Due to the continuous downgrading of diagnosis-related severity (CCL) of malnutrition in the G-DRG system in trauma surgery patients, it is no longer possible to refinance the costs incurred by malnourished patients through the conscientious coding of malnutrition. We assume that the indirect positive effects of nutritional interventions will have to be taken into account more in the costing calculations and possibly lead to indirect cost compensation.It has been internationally recognized that malnutrition is an independent risk factor for patients' clinical outcome. A new mandatory fixed price payment system based on diagnosis-related groups (G-DRG) went into effect in 2004. The aim of our study was to demonstrate the importance of carefully coding the secondary diagnosis of "malnutrition" in the G-DRG system and to highlight how the economic relevance of malnutrition in the G-DRG system has changed from 2014 to 2016. 1372 inpatients at the Berufsgenossenschaftliche Unfallklinik (Trauma Center) in Tübingen were screened for the risk of malnutrition using Nutritional Risk Screening (NRS-2002). Patient data were compared with the NRS values collected during the study and a case simulation was carried out separately for each year. We used the codes E44.0 for NRS = 3 and E43.0 for NRS > 3. The ICD codes were entered as an additional secondary diagnosis in the internal hospital accounting system DIACOS to determine possible changes in the effective weight. In 2014 the highest additional revenue by far was calculated by coding malnutrition. For the 638 patients enrolled in the study in 2014, we were able to calculate an average additional revenue per patient coded with malnourishment of €107. In 2016, we were unable to calculate any additional revenue for the 149 patients enrolled. Although it is well known that malnutrition is an independent risk factor for poor patient outcomes, nationwide screening for a risk of malnutrition when patients are admitted to a hospital is still not required. For this reason, malnutrition in German hospitals continues to be insufficiently documented Malnutrition has been proven by various studies and internationally recognized to be an independent risk factor for patients' clinical outcome  is implemented in German hospitals  Unfallklinik (Trauma Center) in Tübingen were screened for the risk of malnutrition using Nutritional Risk Screening (NRS-2002), an international interview-based questionnaire for detection of malnutrition or a risk of malnutrition . Between 07/2014 and 07/2016, 1372 inpatients at the BG Unfallklinik in Tübingen were screened for the risk of malnutrition using the NRS-2002 screening tool . The clThe NRS score was used to assess the nutritional status. In our study cohort, 19 % of the patients showed a moderate to high risk of malnutrition (NRS ≥ 3) and were therefore classified as malnourished. According to the criteria of Reinbold et al. (2013), the peComparison of the distribution of malnutrition in different age groups indicated the correlation of increasing risk of malnutrition with a higher age. The results are shown in Figure 1e.g.; trauma, septic surgery or arthroplasty), the following tendency could be observed: in septic surgery, 29 % of the patients are malnourished, while the percentage decreases to 16 % in traumatology and to 12 % in arthroplasty. The coding criteria of Reinbold et al.  changesThe coding of malnutrition as a secondary diagnosis leads to a change in the revenue. By coding malnutrition as a secondary diagnosis with E44.0 [moderate protein-energy malnutrition] for NRS = 3 and E43 [unspecified severe protein-energy malnutrition] for NRS > 3, a total additional revenue of € 96,298.39 was calculated from 2014 to 2016. The DRG grouper of the respective year was always used for the calculation. The calculated additional revenue after criteria of Reinbold et al. (2013) amountsGenerally, the highest additional revenues were calculated for male patients. In our study, 65 % of the additional revenue was generated with male malnourished patients. The same tendency was also observed when calculating the average additional revenue. The average revenue generated by each male patient was € 7,768 and increased to € 7,849.72 through the additional coding of malnutrition (NRS ≥ 3) and to € 8,322.05 according to the criteria of Reinbold et al. (2013). Again,No correlation was calculated between average additional revenue from the coding of malnutrition and the age of the patients. However, considering the high number of patients over the age of 65 years in absolute terms, more additional revenue could have been calculated by encoding malnutrition. This correlation is shown in Figure 3Despite differences in patient numbers in the various departments , the total revenue during our study period from 2014 to 2016 was equal in all departments. The highest proportion of 38.66 % in total revenue was achieved in the traumatology department with 619 treated patients. The septic surgery department accounted for 32.88 % of total revenue with 395 treated patients, whereas the arthroplasty department reached 28.46 % with the treatment of 358 patients. The DRG groupers of the respective accounting year were used for the calculations as usual and the results were summed up.By conscientious coding of malnutrition according to the standard criteria, the percentage of revenue shifted to 79 %  for the septic surgery department, while only 21 % of total additional revenue have been earned in the traumatology department and no additional income have been earned in the arthroplasty department. The details are shown in Table 6. In 2015, 585 patients were included into the study, and the average additional revenue had fallen to € 47 (standard coding) or € 167 . For thal. (2013) per patal. (2013), an aveAlthough malnutrition is well known as an independent risk factor in patients' poor outcomes, screening for a risk of malnutrition at patients' admission to a hospital is not mandatory in Germany. For this reason, malnutrition in German hospitals continues to be insufficiently documented. This results in clinical disadvantages for the patients, who can face shortcomings in their treatment as well as in economic disadvantages for hospitals due to lost revenues for additional treatment . The trAmaral et al.  are appe.g.; trauma, septic surgery, or arthroplasty) . We the, 2017[, 2017[Apart from other causes, such as depression and cancer, age is known to be a risk factor for the development of malnutrition . The hi, 2017) showed , 2017; Ihle et17[, 2017; Suarez-17[, 2017). We alsAlthough the prevalence of malnutrition is being reported broadly in the literature, one of the main problems is still a lack of an internationally accepted criterion for the diagnosis of malnutrition and the use of various screening tools (Cruz et al., 2017; OrlandoWe would like to thank Peter Rapp for his support by the case simulation. In addition, we like to thank Mrs. Svetlana Gasimova for editing and proofreading of the final draft.The authors declare that they have no conflict of interest.


Earlier observations on p21 knockout models emphasized the role of this protein in cell cycle arrest under the p53 transcription factor activity. Although tumor-suppressor function of p21 is the most studied aspect of this protein in cancer, the role of p21 in phenotypic plasticity and its oncogenic/anti-apoptotic function, depending on p21 subcellular localization and p53 status, have been under scrutiny recently. Basic science and translational studies use precision gene editing to manipulate p21 itself, and proteins that interact with it; these studies have led to regulatory/functional/drug sensitivity discoveries as well as therapeutic approaches in cancer field. In this review, we will focus on targeting p21 in cancer research and its potential in providing novel therapies. Imbalance between cell proliferation and cell death (apoptosis) leads to tumorigenesis. p21, a well-established cyclin-dependent kinase (cdk) inhibitor, was found to play an important role in controlling cell cycle progression . While pControversial aspects of p21 is decided by p21 location and p53 protein condition . p53  , TALENs −/− mice showed higher S-phase fractions than tumors in MMTV-myc/p21−/− mice. p21 deficiency in these two different mouse models also affected apoptosis levels in different manners. Significant increase in spontaneous apoptosis was reported in MMTV-ras/p21−/− mice tumor, whereas apoptosis levels of MMTV-myc/p21−/− mice tumor did not change significantly. Different oncogene expression patterns caused different cell proliferation profile in p21-deficient mice, pointing out context-dependence function of p21 in cancer progression [Δhepa  mice, which are used as hepatocellular carcinoma model. In this study, p21 deficiency was shown to result in higher DNA damage and higher number of hepatocellular carcinoma (HCC) [FahDexon5 mice are liver failure in vivo model with increased risk for liver cancer. Loss of p21 in C57Bl6-FahDexon5 mice was shown to cause rapid tumor formation and continuous hepatocyte proliferation [+/− mice, an intestinal tumor model. Apc1638+/− /p21−/− mice showed altered cell maturation, increased tumor formation and cell proliferation [p21 has a dual role in cancer . There agression . p21 wasma (HCC) . C57Bl6-feration . Interesferation . Adnane feration . In anotferation .SOCS1 -deficiency in mice, treated with hepatocarcinogen diethylnitrosamine (DEN), was shown to cause large and numerous liver tumor nodules. p21 protein level was shown to increase in SOCS1-deficient mouse liver, causing increased resistance to apoptosis and induced proliferation in response to growth factor stimulation. However, these phenotypes disappeared following p21 knockdown . In anotp21 alteration can have a major effect on cancer susceptibility to chemotherapy, radiation, and targeted therapy. As shown in As we explained above, targeting p21 gene in cancer treatments is disputable; however, p21 is involved in many pathways that could be targeted instead of p21 itself to overcome the controversy. p21 is regulated in different levels: Transcriptionally, post-transcriptionally and post-translationally ; genes oThere are several studies that investigate the effect of drugs and chemicals on p21 expression in cancer cells; we have shown some of these works in Phosphoinositide 3-Kinase-Protein kinase B (PI3K-Akt) signaling pathway is involved in regulation of cell survival, cell cycle progression and cell growth; Akt enhances cell growth and proliferation by phosphorylating p21 . ChemicaManipulating