad hoc networks –31. RelaWith advances in video technology, recent multimedia content contain not only two-dimensional (2D) but also three-dimensional (3D) images. However, because quality measurement techniques for stereo images are not mature, subjective assessments are used. Yang et al. proposed an objective quality metric for stereo images . Yang etThe proposed scheme has the following features. First, the proposed scheme is easily deployed in smart devices without modification of Android because it is implemented in application layer. Second, our approach utilizes WiFi Direct to divide an wireless channel into two or three wireless channel to reduce collisions and increase throughput of each device. It leads to scalability improvement and seamless streaming service.The details of the proposed scheme are presented here. We first present the streaming relay mechanism. Then we discuss the architecture and implementation for the proposed scheme.The proposed streaming scheme has two phases. t2, the buffer of M2 is exhausted, and playback is interrupted until M2 downloads the next video chunk. At t3, playback resumes, but playback interruption occurs again. Our proposed scheme is shown in t1, M1 stops the download and continues to play the video. At t1, M2 can download the requested chunk file faster because M1 stops downloading. Then, M2 stops to download the video chunk at t2. At t3, M1 realizes that the remaining data in the buffer is less than the threshold, so it initiates buffering for seamless video streaming. As the buffered data decrease, M2 also begins to download the next chunk at t5. Because M1 is idle, M2 can download the video file without contention and both devices can avoid inefficiency and unfairness due to channel contention.For example, both member devices M1 and M2 may request a video chunk file from the relay device, simultaneously. In the progressive download scheme, one of the two member devices can experience playback interruption. When several member devices play video, each requests the video file with different resolutions because of different hardware specifications. We assume that a source device has several video files with different bit rates in order to support different resolutions. When a member device requests a video file from the source device, if the source device has a video file whose bit rate matches that of the requested video, it transmits the video chunk to the member device. If not, the source device sends a video chunk file with similar bit rate. For example, a source device has video files with bit rates of 1000, 2000, and 3000 kbps. If a member device requests a video with 1700 kbps, the source transmits the video chunk file with bit rate of 2000 kbps.Stotal, and the total video playback duration, Ttotal, are known. The size of the next chunk to be downloaded, Schunk, is calculated byTrange is the minimum playback time and α is a constant value to guarantee the minimum playback time. For example, when a member device requires a chunk that can be played for 10 seconds, Trange is set to 10. However, Trange does not guarantee the playback duration because the video resolution is an average value. Each scene has a different file size. If the screen changes quickly, the file size of the scene is large. Otherwise, the size of the scene decreases. To deal with the variable length of the chunk, we use the constant value of α. In this paper, we set α to 2, and this is discussed in more detail in the subsection “Variable Chunk Size”.As the video resolution increases, the size of the video file also grows. Therefore, each member device requires different chunk sizes because of the different video resolutions. To determine the size of the next chunk to be sent, we assume that the total size of the video file, In the proposed scheme, a source device is determined depending on its own video files. For instance, two devices A and B have video file A and B, respectively. If the video A is required for streaming, the device A acts as the source device because it has the video file A. The device B becomes the relay or member device, and it requests the chunk of the video A from the source device . Additionally, if the device A has two different videos A and B, it becomes the source device for video A and B. In this case, each member device plays different video. It means that our proposed scheme is able to support P2P like video streaming service because different video files stored in different devices can be transmitted each other.n is the number of chunks for the video.Algorithm 1 presents the pseudo-code for the proposed streaming scheme. If the size of remaining video data in the buffer is less than the threshold, the member device transmits the next video chunk request to relay (or source device). If the relay (or source device) receives the chunk request from the member device (or the relay device) and has the video chunk, it sends the corresponding chunk to the member device (or the relay device). Besides, source, relay, and member devices try to keep playing the video if they have video data in the buffer. This algorithm is repeated until playing all video chunks is finished. Therefore, the time complexity of this algorithm O(n) if the Algorithm 1 Algorithm for the proposed streaming schemeStotal ← the total size of the video fileTtotal ← the total video playback durationTrange ← the minimum playback timealpha ← 2Cn ← 0, as the current chunk number for playing the videoPnext ← 0, as the next chunk number for downloadifStotal mod Schunk ≠ 0 thenCtotal ← Ctotal+1 end ifwhileCn < CtotaldoifPnext < Ctotal and VideoPlayer.RemainingBuffer ≤ threshold then Creq to the source or relay device  transmit the next chunk request Pnext in the video player’s buffer  store the received video chunk Pnext ← Pnext+1  end if if the device receives a chunk request for Cres and Cres < Pnextthen Cres to the relay or member device  transmit the video chunk end if if the video chunk Cn is not playing then Cn  start to play the video chunk else if the video chunk Cn play is finished then Cn ← Cn+1  else Cn  continue to play the video chunk end if end whileTo support scalable video streaming, we present the following architecture of the relay and member devices. α in We evaluated the performance of the proposed scheme by comparing its results with those of the progressive download scheme that transmits the whole video file to the member device , 37. As A video consists of many chunks, and the proposed scheme downloads a chunk for video playback. When a member device downloads chunks of equal size, it experiences different playback durations for each chunk because each scene has a different video resolution. Therefore, the member node downloads a larger chunk size to guarantee the playback duration.α in α from 1 to 3. In the measurements, Trange of α is set to 1, four of the five videos played about 8-9 seconds. This means that the playback duration of the downloaded video chunk was less than expected. As indicated in α. Then, the playback duration of the chunk became larger than the expected duration, Trange. However, when α was large, because the length of the chunk also increased, it caused unnecessary downloading. Based on the measurement results, we set α to 2.To determine We evaluated the performance of the proposed scheme. We measured the total number of playback interruptions when a video was played. We compared the results to those of the progressive download scheme, which has already been implemented in many streaming services . Fig 14 RPI, can be calculated as follows:TP and TI are the durations of the playback and playback interruptions, respectively. A high playback interruption ratio indicates that member devices suffered from frequent playback interruption, but a zero playback interruption ratio means that the video could be played seamlessly. We observed the playback interruption ratio. We measured the playback duration without interruption and the duration of playback interruptions. Then, as given by Xing et al. in , the plaWe measured the duration of the playback interruption experienced by a member device. We evaluated the scalability of both the proposed scheme and progressive download scheme. We counted the maximum number of member devices that could provide streaming service without playback interruption. We have proposed a scalable streaming relay scheme for wireless networks. The main advantages of our proposed scheme are follows. First, to decrease network congestion, a member device decides whether or not to download a video file. Second, our proposed scheme is designed for the application layer, so it does not require driver or firmware updates. Finally, we evaluated the performance through experiments on Android devices. Compared to the progressive download scheme, the proposed streaming relay scheme showed better scalability.