genes that interact with p21 is another alternative to control p21 effect in cancer development. Expression levels of tumor suppressors and oncogenes change in the tumor cells, which leads to cell proliferation and metastasis through PI3K-Akt pathway  and c-MySynthetic lethality was first introduced by Calvin Bridges to describe when certain non-allelic genes are lethal in combination, while parents with a homozygous mutation are viable; in other words, when two genetic events happen together causing cell death, synthetic lethality occurs . This geTranscriptional, post transcriptional, translational and post translational modifications are regulatory checkpoints for protein expression; these regulations decide localization , activitTranslational regulation and post translational modifications could be targeted to manipulate p21 function in cancer cells. DEAD-box  box) RNA helicase DDX41 (DEAD-box helicase 41) binds to 3’-UTR of p21 mRNA and negatively regulates p21 expression . Galecti+/EpCAM+) and highly migratory, derived from non-small cell lung cancer (NSCLC) cell lines (A549 and H2170) [p21 controls expansion of human hematopoietic stem cells and cell cycle progression; p21 knockout stem cells were showed to induce cell cycle and stem cell exhaustion, under normal homeostatic condition ,134. SOXd H2170) . Benzyl d H2170) . Similard H2170) . LincRNA-p21 is downregulated in glioma stem cells (GSCs) following increased expression of Hu antigen R (HuR) through miR-146b-5p downregulation. Overexpression of miR-146b-5p is shown to decrease viability of cell and stemness, increasing apoptosis and radiosensitivity. However, these phenotypes were rescued following lincRNA-p21 knockdown. This study concluded that targeting miR-146b-5p/HuR/lincRNA-p21/β-catenin signaling pathway could be implemented as a co-therapy for glioma cancer patients . In anot+/CD44+/EpCAM+) subpopulation of pancreatic CSCs is higher than triple- negative (CD24−/CD44−/EpCAM−) cells. PAK4 expression correlates with nuclear accumulation and transcriptional activity of signal transducer and activator of transcription 3 (STAT3). PAK4 silencing in pancreatic cancer (PC) cells was shown to decrease tumorigenesis, increasing PC cells sensitivity to gemcitabine toxicity [p21-activated kinase 3 (PAK3) controls Akt-GSK3β (Glycogen Synthase Kinase 3 beta)-β-catenin signaling in pancreatic cancer cells. PAK3 inhibition leads to suppressed tumorigenesis and CSCs expansion . PAK1 watoxicity . PAK4 altoxicity .In this review, we tried to list the available approaches for p21-directed cancer treatment. Not only p21 is involved in many important pathways that are dysregulated in cancer, but the expression of the protein itself is altered in human cancers ,147. As


Surgical education videos currently all use a single point of view (POV) with the trainee locked onto a fixed viewpoint, which may not deliver sufficient information for complex procedures. We developed a novel multiple POV video system and evaluated its training outcome compared with traditional single POV.We filmed a hip resurfacing procedure performed by an expert attending using 8 cameras in theatre. 30 medical students were randomly and equally allocated to learn the procedure using the multiple POV (experiment group [EG]) versus single POV system (control group [CG]). Participants advanced a pin into the femoral head as demonstrated in the video. We measured the drilling trajectories and compared it with pre-operative plan to evaluate distance of the pin insertion and angular deviations. Two orthopedic attendings expertly evaluated the participants’ performance using a modified global rating scale (GRS). There was a pre-video knowledge test that was repeated post-simulation alongside a Likert-scale questionnaire.The angular deviation of the pin in EG was significantly less by 29% compared to CG (p = 0.037), with no significant difference in the entry point’s distance between groups (p = 0.204). The GRS scores for EG were 3.5% higher than CG (p = 0.046). There was a 32% higher overall knowledge test score (p<0.001) and 21% improved Likert-scale questionnaire score (p = 0.002) after video-learning in EG than CG, albeit no significant difference in the knowledge test score before video-learning (p = 0.721).The novel multiple POV provided significant objective and subjective advantages over single POV for acquisition of technical skills in hip surgery. Surgical education has traditionally been on an apprenticeship model in which residents are given increasing autonomy while performing live surgical cases at the discretion of a supervising surgeon , 2, but Outside operating theatres, orthopedic trainees practice technical skills on cadavers and sawbones. However, these teaching techniques are limited by the expense and short supply of cadavers, and the lack of high-fidelity of sawbones. Recently, virtual reality (VR) simulators have provided trainees with the opportunity to practice and overcome the learning curve in a high-fidelity, safe and controlled environment without compromising patient safety –13. But One solution to supplement surgical training has been the use of videos which have become increasingly popular because of their low cost and easy access. Existing video systems all use a single point of view (POV) with 2D or 3D rendering –23 whereThe challenge in surgery teaching resides in teaching both knowledge and spatial perception and gesture transmission. There by, to assess a 3D information such the orientation of a tool in space versus anatomical references, single POV video might reduce the exposure of the student to essential information.Knowledge acquisition of the operative steps with a questionnaireReal-time technical and objective performance metrics achieved after completing the learning session with a GRS score marked by two expert orthopaedic surgeonsAssessing the precision of pin positioningThe objective was to develop a novel multiple POV video system and to evaluate the training outcome of multiple versus traditional single POV. The following three outcomes were observed:The null hypothesis was observing no difference in technical skill and knowledge acquisition between single (control group) and multiple POV  videos.Recording of a procedure performed on a sawbone by an expert orthopedic surgeon with clear explanations of each step,Recording of an actual hip resurfacing procedure in theatre, performed by an expert orthopedic surgeon (>20 years of experience).The following two procedures were recorded:Both videos were recorded by 14 commercial cameras  arrayed around the operating table after getting informed consent from the patient for the second video. Of these, 12 were fixed to the plastic laminar flow hood which surrounds the operating table, with one fixed to the handle of the surgical lamp and the remaining one mounted to the surgeon’s head. After review, 8 cameras considered to have captured optimal viewpoints were retained, whilst the remaining 6 were excluded. Based on the videos recorded from 8 POVs, a novel multiple-view video system was developed, which allowed the subject to switch between viewpoints. For mono-video control group, the view point was the camera attached to the light pole. No identifiable information or image of the patient was retained in the video. The individuals in this manuscript have given written informed consent (as outlined in PLOS consent form) to publish these case details.The recruitment followed the process described in January to March 2016.The study was approved by the Medical Education Ethics Committee at Imperial College and given the reference number MEEC1415-32. Written informed consent from every participant was obtained, in which they agreed on the recording of their procedures and the use of the footage for research purposes only. The individual in this manuscript has given written informed consent (as outlined in PLOS consent form) to publish these case details.Inclusion criteria included undergraduate year 3–6 student doctors naive to both hip resurfacing and related simulations.Exclusion criteria included previous exposure to hip resurfacing or related simulations.View a multiple POV (resp. single POV) video on hip resurfacing procedure composed with a tutorial video captured with several cameras (resp. a front camera) for the simulated sawbone and with a real hip resurfacing procedure as captured a group of cameras  .Watch the video seen by the opposite group before answering a subjective questionnaire to compare their preferences.Participants initially completed a multiple-choice test  which teThe sawbone models were then CT scanned to evaluate the drilling accuracy within the femoral head. The CT data  was obtained and imported into Mimics  software for 3D reconstruction. On each 3D model the actual drilling trajectories were extracted and compared to an ideal virtual drilling path set by the operating surgeon in the video. The difference in the distance (mm) of points of pin insertion into the femoral head, and the axis angle deviation between the actual drilled trajectory and the ideal virtual drilling path were then measured.Both single and multiple video systems included identical verbal commentary about the surgical procedure, including how to achieve an ideal drilling trajectory in the femoral head as demonstrated by the expert surgeon.2 expert surgeons experienced in hip resurfacing surgery evaluated the participants’ performance using the modified global rating scale (GRS) , which wPrimary outcome was the objective performance metrics of the deviation between participants’ trajectory and ideal virtual trajectory of pin insertion into the femoral head. Secondary outcomes were (i) the GRS scores, (ii) knowledge test scores and (iii) questionnaire scores.Based on a previous pilot study (n = 5) modelled on non-inferiority/superiority trials, a minimum number of 9 participants were required in each cohort . In the absence of a figure quoted in current literature, our pilot study suggested that a difference of 30% in the objective test scoring can be construed as being clinically significant, which was agreed by 3 expert orthopedic surgeons.All data were analyzed using SPSS Statistics version 19 . For normally distributed data an independent t-test for dependent variables was adopted, while for data that were not normally distributed, a Mann-Whitney U test was used. Results for testing were calculated as mean (SD) [95% CI] and as median ± IQR (95% Bonett Price CI) for the Likert-scale questionnaire. Results are also displayed using box-and-whisker plots and bar charts. Statistical