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.The CRISPR-associated endonuclease Cas9 from  The Rctively 6612, in lly 66171861012, co2+ ions mediate the connections between Cas9 and DNA, remain elusive in this system11122+ ions and the non-bridging oxygen atoms of the putative scissile phosphate on the ntDNA111011121314152+ ion has been reported. We hypothesize that the catalytic Mg2+ ions induce the conformational change to an active state by facilitating the proximity of Cas9 active sites and DNA. Here, we used molecular dynamics simulations to search for the active state and to test our hypothesis.A wealth of biochemical and structural information has made remarkable contributions to the mechanistic insights into RNA-guided DNA recognition and cleavage by Cas9. However, many details of the cleavage process, such as how the catalytic MgP) or 4 bp apart (−4P)  see 69101120.2+ ions bridge the distance gap between the active sites of Cas9 and ntDNA? 2) Where is the cleavage site on ntDNA? Our simulation results demonstrate that introduction of two Mg2+ ions at −4P remarkably induces conformation changes to bring the active site of the RuvC domain and ntDNA into proximity, forming structurally and energetically favorable coordination in between the catalytic residues and the scissile phosphate for cleavage reaction to occur. Thus, we evidence that Cas9 generates 1-bp 5′-staggered breaks in the dsDNA.Molecular dynamics (MD) simulation has emerged as a powerful tool to explore bio-macromolecule dynamics in complementary with experimental data. To investigate the mechanisms underlying Cas9-catalyzed cleavage process, here, we present the first computational simulation study on the Cas9-sgRNA-dsDNA system at the atomic level to address the following two questions: 1) Could the Mg2+ on conformational change, one Mg2+ ion was placed in the HNH domain on the basis of the one-metal ion catalysis mechanism . Analysis of the root-mean-square deviations (RMSD) of the simulated systems revealed that the overall stability of the entire complex is largely determined by the sgRNA  and between −4P and Asp10 (dP/Asp10-4) (2+ ions (S0), the distances between the ntDNA and the RuvC domain active residues peak at ~10 Å for both measurements (S0), addition of the Mg2+ ions obviously shifted the distance distributions of ntDNA and RuvC active center to smaller values in varying degrees regardless of the Mg2+ ions positions (S1 to S6), indicating the presence of metal ions could indeed induce the conformational change toward the active state and close the distance gap between the ntDNA and the RuvC domain as previously postulated11122+ binding at the −4P in S5 and S6 leads to the largest reduction in dP/His983-3 (by ~4 Å) as well as in dP/Asp10-4 (by ~6 Å) when comparing the highest peaks with that of S0 (We first monitored the proximity of the ntDNA and RuvC active center by measuring the distances between −3P/Asp10)  for eachurements . Compareat of S0 .P and His840 (dP/His840+3) that are largely overlaid with each other  moves toward the −2P from its initial location at −3P  departs from the middle position in between −3P and −4P, entering the negatively charged center enclosed by Asp10, E762 and D986 or −4P (2+ at either −3P (S1/S2) or in between −3P and −4P (S3/S4) all led to the Mg-Mg distance larger than 6 Å  from the experimental and theoretical studies on another two-metal-ion catalytic system with two Mg2+ trapped in between the Bacillus halodurans ribonuclease H (RNase H) and an RNA/DNA hybrid  are involved in coordination with the two metal ions in S5 and S6. Specifically, it is interesting to note that despite the similarity in the coordination configuration, the general base His983 in S6 is directly engaged to the Mg2+ A  and the scissile phosphate17185 and S6 , in whicH system . Unlike e Mg2+ A , whereasdination .2+ at –4 P. Yet it should be mentioned that compared with the canonical two-mental-ion coordination in the RNase H system, there exists a noticeable difference in terms of the scissile phosphate-mediated coordination with the two Mg2+. Both in S5 and S6, the two non-bridging oxygens (pro-Sp and pro-Rp) of −4P together form a bidentate coordination with the double Mg2+ ions . The results showed that the binding of the Mg2+ pair at −4P in S5 and S6 is more stable than those at −3P or in between −3P and −4P in other simulations (S1–S4)  . Moreove5 and S6 . We also MM-GBSA .S2, all of the four active residues hardly contribute to the binding of the two Mg2+, while Asp10 is the sole major contributor in S1  at the RuvC active center for Cas9 catalytic activity6or in S1 . Compareaffinity , accountaffinity . Notablyulations , again s at –4 P . In cont2+ is non-specially bound at the RuvC active site, forming an incomplete coordination due to lack of the involvement of phosphate group , and found very similar results with respect to the two longer simulations here (2 × 200 or 2 × 300 ns)  or −4 phosphate (−4P) (2+-bound RNase H in complex with an RNA/DNA hybrid (PDB code: 1ZBI)17262+ locations between −3P and −4P were also taken into account, in which one Mg2+ ion was placed close to 5′-side of the pro-Sp oxygen of −3P, and the other to 3′-side of the pro-Sp oxygen of −4P 1012142+-bound complex above was solvated with TIP3P water molecules with a thickness of 13.5 Å, leading to a periodic boundary box of 139 × 124 × 187 Å3. To mimic the reaction buffer62+ were added into the water box to generate a concentration of 10 mM, and the ionic strength of KCl was set to 100 mM. As a control, the original system without Mg2+ was also simulated  , respectn of −4P . Given t therein . Meanwhiimulated . The totpmemd engine with GPU acceleration392+ ion is able to reproduce various experimental properties simultaneously; the set of Mg2+ parameters here represent the best possible compromise targeting the experimental Mg-O distance, hydration free energy as well as coordination numberAll the simulations were performed by the AMBER15 2+ coordination were determined through the cluster analysis with the package VMD2+ pair lying between them. In the final trials, the first two clusters were found to account for >90% of total population and the snapshot closest to the centroid of largest structural ensemble was extracted for comparative analysis.The representative (i.e. most-populated) configurations involving the double-Mg2+ pair at respective positions was evaluated via the end-point Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) approach29303233MMPBSA.py in AmberTools143233S1 to S4 and 5,600 structures for S5 and S6, respectively  to the Mg2+ pair. In the final results, we reported the block averaging over even non-overlapping trajectory sections (each containing 400 snapshots) together with the standard error of the mean.The binding stability of the Mgectively . In eachHow to cite this article: Zuo, Z. and Liu, J. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations. Sci. Rep.6, 37584; doi: 10.1038/srep37584 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Refractory coeliac disease (RCD) is a recognised complication, albeit very rare, of coeliac disease (CD). This condition is described when individuals with CD continue to experience enteropathy and subsequent or ongoing malabsorption despite strict adherence to a diet devoid of gluten for at least 12 months and when all other causes mimicking this condition are excluded. Depending on the T-cell morphology and T-cell receptor (TCR) clonality at the β/γ loci, RCD can be subdivided into type 1  and type 2 . It is important to differentiate between the two types as type 1 has an 80% survival rate and is managed with strict nutritional and pharmacological management. RCD type 2 on the other hand has a 5-year mortality of 50% and can be complicated by ulcerative jejunitis or enteropathy-associated T-cell lymphoma (EATL). Management of RCD type 2 has challenged many experts, and different treatment approaches have been adopted with variable results. Some of these treatments include immunomodulation with azathioprine and steroids, methotrexate, cyclosporine, alemtuzumab , and cladribine or fludarabine sometimes with autologous stem cell transplantation. In this article, we summarise the management approach to patients with RCD type 2. It results in both gastrointestinal and extra-gastrointestinal symptoms. Typically, patients have diarrhoea, bloating, weight loss, and anaemia. It is a lifelong condition with a prevalence of around 1% in Europe and the US. The gold standard for diagnosis requires a duodenal biopsy showing villous atrophy and increased intra-epithelial lymphocytes2. The only accepted treatment for CD is to completely remove gluten from the diet; this includes cereal, wheat, rye, and barley. Most cases of CD report symptom improvement within a few weeks of a strict gluten-free diet (GFD). Those who do not respond to this diet are said to have non-responsive CD (NRCD). The difficulty of maintaining gluten elimination means that the majority of NRCD is due to continued ingestion. An alternative diagnosis should be sought in those members of the NRCD group whose continuing symptoms appear to be unrelated to CD. An even smaller group are those with refractory CD (RCD), in which a GFD is being adhered to but the symptoms of malabsorption remain. On biopsy, they will have persistent villous atrophy despite at least 12 months on a GFD. RCD can be classified as primary (no response to GFD) or, more commonly, secondary . RCD can also be classified by its clonality of T-cell receptor (TCR) into RCD types 1 and 2. It is important to make a distinction between RCD types, as type 2 is associated with complications such as ulcerative jejunitis and enteropathy-associated T-cell lymphoma (EATL), giving it a poor prognosis. Identifying the different types of RCD is a complicated process involving polymerase chain reaction (PCR) to determine TCR clonality, histology of the small intestine, and intra-epithelial lymphocyte (IEL) phenotype and morphology (3.Coeliac disease (CD) is an autoimmune response triggered by dietary exposure to gluten in those individuals who are genetically predisposedphology 3.4. A review by Malamutet al. looked at treatment with methotrexate or anti-tumour necrosis factor-alpha in 14 patients with RCD type 1 and 43 patients with RCD type 2 and found that some had a histological response to this treatment5. There have been statistically significant improved survival rates following the use of cladribine , whereas the use of alemtuzumab  has shown only marginal success6. Previously, the use of azathioprine and prednisolone was associated with progression to EATL, but Nasret al. showed that the use of these drugs in combination was not only successful but also safe, especially where the cases of RCD type 2 were identified early3. The results showed 53% histological recovery and 56% transformation from oligoclonal to polyclonal gamma receptor population, and none of the patients developed EATL.RCD type 2 is rare and therefore there are only a few randomised controlled trials to give us treatment recommendations. In some centres, a combination of prednisolone with a thiopurine has been used and shown to give good results for type 1 and variable results for type 2; a clinical improvement was reported in up to 75% of patients with RCD type 212, and folate, and small bowel biopsy will reveal villous atrophy7. The main objective for managing these patients is to find the cause of the NRCD. Those who are diagnosed with NRCD can be classified into one of four groups on the basis of their history, current symptoms, and investigations: (1) patients who are not adhering to the GFD, (2) patients whose symptoms mimic untreated CD but who in fact have a second diagnosis other than CD, (3) patients with CD and complications, and (4) patients with RCD, which comprises 0.1% to 1% of patients with CD8.NRCD requires clinical and histological diagnosis. Following 1 year of a strict GFD, these patients will still be complaining of symptoms like abdominal pain, diarrhoea, and tiredness. Blood tests may show low levels of iron, B9. Nevertheless, when someone presents with NRCD, it is crucial to exclude other diseases which can mimic CD; these include pancreatic insufficiency, lactose intolerance, small bowel bacterial overgrowth, inflammatory bowel disease, hypo-gammaglobulinaemia, tropical sprue, collagenous colitis, and adult-onset autoimmune enteropathy. A detailed investigation is required to rule out these conditions; firstly, it is necessary to make sure the patient is strictly adhering to the GFD. If there are raised CD serology antibody titres, then it can be assumed that there is still ingestion of gluten. Investigations include an upper gastrointestinal endoscopy with biopsy of the small intestine for light microscopy, coeliac serology including IgA antibodies to tissue transglutaminase (tTG) and endomysium and PCR for TCR monoclonality. Immunoglobulin levels, including IgA and IgG titres, are tested by some centres, followed by testing for IgG antibodies to tTG if there is IgA deficiency. Further testing includes HLADQ2 and DQ8 status, colonoscopy, and testing for lactose and fructose intolerance, small bowel bacterial overgrowth, and pancreatic insufficiency. Following this, if CD is confirmed and other causes for the continuing symptoms have been ruled out, a strict GFD must be adhered to and RCD can be considered as the diagnosis.Over 90% of NRCD cases fall into the first group, consisting of those who continue to ingest gluten either by choice or unintentionally10. This subset of individuals will already have been investigated for other causes, as detailed above, and usually nothing else is found. The differentiation into primary and secondary RCD can then be made according to the onset of symptoms. Those with primary RCD will never have had a response to a GFD, whereas those with secondary RCD will have been initially responsive to dietary elimination of gluten but are now experiencing a return of symptoms. There is no time frame for this; some patients develop secondary RCD decades later. Classifying RCD as type 1 or 2 depends on the phenotype of the IELs. Type 1 has a normal IEL phenotype, and type 2 has an abnormal clonal population with loss of CD8 and expression of intra-cytoplasmic CD3 by IELs. Making this distinction between types 1 and 2 is key in being able to manage RCD appropriately. It also aids with prognosis, as RCD type 2 has a 5-year mortality of 55% compared with just 7% in RCD type 14. Ulcerative jejunitis and EATL are the main complications that can occur in RCD, and EATL is responsible for the majority of deaths in patients with RCD. There is a 3:1 female-to-male ratio with RCD type 2, but this is reversed in EATL to result in males being more affected11. HLA DQ2 homozygosity is a risk factor for both RCD type 2 and EATL12.RCD represents a small subset of the NRCD group who continue to have symptoms of malabsorption and have villous atrophy on small bowel biopsy despite not having ingested gluten for at least 1 yearet al.3 describe the strategy used in a tertiary centre for diagnosing RCD type 2:1. Patients must be on a strict GFD with a dietary assessment of compliance. The tTG/endomysial antibody serology is frequently negative in RCD type 2.2. Marsh scoring of small bowel biopsy should be obtained during upper gastrointestinal endoscopy.3. Assessment of IEL phenotyping and PCR for TCR monoclonality at the β or γ loci or both should be carried out. Abnormal  IEL in the small bowel is supportive of an RCD type 2 diagnosis. Transient TCR clonality can be detected in patients at the time of diagnosis and in those with poor compliance.4. Capsule endoscopy should be performed in all cases of RCD type 2 to exclude EATL. However, if there is any suspicion of EATL, magnetic resonance imaging (MRI) must be performed before capsule endoscopy to exclude an obstructing lesion. Capsule endoscopy should be repeated 1 year later to check for the development of EATL. Some have suggested that RCD type 2 be renamed pre-EATL.5. If EATL is suspected, such as when a patient presents with abdominal pain, weight loss, or malnutrition, then cross-sectional imaging, including small bowel MRI, computed tomography scan, and positron emission tomography scan, are recommended. This allows identification of abnormalities within the bowel, abnormal lymph nodes, and involvement of other organs.It can be difficult to diagnose RCD. The process involves clinical assessment of the patient. Pathological, histological, laboratory, and radiological findings can all help, but essentially it is a diagnosis of exclusion in many cases. Nasr13. Nasret al.3 found good results when combining prednisolone with a thiopurine, including azathioprine, mercaptopurine, or thioguanine, and summarised the other trials undertaken in different centres : this has limited data and outcomes• et al. have used this in 32 patients and 18 of those patients had a good response16Cladribine: Mulder• et al. report a 61% histological improvement with the use of cyclosporine to treat 13 patients with RCD type 221Cyclosporine: Wahab• Infliximab has good results reported by single cases, although some of these are for patients with RCD type 1; there needs to be a much larger study of this drug before its value in treating RCD type 2 can be properly establishedThere have been a variety of attempts to manage RCD type 2. A good clinical response has been seen when using budesonide for RCD, but there is not a clear effect on prognosiscentres 3,6,12RCD is a rare but serious condition. Although relatively high numbers of patients present with continuing symptoms of CD despite following a GFD, the majority of them can be attributed to the NRCD group 1, consisting of those who continue to ingest gluten. It is extremely important to be thorough in history taking, examination, and investigations of those presenting with NRCD in order to identify the small group with RCD. Once RCD has been diagnosed, the process of classification into type 1 or 2 can be undertaken by using small bowel biopsy, PCR, IEL phenotyping, capsule endoscopy, and imaging. Knowing whether a patient has RCD type 1 or 2 gives the opportunity to estimate the prognosis more accurately and guides treatment options.Various treatment methods have been used in different centres, and the treatment outcome is variable. RCD type 2 is not common; however, early diagnosis and treatment may play an important role in the prognosis. Future areas for research may help identify other treatment options, particularly for difficult-to-treat cases or cases that have progressed.