significance was set as p<0.05.The dataset of this study is available .30 participants were successfully randomized and completed the experiment. The median age for the participants was 22 (range 19–24) years and their median undergraduate year in medical school was year 4 (range 3–5). Subgroup analysis showed that 47% (n = 14) participants were male and 93% (n = 28) of them were right-handed.The differences in the distance between points of pin insertion into the femoral head, and the axis angle deviation between the actual drilled trajectory and the ideal virtual drilling path in both groups are shown in The GRS scores of participants’ performance in the experiment group (mean score of 26.3) significantly outperformed the control group . The 10 questions in the knowledge test were divided into 3 subcategories: (i) 4 questions regarding spatial awareness , (ii) 4 regarding operation details  and (iii) 2 regarding sequence comprehension . There was no significant difference between cohorts in the baseline test scores (for any category) before video-learning , thus enSimulation is already well-established as an effective training medium in the aviation industry , 25. In A variety of methods are available for capturing video of surgical procedures, but the view is frequently obscured by theatre personnel. In recent years several commercially available head mounted cameras have been used to accurately record surgeons’ view without occlusion, with early positive results –23. TherIn our study, we developed a novel multiple POV video system, which enabled trainees to visualize surgery from numerous viewpoints. Despite equivalent baseline test scores between the cohorts, the experiment group objectively and subjectively outperformed the control group, with an improved performance accuracy, better performance, post-learning test scores and attitude towards the learning experience. This may be attributable to a better appreciation of the technical intricacies of surgery in a 3D setting. Moreover, multiple POVs avoid loss of visualization secondary to camera occlusions. However, in terms of sub-category scores in the knowledge test post-video learning, there was no difference in sequence understanding scores between the two groups. This suggests that both video systems provided adequate information to learn the sequence of operative steps. When asked to perform part of the procedure using a sawbone simulation, the significantly lower angular deviation observed in the experiment group is an objective demonstration that the benefit of multiple POV translates into improved surgical performance, with a higher procedural accuracy. So, the null hypothesis was rejected, and with the exception of sequence comprehension and distance from optimal pin entry site, the multiple POV was found to be superior to the single POV video system.A hip resurfacing procedure was chosen for our study for the following two reasons: (i) it is a relatively new procedure of which student or junior doctors are unlikely to have any former experience or knowledge, thus avoiding bias; and (ii) precise pin positioning in the femoral head (a key element to the success of the operation) requires visuospatial awareness and an appreciation of 3D planes from multiple angles.Despite the advantages of the novel multiple POV, learning to operate using such a system cannot replace the clinical acumen gained from actual hands-on training. Nevertheless, our video system can act as a valuable adjunct to reinforce and enhance the learning experience. Use of this multiple POV video system may help to improve confidence and reduce anxiety before medical students and trainee surgeons move on to apprenticeship-based training in the operating room.Whether the advantage of this multiple POV will be transferred into other surgical specialties still needs to be evaluated and is worth to be investigated in future studies.Firstly, the multiple POV system appeared to stimulate more interactivity by the candidates in terms of button pressing, which may possibly encourage the participants to watch more footage of the operation than with the single POV. This could have contributed towards the higher scores in the multiple POV group. Secondly, as proposed by the participants, the multiple POV video system does not permit interaction between trainees and instructors and there was a lack of zoom function. Additionally, the Likert-scale questionnaire and the knowledge test had not been previously validated, albeit no validated alternatives exist within the medical literature. However, they were validated by four experienced orthopedic surgeons. Finally, the correlation between objective assessment of visuospatial awareness and precision of femoral head pin positioning was not included in our study, which will be conducted in the due course.The benefit of learning surgical procedures using a multiple viewpoint over traditional single viewpoint video has been confirmed in our study. With advances in technology, the next step of our study is to immerse the trainee into a 3D reconstructed theatre setting in virtual world using the Oculus Rift VR headset . This virtual world will allow the trainee to observe an operation from any viewpoint with 3D perception.We developed a novel multiple POV video system to enhance the learning experience of orthopedic operative steps. The multiple POV provided significant objective and subjective advantages over single POV. Whereas multiple POV video cannot replace the experience of learning from actual surgery, it did enhance and reinforce the technical skills and knowledge to improve confidence, competence and performance metrics.S1 FileThis questionnaire evaluates the knowledge of a hip resurfacing procedure.(DOCX)Click here for additional data file.S2 FileThis questionnaire evaluates the surgical video system.(DOCX)Click here for additional data file.S3 FileThis questionnaire allows expert to evaluate the participants’ performance using the modified global rating scale, which was designed using a modified Delphi technique, after watching their performance recorded in the video.(DOCX)Click here for additional data file.S4 FileThis file contains the dataset of the study.(DOCX)Click here for additional data file.


Major categorical disagreements with right heart catheterization were significantly reduced with motion magnification (12%) when compared to unamplified video (25%) or the bedside exam (27%). This novel method of assessing jugular venous pressure improves the accuracy of the clinical exam and may enable accurate remote monitoring of heart failure patients with minimal patient risk.Current remote monitoring devices for heart failure have been shown to reduce hospitalizations but are invasive and costly; accurate non-invasive options remain limited. The EuleriAn Video Magnification ApPLications In Heart Failure StudY (AMPLIFY) pilot aimed to evaluate the accuracy of a novel noninvasive method that uses Eulerian video magnification. Video recordings were performed on the neck veins of 50 patients who were scheduled for right heart catheterization at the Palo Alto VA Medical Center. The recorded jugular venous pulsations were then enhanced by applying Eulerian phase-based motion magnification. Assessment of jugular venous pressure was compared across three categories: (1) physicians who performed bedside exams, (2) physicians who reviewed both the amplified and unamplified videos, and (3) direct invasive measurement of right atrial pressure from right heart catheterization. Motion magnification reduced inaccuracy of the clinician assessment of central venous pressure compared to the gold standard of right heart catheterization (mean discrepancy of −0.80 cm H A critical step to reducing heart failure hospitalizations is identifying which patients will imminently decompensate by evaluating their volume status. While physicians have traditionally relied on the bedside exam of the jugular venous pressure (JVP) to assess volume status, this can be impractical as a primary method for monitoring outpatients. With the recent explosion of telemedicine, the majority of healthcare institutions in the United States employ some form of virtual interaction, paving the way for monitoring tools that can tap this potential.Much of this economic burden is generated by hospitalizations for heart failure, which represent up to 80% of direct costs, making heart failure readmissions a key target for cost reduction.7 While devices such as the CardioMEMS monitor have been demonstrated to reduce heart failure readmissions, they may not be scalable: they are invasively implanted, costly upfront , and demand additional personnel and resources to handle the datastream.8 Furthermore, their cost-effectiveness remains debatable.12Invasive ambulatory hemodynamic monitors have shown promise as early-warning systems in heart failure. A variety of devices have been developed and studied, including right ventricular (COMPASS-HF and REDUCE HF), left atrial (HOMEOSTASIS and LAPTOP-HF), and pulmonary artery (CHAMPION) pressure measurement systems.13 We describe the application of Eulerian video magnification to the jugular venous pulse examination and demonstrate its potential as a novel method of noninvasive monitoring of right-sided filling pressures.In this study, we propose a noninvasive and easily scalable alternative to current invasive remote pressure monitoring systems by combining the bedside examination with modern image processing techniques. Eulerian video magnification is an image processing method by which visually imperceptible periodic motions can be deconstructed and amplified into movements discernible to the naked eye.Participant characteristics are presented in Table 2, respectively. The average right atrial pressure (RAP) by right heart catheterization was 8.56 cm H2O (IQR 5–10). Similarly, average JVP was 7.11 cm H2O (IQR 5–8) when measured at bedside, 7.8 cm H2O (IQR 6.2–8.4) when measured from unamplified video, and 8.8 cm H2O (IQR 7.2–10) when measured from motion-amplified video.A summary of invasive and noninvasive data are reported in Table 2O lower than corresponding right heart catheterization measurements . This discrepancy narrowed when measured via unamplified video . After motion magnification, this discrepancy was no longer significant .All noninvasive modalities yielded JVPs lower than the corresponding invasive right atrial pressures, though the magnitude varied depending on the modality Table . When me2O, “borderline” if greater than 7 but less than or equal to 10 cm H2O, or “elevated” if greater than 10 cm