Dioxin toxicities require binding and activation of the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor. However, after nearly 50 years of study, it remains unknown how AhR activation by dioxin produces toxic effects. Here, using the chick embryo close to hatching, a well-accepted model for dioxin toxicity, we identify NAD+ loss through PARP activation as a novel unifying mechanism for diverse effects of dioxin in vivo. We show that NAD+ loss is attributable to increased PARP activity in thymus and liver, as cotreatment with dioxin and the PARP inhibitor PJ34 increased NAD+ levels and prevented both thymus atrophy and hepatosteatosis. Our findings additionally support a role for decreased NAD+ dependent Sirt6 activity in mediating dioxin toxicity following PARP activation. Strikingly, treatment in vivo with the NAD+ repleting agent nicotinamide, a form of vitamin B3, prevented thymus atrophy and hepatosteatosis by dioxin and increased sirtuin activity, providing a therapeutic approach for preventing dioxin toxicities in vivo.Dioxin (2,3,7,8-tetrachlorodibenzo- TCDD produces a toxicity syndrome in multiple species that includes wasting, thymus involution, hepatosteatosis, and dysregulated glucose metabolism5. Even after many years of study it remains unclear how TCDD produces its diverse spectrum of toxic effects. Activation of the AhR by TCDD increases transcription of a large number of genes6, none of which has yet been able to account for the production of toxic effects by TCDD in multiple organs in vivo.Activation of the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, is required for production of toxic effects by a group of polyhalogenated hydrocarbons, a common class of environmental toxins of which dioxin  that TCDD decreased NAD+ levels while increasing expression of TiPARP (TCDD-inducible poly (ADP-ribose) polymerase, PARP7, ARTD14)7. TiPARP is one of 17 PARP enzymes that consume NAD+ while ADP-ribosylating proteins8 and an AhR target gene11. We used thymus atrophy, an effect of TCDD in all vertebrate species studied12, and hepatosteatosis, another common effect of TCDD15, as endpoints for TCDD toxicity. As thymus atrophy and hepatosteatosis are pathologically distinct we expected that identification of a mechanism that causes both thymus atrophy and hepatosteatosis would shed light on the basis for the diverse effects of TCDD. Using the chick embryo close to hatching, a well-accepted model for TCDD toxicity3, we identified NAD+ loss as a unifying mechanism for these TCDD toxicities. We show that TCDD in vivo decreases NAD+ levels in both thymus and liver and that it does so by increasing PARP activity. NAD+ loss in turn decreases Sirt6 activity in both organs, supporting a role for suppression of sirtuin activities in TCDD toxicities. NAD+ repletion with nicotinamide (NAM) or the PARP inhibitor PJ34 prevented both NAD+ depletion and the production of thymus atrophy and hepatosteatosis by TCDD, supporting a role for NAD+ loss in different TCDD toxicities in vivo.Here we investigated whether NADCYP1A4 and TiPARP in both organs  with TCDD decreased thymus weight significantly after 72 hr and even more after 96 hr Fig.  without + levels (TCDD decreased NAD+ by 38% (p = 0.02) in liver and by 33% (p = 0.03) in thymus .+ depletion in the toxic effects of TCDD we treated CE with nicotinamide (NAM), the amide form of vitamin B3, which undergoes cellular metabolism to NAD+17, as a NAD+ repleting agent. A single dose of NAM increased NAD+ levels in thymus and liver for at least 16 hr 9, we asked whether suppression of NAD+ levels by TCDD in vivo might reflect increased PARP activity. Figures 8) in liver but not in thymus , on the endpoints affected by TCDD to learn whether our findings were specific for TCDD or applied to other AhR ligands. β-NF activated the AhR in both liver and thymus as evidenced by increased CYP1A4 mRNA expression, and, like TCDD, suppressed NAD+ levels in both liver and thymus and  produced steatosis and thymic atrophy  and histone 3 lysine 56 (H3K56)27 consistent with TCDD decreasing Sirt6 activity in both organs. NAM treatment prevented those effects . The possibility that TCDD may increase the activity of the parthanatos pathway, a cell death pathway related to PARP1 activation and involving AIF (apoptosis inducing factor) translocation from mitochondria to nucleus warrants further investigation28.Thymus atrophy involves loss of cells and thus may involve cell death. Consistent with the comment of Virag 29 which could contribute to thymus atrophy by TCDD, we examined effects of TCDD on cellular senescence. TCDD increased cellular senescence in thymus epithelial cells pyrene, PCBs40, and cigarette smoke41 as well as arsenic, which causes thymic atrophy independently of the AhR42.These studies focus on TCDD as a prototype AhR activator and highly toxic chemical, and also provide evidence supporting NAD+ in thymus function independently of effects of dioxin or AhR activation, meriting further study. For example, we provide evidence that NAD+ levels were an order of magnitude lower in thymus than in liver and that PARP activity (ADP ribosylation) and levels of PARP enzymes (PARP1 and TiPARP) were much higher in thymus than liver in both CE and the mouse. The latter finding is consistent with evidence for low levels of PARP1 mRNA in mouse liver43 and shows further the much larger amounts of PARP and PAR activity in thymus, suggesting that there are undeciphered roles for PARPs and ADP-ribosylation in thymus function.Some observations reported here point to roles for PARP activity and NAD44, but there has been surprisingly little attention to forestalling age-related thymus atrophy. The discoveries here that decreased NAD+ has a pathogenic role in thymus atrophy by TCDD and that NAD+ repletion can prevent thymus atrophy provide a basis for investigating the role of NAD+ in thymus atrophy in aging. Further, evidence that declining NAD+ levels accompany human aging45 provides additional impetus to investigate the contribution of exposure to AhR-activating environmental toxicants to adverse effects of aging.Thymus atrophy, an inevitable manifestation of human aging, is thought to contribute to increased autoimmunity and decreased responses to infection in aging adultsGallus gallus)  were maintained at 37 °C at high humidity. Fourteen to 18 day old chick embryos (CE) (hatching is at 21 days) were treated in ovo by injection through a hole in the shell into the fluids surrounding the embryo, as follows: TCDD, 1 nmol per egg in 0.005 ml dioxane , an index of CYP1A activation46); nicotinamide (NAM) , 10 mg/egg in 0.05 ml of distilled water; nicotinamide riboside (NR) , 2 mg/egg in 0.05 ml of distilled water; the pan-PARP inhibitor PJ34 hydrochloride , 2 mg/egg in 0.1 ml of distilled water. Unless otherwise indicated, NAM or NR were administered twice (at the same time as TCDD and 36 hr after TCDD treatment) and PJ34 three times (at the same time as TCDD and 24 and 48 hr after TCDD treatment). TCDD treatment was for 72 hr unless otherwise indicated. All controls received vehicle (dioxane or water) in volumes equivalent to those of the treatments. For experiments in Fig. Fertilized White Leghorn eggs :dH2O) was added to each well and incubated for 10 min. Cells were washed 4 times with dH2O. Images were taken using MagnaFire Software on a Nikon Narishige Diaphot microscope and quantified using Image J software .Pieces of livers and thymus glands removed from CE were promptly placed in 4% paraformaldehyde in 1X PBS at 4 °C for 24 hr fixation, transferred to 30% sucrose for 24 hr, cryoembedded and stored at −20 °C until sectioned (10 μm slices). H&E and Oil red O staining on tissue sections were performed by the Optical Microscopy Core Facility and the Laboratory of Comparative Pathology , following standard procedures. Triglycerides were measured using a Triglyceride Colorimetric Assay kit  following the manufacturer’s instructions.+ levels were measured in liver and thymus homogenates using EnzyChrom NAD+/NADH Assay Kits  following the manufacturer’s directions.NAD7, PARP1 , SIRT6 , total Histone 3 (H3) , acetylated (K9)H3 , acetylated (K56)H3 , histone H2AX , phospho(Ser139)-histone H2AX , β-actin . Peroxidase-conjugated goat anti-rabbit (Sigma-Aldrich) or goat anti-mouse (Santa Cruz Biotechnology) antibodies were used as secondary antibodies.Tissue samples were homogenized in 2X sample buffer  and boiled for 5 min. Proteins were separated on precast Tris-Glycine gels (Invitrogen/Life Technologies) and transferred to nitrocellulose membranes. The following primary antibodies were used: PAR (poly(ADP)ribose)  which recognizes ADP-ribosylated proteins, TiPARP Protein bands were detected using ECL Western Blotting Detection reagents . Band intensities were measured by densitometry using Syngene Software .Chick embryo hepatocytes (CEH) for the experiments shown in Fig. 7. To ensure lipid uptake for Oil Red O staining Albumax (1%)  was added to the medium when cells were treated with TCDD (1 nM) or vehicle. Thymic epithelial cells were cultured as follows: 15–20 thymus glands from CE were placed in 3 ml RPMI 1640 medium containing 2 mg/ml papain and incubated at 37 °C for 15 min, followed by addition of 3 ml RPMI 1640 supplemented with 5% FBS. Thymus glands were chopped with a blade for 3 min and passed through a 100 µm mesh. Cells were plated in 6 cm dishes  in RPMI 1640 supplemented with 5% FBS and 1% pen-strep and allowed to attach for 2 days without changing the medium. On day 3, the medium was replaced with fresh medium, and non-adherent lymphocytes and non-viable cells were removed. The thymic epithelial cells were treated with TCDD at 10 nM.Chicken Sirt6 was cloned as follows: The chicken Sirt6 coding region (from +47 to +1120 bp) was amplified by PCR with primers designed using the predicted chicken Sirt6 mRNA sequence : forward primer, 5′- CACCatggcggtgaattacgcg-3′; reverse primer, 5′-ggtgaggagaggctctacc-3′. cDNA from CE liver was used as a template. The PCR amplification product was purified using Wizard SV Gel and PCR Clean-up System  and cloned into pcDNA™3.1 using a pcDNA™3.1 Directional TOPO Expression Kit  following the manufacturer’s directions. Nucleotide sequencing confirmed the PCR product as chicken Sirt6 (Gene Bank #: KX395903).7 using the A023 and T-030 programs, respectively. Transfection efficiency in CEH and TEC was >80% as detected by fluorescence using a plasmid containing GFP.CEH and thymic epithelial cells (TEC) were transfected using AMAXA Nucleofection technologyThymus sections: Thymus glands were removed from CE, fixed in 1X PBS containing 2% v/v formaldehyde and 0.2% v/v glutaraldehyde for 15 min at 4 °C, and incubated in 30% sucrose for 2 hr at 4 °C, then embedded in 30% sucrose:OCT (1:2) and cryosectioned in 10 µm slices. SA- β-gal assay: After drying for 30 min, sections were placed in a SA-β-gal staining solution  in a 37 °C incubator with ambient CO2. After overnight incubation, blue cells (senescent) were counted using Image J software. (b) Thymic epithelial cells: TEC obtained as described above were washed twice with 1X PBS, fixed for 5 min in formaldehyde, washed twice with 1X PBS and placed in the SA-β-gal staining solution. Blue (senescent cells) were counted after overnight incubation at 37 °C. Fixing and SA-β-gal staining solutions were as described above.(a) 7. For PCR, primers and annealing temperatures were as follows: 18s, 5′-gaccataaacgatgccgact-3′ and 5′-agacaaatcgctccaccaac-3′ (55 °C); TiPARP, 5′-ccagctccagctccaactac-3′, 5′-ctgtaagaacggcatcagca-3′ (57 °C); CYP1A4, 5′-ggacggaggctgacaaggtg-3′, 5′-tgctgcaggatggtggtgag-3′ (59 °C); PARP1 5′-gcaggaaaaacagctgaagg-3′, 5′-gcatcgctcttgaacacaaa-3′(54 °C), Sirt6, 5′- ctactcggataagggcaagt-3′, 5′- cttccatagtccagacacca-3′. An Eppendorf Mastercycler ep gradient machine was used for 40 cycles of amplification. Fold-changes in mRNA were calculated by the standard 2−ΔΔCt method47 using 18S mRNA as a reference for normalization.Total RNA was extracted from small pieces of liver or thymus and retrotranscribed as reportedProtein concentration was measured using Biorad-DC or RC-DC Protein assays .p values ≤ 0.05 were considered statistically significant. All experiments were replicated with the number of independent experiments and numbers of CE in each experiment indicated in the Figure legends.Differences between group means were evaluated using GraphPad software by unpaired, two tailed t-tests; Supplementary Figures 1–11


Notably, lincRNA-p21 overexpression contributed to the suppression of HSC activation. lincRNA-p21 suppressed HSC proliferation and induced a significant reduction in α-SMA and type I collagen. All these effects induced by lincRNA-p21 were blocked down by the loss of PTEN, suggesting that lincRNA-p21 suppressed HSC activation via PTEN. Further study demonstrated that microRNA-181b (miR-181b) was involved in the effects of lincRNA-p21 on HSC activation. The effects of lincRNA-p21 on PTEN expression and HSC activation were inhibited by miR-181b mimics. We demonstrated that lincRNA-p21 enhanced PTEN expression by competitively binding miR-181b. In conclusion, our results disclose a novel lincRNA-p21-miR-181b-PTEN signaling cascade in liver fibrosis and suggest lincRNA-p21 as a promising molecular target for antifibrosis therapy.Previously, we found that long intergenic noncoding RNA-p21 (lincRNA-p21) inhibits hepatic stellate cell (HSC) activation and liver fibrosis via p21. However, the underlying mechanism of the antifibrotic role of lincRNA-p21 in liver fibrosis remains largely unknown. Here, we found that lincRNA-p21 expression was significantly downregulated during liver fibrosis. In LX-2 cells, the reduction of lincRNA-p21 induced by TGF- Liver fibrosis, characterized by enhanced extracellular matrix (ECM), represents the common responses of the liver to various chronic injuries. It is well known that hepatic stellate cells (HSCs) are crucial mediators of liver fibrosis , 2. In nMicroRNAs (miRNAs) are highly conserved noncoding RNAs of ~22 nucleotides that regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of the target gene mRNAs to repress protein translation or induce mRNA degradation . NotablyLong noncoding RNAs (lncRNAs) are transcribed RNA molecules (>200 nucleotides in length) that structurally resemble mRNAs but do not encode proteins . Emerginβ1 (TGF-β1) was purchased from R&D Systems . Adenoviral vectors expressing a control scrambled sequence (Ad-Ctrl) and adenoviral vectors expressing lincRNA-p21 (Ad-lincRNA-p21) were purchased from GenePharma biotechnology . Antibodies against type I collagen, α-smooth muscle actin (α-SMA), PTEN, and GAPDH were obtained from Abcam . Chemically synthesized RNAs including negative control (miR-NC), miR-181b mimics, and miR-181b inhibitor were obtained from GenePharma biotechnology. For transfection, cells were transfected with 1 μg of the chemically synthesized RNA using Lipofectamine 2000 .Transforming growth factor-Written informed consent was received from all patients prior to liver tissues. In this study, 15 healthy controls and 15 liver cirrhosis patients undergoing partial liver resection or liver biopsy were selected from the First Affiliated Hospital of Wenzhou Medical University. Liver cirrhosis was diagnosed by liver biopsy and/or a typical appearance of the liver on abdominal ultrasound and/or computed tomography scan. This study was performed in compliance with the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University.2. Exponentially growing cells were seeded in a six-well plate at a density of 1 × 106 cells/well; then the cells were transduced with the Ad-lincARNA-p21 or Ad-Ctrl for 48 h. Cells were also treated with TGF-β1 for different experiment purposes. Cells were harvested for RNA/miRNA isolation, and whole cell extracts were subjected to western blot analysis.Human LX-2 cell strain was obtained from JENNIO Biological Technology . It was cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin G sodium salt, and 100 U/mL streptomycin sulfate . The cells were grown in a 37°C incubator with 5% CORNA interference experiments were performed using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's instructions. PTEN siRNA1 , PTEN siRNA2 , and scrambled siRNA (negative control) were designed and synthesized by GenePharma biotechnology. siRNAs were transfected into cells at a final concentration of 100 nM.