H2O. “Agreement” with right heart catheterization was achieved if both categories were the same ; partial disagreement was noted if there was a one-category discrepancy ; and complete disagreement was noted if there was a two-category discrepancy . These pooled data are presented in Fig. p = 0.44 and p = 0.10, respectively; Table p = 0.0034) than the other noninvasive modalities.To better understand the practical clinical utility of the video magnification, we compared the noninvasive to the invasive measurements by characterizing them qualitatively. Each invasive and noninvasive measurement was designated “normal” if it was less than or equal to 7 cm H15 Our data confirm this tendency. Furthermore, our data suggest that the bedside exam is inaccurate at characterizing right-sided filling pressures: 27% of characterizations made at the bedside by two independent cardiologists were completely erroneous, which may reflect the high proportion of obesity in this study. Despite the challenging population, motion magnification resulted in less central venous pressure discrepancy with right heart catheterization as compared to bedside or unamplified video assessments. Perhaps more relevantly, it reduced significant categorical disagreements with right heart catheterization compared to the other noninvasive modalities.The AMPLIFY pilot study suggests that computerized motion amplification can improve the accuracy of clinical bedside JVP measurement. Historically, studies of the bedside exam have reported variable accuracy, and some suggest that it tends to underestimate the central venous or right atrial pressure.13 or localizing and labeling major blood vessels during surgeries in real time.16 This method is the first published application of Eulerian phase-based motion amplification to heart failure diagnostics.Since its first description in 2012, Eulerian phase-based motion amplification has been re-tooled for a variety of purposes, such as measuring heart rate by amplifying the imperceptible color shifts of the skin with each pulse17 With that in mind, we designed AMPLIFY to emulate the telehealth setting: all video assessments were completed by cardiologists who never made physical contact with study participants. Unlike current invasive monitoring tools, which generate overwhelming amounts of data that do not integrate into existing electronic health record systems, the on-demand nature of motion-amplified monitoring should yield a more manageable datastream, generated at clinically important time points. Finally, as a software-based technique, motion amplification has great potential for scalability as it requires no specialized equipment beyond what is already available in many telehealth ecosystems. Given our use of commercially available video equipment, we speculate that this technology could also be eventually adapted for use with video equipment available on most modern mobile phones.Perhaps most importantly, the AMPLIFY ambulatory heart failure monitoring strategy has the potential to expand modern telehealth capabilities. Telehealth adoption has been estimated to exceed 60% across all U.S. healthcare institutions, and virtual interactions  already outnumber in-person visits in some healthcare systems.AMPLIFY has several limitations. As a pilot study, the cohort is small and derived from a predominantly male Veterans Affairs population, and the results should not be generalized beyond the scope of the study. AMPLIFY was intentionally conducted on an outpatient population to emulate the telehealth setting and therefore does not apply to inpatient monitoring. The study was not designed to examine clinical outcomes such as heart failure decompensations or cost-effectiveness. From a technical standpoint, the method itself is reliant on detecting changes in local contrast and may have theoretical limitations when applied to patients with darker skin tones, though our study did capture a variety of skin tones. Finally, the clinical assessment of JVP in general can be inaccurate when it is very high ; as such, we do not consider it a quantitative technique in its current form.The next steps will be to refine, automate, and implement this novel technique in the telemedicine setting to compare it with modern invasive monitoring systems on a larger scale. Visual amplification techniques are heavily reliant on image contrast, and improvements to acquisition technique, such as using off-camera point light sources to improve local contrast and signal-to-noise yield, may improve accuracy. This may be especially relevant to patients with darker skin tones and naturally lower tonal contrast. Furthermore, standardizing acquisition position, scale, and distance from the patient, as well as identification of landmarks from which JVP is clinically measured  are potential targets for optimization. In the future, advances in mobile hardware capabilities may improve clinical usability by allowing for real-time visualization of the jugular venous pulse . Finally, automation of the measurement process using computer vision techniques could further decrease the need for human supervision and improve cost-efficiency.In conclusion, AMPLIFY serves as a pilot to demonstrate the potential utility and scalability of motion magnification for providing clinically relevant remote monitoring of patients with heart failure.The AMPLIFY study was a pilot clinical trial evaluating the efficacy of using video image processing as a novel method for measuring JVP. This study was approved by both the Stanford Institutional Review Board and the Veterans Affairs Research and Development Board. Written informed consent was obtained from each participant.Eligible participants were screened through the VA electronic medical record system. Patients were eligible for inclusion if they were over 18 years of age and were scheduled to undergo right heart catheterization for any indication at the Palo Alto Veterans Affairs Hospital. Exclusion criteria included existing right internal jugular vascular access, ventilatory support or airway/cervical deformity interfering with neck visualization, inability to tolerate lying at a 30 or 45-degree angle, or inability to lie still for the duration of the video recording.The AMPLIFY protocol is outlined in Fig. Using commercially available video and lighting equipment, two 60-s video clips of the right side of the participant’s neck were acquired: one with the participant lying on a standard wedge pillow at a 45° angle, and the second while sitting at a 90° angle. During all video acquisition, the patient was monitored with a continuous three-lead electrocardiogram  with a commercially available portable ECG machine . The acquisition system consisted of a commercial digital camera connected to a laptop PC. The camera system consisted of a Basler aCA1920-155uc  with a Kowa LM25HC 1″ 25 mm F1.4 lens. A battery-powered LED light source was mounted above the camera facing the same direction to provide illumination. A laptop computer running custom software streamed uncompressed, 1920 × 1280, 8-bit RGB at 155 frames per second to a solid-state drive. The acquisition system was stabilized on a tripod cart to reduce artifact from translational and rotational vibrations.Two cardiologists independently performed bedside examinations of each participant’s JVP and recorded their measurements prior to right heart catheterization. Clinicians qualitatively assessed venous pressure by estimating the orthogonal height of the JVP column, which appears as a subtle, transient movement of the skin. Copies of the pre-catheterization ECG and the participant’s most recent echocardiogram report within the last year were collected. Upon completion of right heart catheterization, copies of all pressure tracings were obtained, from which pressure measurements were extracted by an independent cardiologist.The de-identified video recordings were then sent to Google LLC for image processing. The video streams were processed as shown in Fig. 13 and Wadhwa et al.18 The basic principle of the approach is to decompose a standard video sequence into a time series of color values at a given pixel. This time series is then filtered for a prespecified band of temporal frequencies ; the resultant signal is then amplified by a specified magnification factor and reconstituted back into video format. Our code was built off the publicly available MATLAB code; signal processing was performed by commercially available personal computers. A sample video is included in the supplementary materials  and anthropometric  data were collected from each participant during the study visit. Participants’ body mass indices were calculated from the height and weight and categorized using the CDC definitions for underweight (<18.5), normal weight (18.6–24.9), overweight (25–29.9), and obese (≥30).Each bedside cardiologist’s assessment included an estimation of the JVP at bedside (reported in centimeters of water) and the angle at which it was assessed  right heart catheterization, (2) bedside assessment, and (3) video assessment.2O, greater than 7 but less than or equal to 10 cm H2O, or greater than 10 cm H2O, respectively.22 Mean differences and 95% CI of the mean difference was calculated between each of the averaged JVPs and RHC right atrial pressure. All analyses were conducted using SAS 9.3 .JVP assessments from the noninvasive techniques and RHC measurements were descriptively summarized for all participants who completed the study. The average of the bedside JVP assessments was calculated for each participant to obtain an averaged JVP to compare against the RHC RAP. The average JVP assessed by video (unamplified and amplified) was also compared against the RHC RAP. To translate the measurements to clinically actionable categories, we converted them to “normal,” “borderline,” or “elevated” if they were less than or equal to 7 cm HFurther information on research design is available in the Reporting Summary ChecklistSupplementary Information.Supplemental Video 1