α-SMA, GAPDH, and U6 were designed as described previously [−ΔΔCt) of lincRNA-p21, mRNAs, and miRNAs were calculated as described previously [Total RNA was extracted from human liver tissues and LX-2 cells using miRNeasy Mini kit  according to manufacturer's instructions. Fifty nanograms of total RNA was reverse-transcribed to cDNA using the ReverTra Ace qPCR RT Kit . Gene expression was measured by real-time PCR using cDNA, SYBR Green real-time PCR Master Mix . The primers of lincRNA-p21, alpha-1 (I) collagen (Col1A1), eviously , 14. Theeviously .μg) were separated by SDS-PAGE and blotted onto a PVDF membrane . After blocking, nitrocellulose blots were incubated for 1 h with primary antibodies diluted in TBS/Tween20 (0.075%) containing 3% Marvel. Rabbit polyclonal antibodies against type I collagen and PTEN were diluted 1 : 1000, and mouse monoclonal antibodies against α-SMA and GAPDH were used at 1 : 2000. The membranes were washed 3 times with TBS/Tween20 (0.075%) containing 3% Marvel, followed by incubation with HRP-conjugated secondary antibodies (1 : 5000) at 37°C for 1 h. The antigen-antibody complex was developed by enhanced chemiluminescence, exposed in the dark room and analyzed for integral absorbance (IA) of the protein bands using quantitative software, Quantity One 4.4.The protein concentration of samples was determined by a BCA protein assay kit . Total proteins  according to manufacturer's instructions. Absorbance values for each well were determined at 450 nm using a microplate reader . All experiments were performed in triplicate and repeated at least three times.Cells were seeded in 96-well plates at a density of 1 × 10According to RNA22 analysis, oligonucleotides containing human lincRNA-p21 3′UTR target sequence were annealed and cloned into the pmirGLO plasmids  to generate the pmioGLO-lincRNA-p21 vector: lincRNA-p21-3′UTR for miR-181b (position of 1296–1318) forward, 5′-CCCTCCGACAGGAGTCTCA-3′, and reverse, 5′-TTGAGAGAGATGCACAGCCAG-3′. Empty plasmid pmirGLO was regarded as a negative control. Luciferase reporter plasmids plus miR-181b mimics or miR-NC were cotransfected into LX-2 cells using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, relative luciferase activity was examined in a luminometer using a Dual-Luciferase Reporter Assay System (Promega). t-test and one-way analysis of variance, respectively. Correlation between the expressions of miR-181b and lincRNA-p21 in liver tissues was examined by Pearson's correlation coefficient. P < 0.05 was considered significant. All statistical analyses were performed with SPSS software .Data from at least three independent experiments were expressed as the mean ± SD. Comparisons between two groups and multiple groups were made using Student'sβ1 is recognized as the main profibrogenic mediator [β1, and the expression of lincRNA-p21 was detected. There was a significant reduction in lincRNA-p21 level as the increased concentrations of TGF-β1 (from 0 to 15 ng/mL), suggesting that lincRNA-p21 was reduced by TGF-β1 in a dose-dependent manner , both PTEN siRNA1 and PTEN siRNA2 caused a significant reduction in PTEN protein level. Then, the expressions of α-SMA and collagen were examined in lincRNA-p21-overexpressing cells with PTEN siRNA. It was found that reduced HSC proliferation caused by lincRNA-p21 was blocked down by PTEN siRNA , PTEN is predicted as a target of miRNAs. In theory, PTEN can be regulated by them. Many studies have demonstrated that miRNAs including miR-21, miR-24, miR-32, miR-93, miR-153, miR-205, and miR-214 can regulate PTEN expression as a target in human diseases –25. Our  Figures . All ther = −0.961, P < 0.001)  . There m< 0.001) . Using p< 0.001) . miR-181< 0.001) . lincRNAα-SMA expression. Without the enhanced PTEN caused by lincRNA-p21, all these effects were blocked down, suggesting that lincRNA-p21 suppresses liver fibrosis, at least in part, via PTEN. Notably, further studies showed that enhanced PTEN protein level and the antifibrotic effects induced by lincRNA-p21 were blocked down by miR-181b. As confirmed by luciferase activity assays, lincRNA-p21 was confirmed as a target of miR-181b. Combined with the data above, we demonstrated that lincRNA-p21 contributes to the suppression of liver fibrosis via miR-181b-mediated PTEN.The results of the present study showed that PTEN expression was enhanced by lincRNA-p21 via the regulation of miR-181b. HSC activation was significantly suppressed by overexpression of lincRNA-p21, including the reduction in HSC proliferation, ECM protein, and 1/S checkpoint is reported to be regulated by lincRNA-p21 via p21 in cis [β-catenin signaling and tumorigenicity of colorectal cancer stem cells [LncRNAs, lacking significant protein-coding capacity, can regulate a wide range of biological processes through diverse molecular mechanisms including chromatin modification, transcriptional regulation, and posttranscriptional regulation , 27, 28.1 in cis . miRNA-rem cells . Our preem cells . In our PTEN, as a tumor suppressor, is often deregulated in various cancers. Both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways can be regulated by PTEN through its lipid phosphatase and protein tyrosine phosphatase activity, respectively , 33. TheIn conclusion, our results provide new insights that lincRNA-p21 inhibits the progression of liver fibrosis via PTEN. Moreover, our data disclose a novel lincRNA-p21/miR-181b/PTEN signaling cascade in liver fibrosis. The downregulation of lincRNA-p21 in patients with cirrhosis suggests that it may be potential diagnostic biomarkers for cirrhosis.Fig.S1 The effects of PTEN siRNA, Ad-lincRNA-p21 or miR-181b inhibitor on the protein expression of PTEN. (A) PTEN protein was reduced by PTEN siRNA1 or siRNA2. Cells were transfected with PTEN siRNA for 48 h. (B) PTEN protein was increased by Ad-lincRNA-p21, which was further enhanced by miR-181b inhibitor. Cells were transduced with Ad-lincRNA-p21 for 48 h and treated with miR-181b inhibitor for additional 48 h. GAPDH was used as internal control. Each value is the mean ± SD of three experiments. ∗P<0.05 compared with the control and #P<0.05 compared with Ad-lincRNA-p21 group.