The role of microglial-mediated sustained neuroinflammation in the onset and progression of Parkinson’s disease (PD) is well established, but the mechanisms contributing to microglial activation remain unclear. LincRNA-p21, a well studied long intergenic noncoding RNA (lincRNA), plays pivotal roles in diverse biological processes and diseases. Its role in microglial activation and inflammation-induced neurotoxicity, however, has not yet been fully elucidated. Here, we report that lincRNA-p21 promotes microglial activation through a p53-dependent transcriptional pathway. We further demonstrate that lincRNA-p21 competitively binds to the miR-181 family and induces microglial activation through the miR-181/PKC-δ pathway. Moreover, PKC-δ induction further increases the expression of p53/lincRNA-p21 and thus forms a circuit. Taken together, our results suggest that p53/lincRNA-p21, together with miR-181/PKC-δ, form a double-negative feedback loop that facilitates sustained microglial activation and the deterioration of neurodegeneration. Microglia is believed to exacerbate the loss of dopaminergic neurons within the SN once they are persistently activated5. Thus, understanding the regulation of microglial activation is critical for comprehending the extant inflammatory response during the onset and progression PD.Parkinson’s disease (PD) is the second most common neurodegenerative disorder, with evolving layers of complexity. The underlying molecular mechanisms of PD are still poorly understood. In recent years, neuroinflammation, essentially mediated by chronic and sustained activated microglia, has been receiving particular attention6 and are associated with diverse physiological and pathological responses7. Significant insight has been gained regarding their potential importance in the immune system9. For example, in a recent report, lncRNA GAS5 has been reported to suppress microglial M2 polarization by inhibiting the transcription of TRF4, a key factor controlling M2 macrophage polarization10. Although numerous lncRNAs are induced in innate immune cells, many of them remains functionally uncharacterized.LncRNAs are considered to serve as a cryptic, but critical layer in the genetic regulatory code11. Specifically, a new hypothesis, namely competing endogenous RNAs (ceRNAs), has been proposed for the layer of gene regulation mediated by RNA transcripts with shared miRNA binding sites (MREs)12. Based on this hypothesis, lncRNAs are believed to fine tune gene expression through this new “RNA language”. Increasing evidence indicates that disruption of the equilibrium of ceRNA networks can be critical for various diseases and developmental stages13. As one of the ceRNA protagonists, microRNAs (miRNAs) are also well known key modifiers for fine tuning key genetic pathways in microglial polarization and function15. Therefore, we propose that some lncRNAs may interact with miRNAs and act as ceRNAs in the post-transcriptional network in microglia.Four archetypes of molecular mechanisms have been proposed to illustrate the myriad functions of lncRNAs-signals, decoys, guides, and scaffolds, in which lncRNAs interact with DNA or RNA through nucleic-acid base pairing or with proteins through modular domains16. However, the functions and mechanisms utilized by lincRNA-p21 during microglial activation and the potential impact of lincRNA-p21 on the inflammatory component of PD have not as yet been fully elucidated. In this study, we thus focused on the role of the lincRNA-p21-mediated ceRNA network in microglial activation and inflammatory responses in PD.LincRNA-p21 was previously identified as a p53-inducible lncRNA and functions as a component of p53-dependent transcriptional responsesWe began by assessing lincRNA-p21 levels in LPS-treated BV2 microglia cells. LPS treatment induced lincRNA-p21 levels in a time-and dose-dependent manner Fig. . Increas18 have demonstrated that p53 mediates microglial activation and triggers microglial-induced neurotoxicity in many neurodegenerative diseases. Indeed, we found that exposure of BV2 microglia cultures to LPS led to significantly increased p53 protein expression  suggested that lincRNA-p21 presented a putative binding site for the miR-181 family (miR-181a/b/c/d)  Fig. . Importamut Fig. . We alsomut Fig. , while amut Fig.  in respoTo further validate the direct binding between miR-181 family and lincRNA-p21 at endogenous levels, we performed an anti-Ago2 RNA immunoprecipitation (RIP) in BV2 microglia cells extracts. As expected, we found that both miR-181a/b/c/d and lincRNA-p21 were specifically enriched in the Ago2 group Fig. . MoreoveWe next validated the role of miR-181 family in lincRNA-p21-induced microglial activation. As shown in Fig. Next, we employed rescue experiments by transiently co-transfecting miR-181a/b/c/d mimics and lincRNA-p21-overexpressing vector into BV2 microglia cells, respectively. Phenotype analysis showed that all the miR-181 members prevented the activation of microglia cells resulting from the overexpression of lincRNA-p21, with a reduction of iNOS, NO and ROS Fig. . These d24. Indeed, dual-luciferase assays showed that all the miR-181 family members could greatly reduce the luciferase activities of Luc-PKC-δ-wt but not Luc-PKC-δ-mut , the microglial pro-inflammatory response to neuron damage can drive the progressive inflammation and cumulative loss of neurons over time. Thus, knowledge of how they are maintained in the sustained activated state and associated continuous neuron damage is of great importance. Recently, studies have yielded important insights into the regulation of inflammation by non-coding RNAs, such as lincRNAs and miRNAs32. However, such lincRNA and miRNA interactions mediating the key molecular signaling events in microglial activation have not yet been clearly defined. Here, we report that lincRNA-p21 functions as a ceRNA to promote microglial activation and exerts dopaminergic neurotoxicity in both cell culture and murine models of PD.Increasing evidence has linked chronic inflammation mediated by activated microglial cells to a number of neurodegenerative disorders including PD16. In this study, we focused on the novel role of lincRNA-p21 in promoting inflammation-induced neurotoxicity, which has not yet been clearly illustrated. Indeed, inhibition of endogenous lincRNA-p21 effectively blocked LPS-induced microglial activation (M(LPS) state33), while exotic expression of lincRNA-p21 further facilitated microglial activation, skewed the microglia cells toward a pro-inflammatory phenotype, and thus increased the expression of inflammatory mediators and accelerated the death of dopaminergic neurons. Notably, earlier report34 demonstrated that methotrexate reduced inflammatory responses in rheumatoid arthritis (RA) patients through the induction of lincRNA-p21. We inferred that this discrepancy could be attributed to the different stimulators and specific disease context used in the two studies36.lincRNA-p21 is well known to be induced by p53 and exerts diverse functions in multiple diseases37, as observed for the expression pattern of lincRNA-p21 in the acute MPTP mouse model of PD, indicating a potent critical role for lincRNA-p21 in mediating the microglial activation response and aggravating TH + neuron loss in PD. In agreement with our in vitro data, we found that ablation of lincRNA-p21 limited inflammatory responses in the SN and protected TH + neurons from MPTP-induced toxicity. Thus, strategies to suppress the expression of neurotoxins by interfering with lincRNA-p21 may have therapeutic utility.Interestingly, a previous study has demonstrated that the maximum microglia activation response occurred 24–48 h after the last injection of MPTP in the mouse SN38. Thus, we propose that this lincRNA-p21-mediated regulatory loop facilitates the conversion of microglia from a steady state to a pro-inflammatory state, eliciting rapid response to the changes in their surroundings in the CNS.The present studies demonstrate a potent pro-inflammatory activity of lincRNA-p21 in microglia. We propose that this pro-inflammatory activity is mediated by a p53/lincRNA-p21/miR-181 family/PKC-δ dual-negative feedback loop that further augments this effect Fig. . It is k39, apoptosis16, and somatic cell reprogramming40. Of interest, we also showed that lincRNA-p21 was functionally dependent on p53 during the regulation of microglial activation, consistent with the important role of p53 in the regulation of chronic inflammation41. Moreover, considering the association between PKC-δ and p53, we wondered whether PKC-δ could, in turn, affect the expression of p53/lincRNA-p21 in turn. Notably, we showed that PKC-δ induction could further increase the expression of p53 and lincRNA-p21 in microglia cells, thus ensuing LPS-induced p53/lincRNA-p21 expression. Therefore, we conclude that lincRNA-p21, together with miR-181 family and PKC-δ, forms a regulatory circuit consisting of autoregulatory and dual-negative feedback loops during the progression of microglial activation. Collectively, our data identify a novel model of lincRNA-miRNA interactions during microglia activation. Considering the multiple targets of miRNAs, we also hypothesize that there may be many other lincRNAs that function as ceRNAs to regulate the expression of key genes in microglia cells42. Thus, the identification of these ceRNAs may improve our understanding of immune and inflammatory alterations and many related diseases, such as PD.Mechanistically, we provide evidences that lincRNA-p21 serves as an endogenous miRNA sponge for the miR-181 family, indirectly upregulating PKC-δ, and then inducing microglial activation during inflammation. In our in vitro system and in vivo models of neuroinflammation, we observed that overexpression of lincRNA-p21 was sufficient to increase PKC-δ and induce microglial activation. Remarkably, this role depends on the competitive binding of miR-181 family, indicating that lincRNA-p21 functions as a ceRNA. Reduction of PKC-δ or ectopic expression of all the miR-181 family members severely compromises the pro-inflammatory activity of lincRNA-p21. It is widely recognized that lincRNA-p21 tends to act as a downstream effector of p53-dependent transcriptional pathways, regulating cell proliferationBased on our data, we propose a model depicting a pivotal role for lincRNA-p21 in the regulation of inflammation-induced neurotoxicity. We identify a regulatory circuit, composed of p53, lincRNA-p21, the miR-181 family and PKC-δ, which mediates a regulatory signal that is critical for eliciting innate immune responses in microglia. Considering that most of our findings were obtained using a single BV2 microglia cell line, further validation in primary microglia cells and other disease models is urgently needed in our future work. These results of such analyses might lead to the development of potential therapeutic agents for the treatment of neuroinflammatory diseases in the central nervous system.Escherichia coli, serotype 026:B6; Sigma-Aldrich, St. Louis, MO), MPTP , lipoteichoic acid , PamC3sk4 (R&D Systems) and mouse interferon-gamma  were used in this study. pcDNA3.1-licRNA-p21 and PKC delta WT (Addgene plasmid # 