The idea of a Video Corner in our Journal has been discussed in the last 2 years, inspired by numerous video sessions introduced by many ObGyn societies during their meetings. Technological advances matured and provide today uninterrupted and high clarity videos. Based on the moto that one picture is worth thousand words you can imagine the impact on teaching ability of a short video when a new operative technique or the management of an obstetrical complication and its treatment can be presented.This Journal embraces all ObGyn disciplines from the beginning and it always showed a major interest in images, art contributions and visionary ideas. Therefore we believe that it provides the perfect scientific environment and conditions to initiate and support the first of its kind video journal in medical profession.Traditional educational methods and presentation of academic work by written articles definitely have a great value, perspective and of course limitations. Our Video Corner aims to facilitate further learning and understanding, especially when complex matters are involved. High definition audiovideo presentations, accompanied by clear and evidence based articles or case-reports provide, in our opinion, the best option to show and explain complicated cases and pathologies that normally need extensive writing to describe and fully understand the case. Within this context a video corner with ObGyn articles can improve educational skills and shorten learning curves.In addition, important details of an operation, rare pathologies, anatomical deviations and surgeon ergonomy can be demonstrated in a better way. Many operative procedures are poorly described, due to the limitations inherent to written papers. Hence, video articles have an added value in learning difficult tasks and open a new way to transfer medical information and knowledge which is not possible without a video presentation. We are also open for new techniques and innovative methods, setting up unusual projects etc., as long as the guidelines are followed. Video articles presenting the management of complications during surgical and medical treatments are urgently needed. It is of imperative value since concerns patients’ safety and surgery outcome.It is our aim that the guidelines to authors considering abstract, conclusions and references remain the same as for the written format articles in our journal. However, the paper should be presented with restricted word numbers and accompanied with video sessions. It will be mandatory to present in each video article the learning objectives in a constructive and clear way and to discuss crucial scientific matters and conclusive remarks.The video articles will be reviewed by a group of experts of each discipline in ObGyn. The important factors to accept a video article is the video content fulfilling the written learning tasks and the importance of the presenting objectives.In the next issue of FV&V we will present the editorial board, the guidelines to follow and an example of a video article. From November on it will be possible to submit your contributions to the Journal.


Malnutrition is a significant issue in the hospital setting. This cross-sectional, observational study determined the prevalence of malnutrition amongst 189 adult inpatients in a teaching hospital using the Patient-Generated Subjective Global Assessment tool and compared data to control groups for coding of malnutrition to determine the estimated unclaimed financial reimbursement associated with this comorbidity. Fifty-three percent of inpatients were classified as malnourished. Significant associations were found between malnutrition and increasing age, decreasing body mass index, and increased length of stay. Ninety-eight percent of malnourished patients were coded as malnourished in medical records. The results of the medical history audit of patients in control groups showed that between 0.9 and 5.4% of patients were coded as malnourished which is remarkably lower than the 52% of patients who were coded as malnourished from the point prevalence study data. This is most likely to be primarily due to lack of identification. The estimated unclaimed annual financial reimbursement due to undiagnosed or undocumented malnutrition based on the point prevalence study was AU$8,536,200. The study found that half the patients were malnourished, with older adults being particularly vulnerable. It is imperative that malnutrition is diagnosed and accurately documented and coded, so appropriate coding, funding reimbursement, and treatment can occur. Malnutrition has been identified as a significant clinical problem in hospital settings both nationally and internationally , 2. MalnMalnutrition has been defined by the American Society for Parenteral and Enteral Nutrition as cited in Mueller et al.  as “an aMalnutrition directly impacts health care costs and is captured in the Activity-Based Funding model used for the funding of hospitals in Australia . ActivitSeveral previous Australian studies have reported estimations of unclaimed reimbursements for patient admissions, where malnutrition was not recorded as a comorbidity as part of the DRG , 13–15. The Australian Capital Territory (ACT) is a territory within Australia, where Canberra the capital city of Australia is situated. The estimated population of the ACT is currently 390,800 . HoweverAs there are currently no data on malnutrition in adults residing in the ACT, the aim of this study was to determine the prevalence of malnutrition in a sample of adult inpatients at a tertiary teaching hospital in the ACT region to provide a baseline for effective monitoring and outcome measures, to determine characteristics associated with malnutrition in this group of patients, and to compare data to historical and subsequent cohorts for prevalence of malnutrition and coding of malnutrition. Based on these data we were then able to estimate the unclaimed annual financial loss of hospital income due to undiagnosed and/or undocumented malnutrition.This cross-sectional, observational study involved the collection of nutritional status data of adult inpatients in the aged care, rehabilitation, surgical, medical, critical care, acute care, orthopaedic, gastroenterology, vascular, neurology, renal, respiratory, cardiology, mental health, and oncology wards at a tertiary teaching hospital.Participant recruitment was on a voluntary basis. Prior to data collection all participants received a copy of an information sheet and consent form. Patients over the age of 18 years admitted to the hospital over the study days were eligible for inclusion. Exclusion criteria were moderate-severe cognitive problems , no conversational English, day patients, terminal medical illness, and patients who were antenatal, postnatal, or determined as unfit by medical officers. If a participant was identified as malnourished then this was documented in their medical notes and they were referred to the nutrition department for nutrition intervention. Data collection took place over two consecutive days in June 2012. All eligible patients were invited to participate. The research was approved by the ACT Government Health Directorate Human Research Ethics Committee (ETH6.11.126 and ETHLR.13.222), and informed written consent was obtained from all participants.p < 0.001).Nutritional status of subjects was assessed using the scored Patient-Generated Subjective Global Assessment (PG-SGA) tool  which isFollowing the data collection, a retrospective review of the medical records of study participants was conducted by the authors to provide information regarding length of stay, DRG allocation, documentation of malnutrition, and dietetic intervention. This was to determine if the study participants who were diagnosed as malnourished in this study were documented as malnourished by the dietitians and coded as malnourished by the medical coders. These data were then compared to the medical records of two historical control groups (June 