16386)43 were kind gifts from Nadya Dimitrova and Bernard Weinstein, respectively.DMEM, DMEM/F12, fetal bovine serum (FBS), penicillin, streptomycin and other cell culture reagents were purchased from Invitrogen . LPS  supplemented with 10% heat-inactivated FBS , penicillin (100 IU/ml) and streptomycin (100 mg/ml) at 37 °C in a humidified incubator with 5% CO2. FBS was reduced to 5% when the experiments were performed. LPS (1 μg/ml), LTA (100 µg/ml), PamC3sk4 (100 ng/ml), and IFN-γ (10 ng/ml) were used in this study.44. A CM transfer system was used to determine the impact of LPS-treated BV2 cells on SH-SY5Y cells45. CMs from control and lincRNA-p21-overexpressed BV2 cells were filtered through 0.45-μm filters and frozen at −80 °C. SH-SY5Y cells were cultured with CMs (diluted with fresh medium at a 1:1 ratio) for 24 h and apoptosis assays were analyzed by flow cytometry  using an Annexin V-FITC and propidium iodide (PI) kit .SH-SY5Y cells were obtained from the Central Laboratory of Nanfang Hospital . Cells were maintained in DMEM/F12  supplemented with 10% FBS  as described46. Briefly, the purified cells were incubated with 5 μl of anti-mouse Fc receptor block antibody  for 15 min on ice. The cells were then surface-stained with fluorescein isothiocyanateconjugated anti-CD11b-APC-eflour780, anti-CD45-PE-Cyanine7, anti-MHC class II(I-A/I-E)-PE, anti-CD86(B7-2)-PE-Cyanine5, and anti-CD206(MMR)-APC  for 20 min on ice in the dark. Data were acquired with an LSRII cytometer  and analyzed with the FlowJo software program . Positive and negative gates were drawn based on the intensity of an unstained control sample.Surface antigens changes in primary mouse microglia were assayed by flow cytometry, as described2), a stable end product resulting from the reaction of NO with molecular oxygen, was measured in supernatants collected from treated and untreated microglia using a Griess reagent system  according to the manufacturer’s instructions. A synergy Mx fluorescence plate reader (Bio-Tek Instruments) was used to detect the absorbance at 550 nm. The concentration of nitrite was calculated from a standard curve using sodium nitrite solution.The expression of nitrite  and washed three times with PBS. ROS were detected immediately by flow cytometry  using the FL1 green fluorescence channel. Data was collected as the mean fluorescence intensity and analyzed using the FlowJo software program .The levels of intracellular ROS was determined using DCFH-DA dye  as described previously−(ΔΔCt) method48. U6 snRNA premier set was purchased from RiboBio . Other primer information provided in Supplementary Table Total RNA was extracted from cells and mouse brain tissue samples using RNAiso Plus . A NanoDrop ND-2000 Spectrophotometer  was used to quantify the RNA concentration. Stem-loop qRT-PCR for mature miRNA expression was performed. For assessments of mRNA expression, 1 μg total RNA was used to synthesize the complementary DNA using a PrimeScript™ RT reagent kit with gDNA Eraser . cDNA products were then diluted 1:10 in ddH2O. qRT-PCR was performed using SYBR® Premix Ex Taq™ II and a CFX96 real-time PCR system . U6 snRNA (for miRNAs) or β-actin expression (for mRNAs) were used as control for normalization and the relative gene expression levels were calculated using the 249. Briefly, equal amounts of the proteins were loaded on 10% or 15% SDS-PAGE gels for separation under reducing conditions, followed by blotting of the protein on a PVDF membrane  in a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Membranes were then blocked with 5% non-fat dry milk in TBS with 0.1% Tween and probed with appropriate primary antibodies against PKC-δ , iNOS , p53 , Ago2 , histone H3 , and GAPDH  overnight at 4 °C. HRP-conjugated secondary antibodies (goat anti mouse/rabbit IgG 1:5000) were used for antibody detection with the immobilon ECL kit (Merck Millipore) in a ChemiDoc XRS + system (Bio-Rad) and band intensities were quantified using ImageJ software 50. Full blots are presented in Supplementary Fig Brain tissues and microglial cell lysates were prepared in standard RIPA buffer  containing 1 × protease and 1 × phosphatase inhibitor cocktails . Western blotting was carried out as previously described51. The efficiency of the nuclear and cytoplasmic fractions was determined by comparing the abundance of nuclear RNA (U2 snRNA) or cytoplasmic mRNA (18S rRNA) using qRT-PCR. It was also assessed by quantifying the known cytoplasmic protein GAPDH and nuclear protein histone H3.To determine the subcellular distributions of lincRNA-p21, miR-181 family and Ago2, we performed subcellular fractionation using a PARIS kit , according to the manufacturer’s protocols. After adding an exogenous spike-in of cel-mir-39  to each sample as a normalizer for the qPCR analysis, RNA was extracted using RNAiso Plus , and equal volumes of ddH2O were used to resuspend the resulting RNA pellets to obtain cell-equivalent concentrationsThe sequence from wild-type or mutants 3′-UTR of PRKCD and lincRNA-p21 containing the common miR-181 family binding site were synthesized and cloned downstream of the luciferase reporter gene into the pmirGLO vector  using the SacI and SalI restriction enzymes. For mutated 3′-UTRs, six or eight bases of each seed region were replaced with complementary bases. For the luciferase reporter assays, HEK293T cells were co-transfected with the indicated luciferase constructs with synthetic miRNA mimics , and luciferase activity was measured using a dual reporter luciferase assay kit , according to the manufacturer’s protocol. Renilla luciferase activity was normalized to the corresponding firefly luciferase activity and plotted as a percentage of the controls.52. Briefly, BV2 cells were harvested and lysed with the buffer provided in the kits and sonicated six times for 30 s to facilitate lysis. Immunoprecipitation was performed using anti-Ago2 or control IgG (negative control) and snRNP70 (positive control). The RNA fraction isolated was then treated with RNAiso Plus  for further purification and qRT-PCR analysis. Total RNAs from cell lysate (Input) were detected simultaneously as controls.For the anti-Ago2 RIP experiments, a EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit  and mouse anti-Ago2 antibody  were used according to the manufacturer’s instructions53 with modifications. In brief, the DNA fragment covering the entire mouse lincRNA-p21 sequence was PCR-amplified from vector pcDNA3.1-lincRNA-p21 using T7-containing primers  and then cloned into pGEM-T . After linearization with the restriction enzyme NotI, the full-length of mouse lincRNA-p21 was labeled with biotin using Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), then treated with RNase-free DNase I (Roche), and purified with a RNeasy Mini Kit . The probes were then incubated with streptavidin-coated magnetic beads  at 4 °C over-night to generate probe-coated magnetic beads. Next, 1 mg whole-cell lysates from BV2 cells were incubated with 3 μg of probe-coated beads for 1 h at room temperature, and the isolated complexes were used either for extraction of the RNA for qRT-PCR or for western blotting according to standard procedures.The RNA pull-down assay was performed as previously describedsiRNA oligos targeting mouse lincRNA-p21 , mouse p53 (#1 AGAAGAAAAUUUCCGCAAATT), mouse PKC-δ  and negative control (NC) siRNA (UUCUCCGAACGUGUCACGU) were synthesized by GenePharma biotechnology . LncRNA Smart Silencer, synthesized by RiboBio , was also used to knock down the expression of lincRNA-p21. MiR-181a, b, c, d mimics, inhibitors and negative controls  were purchased from RiboBio . These oligonucleotides were all transfected into BV2 cells using Lipofectamine 2000 (Invitrogen) at a final concentration of 100 nM. For overexpression, pcDNA3.1-licRNA-p21 and PKC delta WT vector were transfected into BV2 cells using Lipofectamine 2000, according to the manufacturer’s instructions.TM adenovirus purification kit . All the adenoviruses were tagged with HA epitope.The lincRNA-p21 related adenoviruses were constructed using the Adeno-XTM expression system (Clontech). Briefly, full length of mouse lincRNA-p21 was subcloned into adenoviral shuttle vector pDC315  to generate pDC315-lincRNA-p21 (Ad-lincRNA-p21). LincRNA-p21 shRNA harboring lincRNA-p21 siRNA #1 (Ad-lincRNA-p21-shRNA) was designed, synthesized and inserted into pDC315-U6-shRNA. Adenoviruses were then produced by co-transfecting HEK293T cells with each adenoviral construct together with the packaging vectors pBHGloxdelE13cre  using Lipofectamine 2000 (Life Technologies), according to the instructions of the manufacturer. Ad-lincRNA-p21, Ad-lincRNA-p21-shRNA and Ad-control (used as control) were then amplified and purified using the ViraTrap25 was used for the neuroinflammation and neuroprotection studies because the kinetics of microglial neuroinflammation is well characterized in this model. The mice from the MPTP treatment group received 4 i.p. injections of MPTP-HCl (18 mg/kg free base) at 2-h intervals and control mice received equivalent saline injections. The mice were killed at indicated time points: 0 (immediately after the last MPTP injection), 1, 2, 4, and 7 days after the last MPTP injection. The nigral neuroinflammatory response was also studied using the systemic LPS injection model, which was used to induce chronic neuroinflammation and delayed dopaminergic degeneration in mice54. The mice received a single injection of LPS  or equivalent injections of saline. The mice were killed 6 h later.C57BL/6 mice  were purchased from the Laboratory Animal Centre of Southern Medical University. Animal care and procedures were carried out with the approval of the Southern Medical University Ethics Committee and strictly complied with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Two animal models were used. The well-characterized acute MPTP mouse model of PD10 IFU/μl per construct) and micrON® miRNA agomir  from RiboBio were stereotaxically delivered into the right substantia nigra region  as previously reported26. One week later, the mice were treated with either saline or MPTP/LPS as described above. All mice were killed at the indicated time points.Stereotaxic surgery was carried out with a stereotaxic frame  and a 5 µl Hamilton syringe fitted with a pulled glass capillary tube. After anesthetization, the mice were head immobilized in a flat-skull position for stereotaxic surgery. Adenoviruses  were collected using a freezing microtome (Leica) and mounted on PDL-coated slides. Sections were incubated with the indicated primary antibodies. For Iba-1 immunostaining, sections were probed with rabbit anti-Iba1  over-night at 4 °C and then incubated with Cy3-conjugated goat anti-rabbit IgG . To analyze the interplay between Iba-1 and TH cells, double-labeling immunofluorescence was performed. Brain sections were incubated with mouse anti-TH  and rabbit anti-Iba1  followed by an incubation with goat anti-mouse IgG conjugated to Alexa Fluor 488  and Cy3-conjugated goat anti-rabbit IgG , respectively. Mouse anti-HA  was used to determine the efficacy of lincRNA-p21 related adenoviruses transduction in vivo. The 4,6–diamidino-2-phenylindole  was used to identify nuclei. Fluorescence images were obtained with a Zeiss LSM 880  laser-scanning microscope and confocal images were acquired and analyzed using a ZEN lite 2012 software. For each animal, eight sections covering the entire antero-posterior ventral mesencephalon were analysed. Total numbers of TH-stained neurons and Iba-1 positive microglia in the substantia nigra region were counted using the Optical Fractionator method with Microbrightfield Stereo-Investigator software  as described previously56.Immunofluorescence was performed as described previously58. Briefly, brain tissue was gently dissociated by grinding in a 15-ml dounce homogenizer  containing DNase I  and passed through 70-μm filtration . After centrifugation, the supernatants were discarded, and the cell pellets were resuspended in 40% Percoll , followed by spining at 800 g for 30 min with discontinuous Percoll gradients (HBSS/30%/40%/70%). Microglia cells were collected at the 40%/70% interface. Cells were washed and then resuspended in FACS buffer (1X PBS with 2% FBS). Quantification of CD11b + cells with CD45, MHC class II, and CD86 was performed with a FACSCalibur flow cytometer (Becton Dickinson) using standard procedures.At 6 h after LPS treatment, microglia cells from the adenovirus injected groups were isolated from whole brain homogenates by Percoll gradient centrifugation according to previous reportst-test. Comparisons between multiple groups were performed by one-way or two-way analysis of variance ANOVA with Bonferroni post hoc tests. P < 0.05 was considered statistically significant.All data are presented as means ± standard error (SEM). Statistical analyses were performed using SPSS version 19 . Significant differences between the two groups were carried out using the two-tailed unpaired Student’s Supplementary figure legendssupplementary figure 1supplementary figure 2supplementary figure 3supplementary figure 4supplementary figure 5supplementary figure 6supplementary figure 7supplementary figure 8supplementary figure 9supplementary table 1


Background: In patients with alcohol use disorder, novel interventions to increase abstinence have attracted growing attention. Interventions aimed at modifying cognitive biases linked to alcohol use [i.e. cognitive bias modification (CBM)] may serve as an add-on to standard therapy. This systematic review thoroughly aggregates existing data on the effects of three alcohol-specific computerized interventions, namely attentional bias modification (AtBM), approach bias modification (ApBM), and inhibition training (IT). In doing so, each CBM’s effects on experimental tasks assessing the relevant biases, drinking behavior, and neurophysiology are summarized. Also, the influence of drinking behavior severity and motivation to change drinking behavior are discussed.Methods: A literature search was conducted in four databases for original research articles published between 2000 and May 2019. Studies were eligible if investigating the effects of alcohol-specific computerized interventions  on drinking behavior, bias change, and/or neurophysiology. Forty eligible articles were classified as being either a non-clinical experimental lab study (ELS) or clinical randomized-controlled trial (RCT) and summarized.Results: While AtBM seems to influence attentional bias, its effects on drinking behavior are inconsistent. As for ApBM, the best effects on drinking behavior are obtained in clinical samples. Effects of ApBM on approach bias are mixed. Interestingly, those clinical RCTs which investigated ApBM effects on bias change as well as on drinking outcome, reported consistent effects in both measures (i.e. either effects on bias and drinking or no effects). Studies on IT are limited to non-clinical samples and show inconsistent effects on drinking behavior. Considering ITs effects on implicit semantic associations, most studies do not support the conceptualization of IT as a form of memory bias modification, while reports on IT’s effects on inhibitory control are still incomplete. Conclusions about the overall influence of drinking behavior severity are hampered by the non-uniform use of sample descriptions.Conclusions: In clinical samples, ApBM has shown more consistent beneficial effects, while evidence on AtBM is more inconsistent, and data on IT still lacks important information. Conclusions about the influence of drinking behavior severity would be facilitated by a uniform use of clearly defined sample descriptions. Alcohol use disorder (AUD) is a chronic health problem with serious biological, psychological, and social consequences . DespiteCertain alterations in the cognitive processing of alcohol-specific cues have been identified as being relevant in AUD. While the automatized tendency to process, memorize, and approach alcohol-related cues , 5 is prThe first of these processes, attentional bias (AtB), describes that alcohol-specific cues rapidly attract and subsequently hold selective attention . The extMotivated by cumulative evidence of altered cognitive processes in AUD, research has tested various interventions aimed at modifying attentional bias, approach tendencies, and inhibitory control impairments. The modification of such processes is commonly referred to as cognitive bias modification (CBM). Recently, there has been greater interest in alcohol-specific computerized interventions that target altering these deficits. On the one hand, the focus on alcohol-specific interventions is justified from a clinical perspective, since drinking behavior is often triggered by alcohol stimuli . On the Several reviews and meta-analyses have covered literature on one or more of the three aforementioned alcohol-related CBMs, with some of them reaching a rather positive conclusion , 18, whiThis systematic review aims to extend and update those summaries as follows: First, the current database will be expanded by including the most recent research articles. Second, evidence will be thoroughly summarized separately for each of the computerized interventions targeting either attentional bias, approach bias, or inhibitory control processes. Conclusions are drawn for three outcomes, namely drinking behavior, experimental tasks, and neurophysiological effects, while distinguishing between non-clinical experimental lab studies and clinical randomized-controlled studies. In doing so, the potential role of motivation to change drinking behavior is acknowledged and systematically assessed. By visualizing whether an intervention affected the targeted bias and at the same time drinking behavior, light is shed on the assumption that drinking behavior change is associated with and driven by a bias change see 3.Since the predictive value of non-clinical studies for potential benefits in a clinical sample is subject to question, we will compare the different studies’ effects while considering the investigated sample’s severity of drinking behavior. On the one hand, this will be a focus in the summary of each intervention. On the other hand, this review comprises an overall insight into the link between severity of drinking behavior and training effects across all three interventions.Thorough literature searches in the databases PubMed Medline, Google Scholar, and PsycInfo were conducted in January and May 2019 to identify eligible publications papers on computerized alcohol-specific interventions for attentional and approach bias modification and inhibition training. The sample was to be alcohol-consuming, and studies were to include alcohol-specific interventions with the aim to alter the alcohol-specific biases . Both clinical and non-clinical samples were represented. The following keywords and all possible combinations thereof were entered into the search masks. Alcohol*, alcohol depend*, addict*, alcohol use disorder, harmful drinking, heavy alcohol use, heavy drink*, hazardous drink*, drink*, alcohol-specific computerized intervention, attention* bias, approach bias, approach-avoidance bias, cognitive bias modification, computerized training, inhibition, inhibitory control, and inhibition training. In order to identify additional papers, a search within Google Scholar was conducted for papers, in which already detected eligible papers for this review had been cited. Furthermore, the bibliographies of the included studies were reviewed. The literature search was restricted to original research articles including a computerized and alcohol-specific CBM intervention that had been published from 2000 through May 2019. Eligible studies met the following criteria: the publications (a) were peer-reviewed and published quantitative research studies; (b) involved computerized interventions aimed to alter either attentional or approach bias or inhibitory control; c) implemented alcohol-related stimuli in the computerized intervention; and (d) examined either the effect of interventions on experimental and neurophysiological outcomes in a longitudinal design including a pre- and post-measurement, or the effect of the intervention on drinking outcomes at post-intervention only , and e) were written in English. Studies assessing bias, drinking, or neurophysiology cross-sectionally only were excluded.Articles found in the literature search were assessed for eligibility by the first author (HB). Once all duplicates were removed, the remaining articles were screened for eligibility based on the inclusion and exclusion criteria. If inclusion was debatable, another author (MS) also assessed the paper and the case was discussed until an agreement was reached.Studies were categorized as either experimental laboratory study (ELS) if they investigated non-clinical samples, or clinical randomized-controlled trials (RCT) if they described an RCT conducted in a clinical sample containing treatment-seeking patients with AUD. For all three CBMs, this review will initially summarize effects on experimental tasks, i.e. whether the CBM intervention affected the according bias. Thereafter, effects on drinking behavior are reported, as are