2011 and March 2012) and two subsequent cohorts (September 2012 and June 2013) to determine the proportion of patients coded as being malnourished prior to our study and after our study. All patient separations within these months using the same inclusion criteria were included in the data collection. A patient separation can be defined as, “the process by which an episode for an admitted patient ceases .”Unclaimed potential reimbursements to this hospital (per annum) were calculated based on previous studies , 14, whe t-test. Associations between continuous variables and malnutrition prevalence were assessed using the One-Way ANOVA test. The Mann–Whitney U test was used to examine differences in variables between the groups of patients according to malnutrition status as assessed by the PG-SGA. For this purpose groups SGA B and SGA C  were combined for statistical purposes. A p value of less than 0.05 was considered to be statistically significant. Intraclass correlation was used to show the measure of agreement between multiple raters.Data were entered into IBM SPSS Version 21.0 software . Patient characteristics were reported as frequencies, means, and standard deviations. Nonparametric techniques were principally used to describe patient characteristics. Associations between gender and continuous variables were assessed using the independentOne hundred and eighty-nine patients participated in the study over the two days. There were 434 adult inpatients at the hospital over the study period, and 163 were excluded . Of the 271 eligible patients approached to participate, 75 declined and 7 were away from their room at the time of data collection. Overall the response rate was 73.1%.One hundred and seventeen participants (62%) were male, and 72 (38%) were female. The age range of the 189 participants was 18–97 years (mean age 62.9 ± 17.9). Fifty-two percent of the participants were over the age of 65 years. The baseline characteristics of our participants are summarised in n = 88) of patients were classified as well-nourished, 47% of patients (n = 89) were classified as moderately or suspected of being malnourished, and 6% (n = 12) of patients were classified as severely malnourished. By ward, 96% of the participants from the oncology ward, 83% of the participants from the aged care ward, and 79% of the participants from the respiratory ward were found to be moderately or severely malnourished.Forty seven percent (p = 0.051). There was a significant association between malnutrition and increasing age (p = 0.040). There was a significant difference between mean PG-SGA scores for each of the SGA classifications (p < 0.001), with the severely malnourished having the highest scores. There was also an association between SGA groups and body mass index (BMI), with the higher the SGA classification, the lower the BMI (p < 0.001). There was a significant difference in length of stay between well-nourished patients (SGA A)  and malnourished patients (SGA B and SGA C) , U = 2961, Z = −3.96, p < 0.001, and r = −0.28. On the study days in June 2012, malnutrition was coded in 98.2% (n = 99) of patients assessed as malnourished. Eighty-five percent of malnourished patients were coded as having been seen by a dietitian.The characteristics of patients according to malnutrition status were assessed according to the Patient-Generated Subjective Global Assessment (PG-SGA) tool as shown in The results of the medical history audit of patients that was conducted on patient separations in June 2011, March 2012, September 2012, and June 2013 are shown in Extrapolating from Agarwal et al. , we can In this observational study, we found the prevalence of malnutrition to be 53% amongst the adult inpatients, which is the highest prevalence of malnutrition recorded in an Australian study, equivalent to research conducted by Thomas et al. in 2007 . PreviouWhen we compared our study data to two historical control groups and two subsequent control groups we found that between 0.9% and 3.8% of patients were coded as being malnourished before the study and 4.4%–5.4% of patients were coded as being malnourished in the subsequent control groups. These figures are significantly lower than the 52% of patients that were coded as malnourished from the point prevalence study data  were coded as being malnourished in the medical records). It is difficult to ascertain whether this difference in malnutrition coding figures was due to lack of identification, documentation, or coding in the control groups, but considering the total number of occasions of service for dietitians documented in the medical notes  and the It is also important to recognise that the point prevalence study was conducted under study conditions, where 16 dietitians were conducting assessment, all eligible patients were assessed, and malnutrition stickers were used. These are ideal conditions and may vary to “usual care,” where factors affecting the identification of malnutrition may include poor completion of malnutrition screening by nursing staff, poor referral rates to the dietitian, and patients who are discharged before being seen by the dietitian for assessment. The historical and subsequent cohorts were representative of “usual care” which may also account for some of the differences in coding of malnutrition between groups.We estimated the unclaimed potential reimbursements per annum to this hospital to be AU$8,536,200. This additional funding to the health facility could make a valuable contribution to the increased costs associated with treating these malnourished patients. For example, if these costs were recovered, a portion of this additional funding could be directed to funding the employment of additional Accredited Practising Dietitians (APDs) to assist with the identification and management of patients with malnutrition, which may improve clinical outcomes.Malnutrition as a health issue in the hospital setting was first documented in the medical literature over forty years ago . Since tThe reported prevalence rate of malnutrition in this study may be an underestimation of this clinical issue, as a result of the exclusion criteria used. Patients with dementia were excluded who are a high-risk group for malnutrition . IncludiA contributing factor towards the high prevalence of malnutrition found in this study may be a result of the increase in acuity of patient admissions over time. For example, in 2000-2001, the average length of stay in an Australian Public Hospital was 3.7 days . The mosUsing validated nutrition assessment tools to diagnose malnutrition in the acute care setting is of paramount importance. In the acute care setting, valid nutrition assessment tools include the Subjective Global Assessment (SGA) , the PG-Mitchell and Porter  highlighIn conclusion, this study has highlighted that malnutrition continues to be underdiagnosed in the acute care setting in Australia, with older adults being particularly vulnerable. Diagnosing malnutrition using validated assessment tools and documenting and coding accordingly may increase potential reimbursements for hospitals which enables funding to be directed towards better care. Using standardised preprinted stickers may assist in identifying malnourished patients to coders and standardising documentation by dietitians. Malnutrition has been strongly associated with adverse clinical outcomes such as an increased length of stay and higher rates of medical complications. Not diagnosing malnutrition presents a high risk to patients and is a lost opportunity for financial reimbursement for the increased costs associated with the care of these vulnerable patients.