neurophysiological effects, whenever applicable.For all studies included in this review, the following data were extracted see Table 1:name of the first author(s) and publication yearthe sample description , the sample’s mean age and standard deviationthe type of CBM intervention: attentional bias modification (AtBM), approach bias modification (ApBM), or inhibition training (IT)the study design: non-clinical experimental lab study (ELS) or clinical randomized-controlled trial (RCT)the different groups, to which participants were allocated (including size)which task the intervention is based on the number of training sessions conductedthe outcomes, separated into experimental tasks, drinking behavior, and neuronal activity. As for experimental tasks, all assessed tasks were extracted. Drinking behavior outcomes were summarized according to how long after the intervention they were assessed.remarks on important characteristic of study (e.g. no control group)Table 1A–CDetailed overview of original research articles included in review according to CBM type , and study design/sample included [non-clinical (ELS) or clinical (RCT)]. Abbreviations are summarized below each sub-table.The quality of the included studies was assessed based on the Cochrane Collaboration’s Risk of Bias guidelines  by the fSample composition , mean age (+ standard deviation), sex ratio Number of sessions and setting (e.g. lab or web/home-based intervention)Whether motivation (regarded as moderator for CBM effectiveness) was assessed, and if so, if it was included in the analysesWhether a power analysis was conducted prior to the study onset, and whether the calculated N was achievedAdequate generation of allocation to conditions (randomization bias)Blinding procedure Attrition bias: Initial N and N for (sub-)analysesResults are summarized first for each CBM separately, before an overall summary is given, which also addresses aspects of potentially moderating variables, such as drinking problem severity and motivation to change alcohol consumption. Both a detailed summarizing table Table 1,AtBM aims to alter AtB by training to direct attention away from alcohol-specific cues . The intSome studies , 34, 41 Research investigating the effects of VPT-AtBM has reported changes in VPT-AtB in the expected direction in young adult drinkers , heavy sRegarding the effect of VPT-AtBM on alcohol consumption in experimental lab studies, two studies reported less drinking during  or immedTwo studies reported a decrease in alcohol consumption in non-clinical samples 1 week or 3 months after Stroop-AtBM, however these studies either lacked a control group  or the eGenerally, motivation to change is assumed to be higher in populations with more problematic drinking patterns. When considering clinical trials on AtBM in AUD, however, two of four studies reported that AtBM increases the time to first drink at 3-month follow-up  or reducTo the best of our knowledge, there are no studies investigating neurophysiological effects of AtBM.Considering effects on experimental tasks, two-thirds of the studies showed that AtBM influences AtB when measured with a similar task. Support for generalization to other cognitive tasks is limited, with only one-third of the studies reporting such effects. It is possible that these tasks measure different and only slightly intercorrelated incentive-motivational aspects of alcohol-related cues that remain unaffected by the intervention.Beneficial effects of AtBM on drinking behavior were only observed in the minority of experimental lab studies. In clinical RCTs, where the motivational prerequisites for the translation of bias to drinking behavior change might be given more often, the two studies observing a bias change also observed changes in drinking behavior, while the two studies that failed to change the bias reported no drinking changes. Note however, that the only study analyzing specifically whether bias change mediates changes in drinking behavior did not show such a mediation .It has been suggested that drinking behavior change should only be expected if a bias change was achieved and if participants are motivated to change their drinking behavior . ConsideRegardless of this controversy, it remains to state that in clinical trials, the intended effects on bias change or drinking behavior, respectively, could not be reliably replicated. Factors influencing this variability must be identified, experimentally examined, and optimized if AtBM is to truly help clinical populations with AUD.Alcohol approach/avoidance inclinations can be assessed with the Approach Avoidance Task (AAT), which has also been the basis for the development of an intervention targeting action tendencies . In thisApBM was shown to modify alcohol avoidance and approach bias (AvB/ApB) in the expected direction in social drinkers , hazardoRegarding generalization of effects to new tasks, two studies showed a generalization to the IAT , 55. YetWith respect to working mechanisms, there is some evidence that the effect of ApBM on ApB is moderated by strong baseline ApB , and thaExperimental lab studies conducted in non-clinical samples supposedly not motivated to change drinking behavior reported no effects on drinking during a post-intervention taste test in social , heavy  or hazarContrary to these findings in experimental lab studies, the majority of clinical RCT trials did report effects of ApBM on drinking behavior. One study in AUD showed a 21.4% lower relapse rate in the ApBM compared to the control group at 2-week follow-up , but did not report differences regarding time to relapse, mean drinking days, and mean standard drinks per drinking day . Long-tefMRI studies showed ApBM to decrease amygdala activation during a cue-reactivity task, an effect also related to decreased subjective craving . Comparivia ApB changes  this bias change as well as changes in drinking behavior could quite consistently be shown in clinical populations. However, c) effects regarding bias change as well as drinking behavior change are inconsistent in non-clinical experimental studies. Earlier reviews  the evidence speaks for the proposed working mechanism of ApBM to work ews e.g.  explaineNeurophysiological effects were reported using fMRI 47 but nTwo tasks measuring different aspects of inhibitory control , the Go-IT: Effects on Experimental Tasks, we will review whether existing data speaks in favor of one or the other hypothesis and whether the conceptual framing of IT as a form of memory bias modification is still justified. The discussion about the potential working mechanisms and the conceptual framing of IT followed mainly two lines: One assumption is that IT improves inhibitory control over a patient’s reaction to alcohol-specific cues . FollowiConsidering the mechanism of action of IT, two main hypotheses have guided the selection of experimental tasks employed in these studies. The first and more straightforward hypothesis is that IT increases inhibitory control  and shouThe effects of alcohol-specific IT on inhibitory control have been investigated in six studies: Regarding SST-based IT, one study reported a selective decrease in inhibition errors in alcohol-related stop trials when comparing the first to the last training block which might indicate an improvement of inhibitory control , while aThe effects of alcohol IT on implicit associations have been examined in seven studies. Two studies reported that GNG-based IT decreased positive implicit associations to alcohol , 58, supNo generalization was observed on automatic action tendencies  nor on aTo date, effects on alcohol-specific IT on drinking behavior have so far only been investigated in non-clinical samples, and as such, have primarily included participants who lack the motivation to change their drinking behavior. Motivation to change is discussed to be a necessary prerequisite for any CBM to affect drinking behavior . Even ifTo date, the only study investigating neurophysiological effects of IT in HD  concentrvia stimulus devaluation, at least not when assessed with the IAT  were aligned according to the mean AUDIT value Figure 4What also becomes evident in Generally, there is still limited research elucidating computerized CBM’s effects. The abundance of cross-sectional studies reporting altered cognitive processing in HD/AUD, which led to the development of computerized interventions, is contrasted by a limited number of longitudinal studies that investigate computerized interventions’ effectiveness, let alone working mechanisms or neuronal effects. We reviewed 40 papers on alcohol-specific computerized CBM interventions. In doing so, we set out to analyze all studies available to date on three alcohol-specific CBMs, namely AtBM, ApBM, and IT, and to summarize this data regarding effects on drinking behavior, experimental tasks, and neurophysiological effects. In contrast to earlier analyses of several , 73 or oSeveral limitations when comparing CBM studies and in terms of this review should be addressed. Firstly, despite having carefully searched several databases and articles, we cannot fully rule out having missed a paper that should have been included in this review. Secondly, because the non-uniform use of terminology regarding the subjects’ drinking behavior complicates conclusions with respect to severity, we strongly encourage future studies to lean on the WHO’s definitions depending on the achieved AUDIT score across the sample . FurtherFurther, regarding methodological variation, the number of sessions ranged between one and 14 in the articles reviewed, and within certain studies, participants completed different numbers of interventions. Earlier research suggests that it is unlikely for one session to have a therapeutic effect . For ApBOverall, it seems advisable for future research to concentrate on clinical populations, include the assessment of motivation and baseline bias levels, analyze mediation, and neurophysiological effects and use longer follow-up periods to augment the understanding of computerized interventions’ effects.HB conducted the literature search in January and May 2019. HB and MS equally contributed to tables, figures, and writing the initial manuscript. HB, MS, and FM elaborated conclusions. HB, MS, RT, LS, and FM contributed to revising the manuscript and finalizing it. HB and FM assessed each study for quality criteria. Whenever unclear, MS was asked for her opinion.This research was supported by a grant from the Swiss National Science Foundation (grant-nr: 105319_159286) to MS, LS and FM.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.