People with severe mental illness (SMI) face discriminatory situations because of prejudice toward them, even among health care personnel. Escape rooms can be a novel educational strategy for learning about and empathizing with SMI, thus reducing stigma among health care students.This study aimed to examine the effect of the Without Memories escape room on nursing students’ stigma against SMI.A pre- and postintervention study was conducted with a control group and an experimental group. A total of 306 students from 2 Andalusian universities participated in the study. Data were collected through a pre-post study questionnaire, consisting of an adapted version of the Attributional Style Questionnaire and a questionnaire on motivation for cooperative playful learning strategies. The control group carried out an escape room scenario without sensitizing content, whereas the experimental group carried out an escape room scenario on SMI, with both escape rooms being carried out in a 1-hour session of subjects related to mental health. To answer the research questions, a 2-way analysis of variance with repeated measures, a linear regression, and a 2-way analysis of variance were performed.P<.001) was observed in the experimental group in stigmatizing attitudes compared with the control group, in which no statistically significant changes (P>.05) were observed. In contrast, the linear regression  showed that there was an inverse relationship between flow and the level of reduced stigma. When controlling for having or not having a close relative, the intervention was also shown to be effective (P<.001) in reducing the stigma displayed, both for people with affected and unaffected relatives.After the intervention, a significant reduction (Our findings suggest that the Without Memories escape room can be used as an effective tool to educate and raise awareness about stigmatizing attitudes toward SMI in university students studying health care. Future testing of the effectiveness of educational escape rooms should be designed with new programs through playful strategies of longer duration to evaluate whether they can achieve a greater impact on motivation, acquisition of knowledge, and awareness. In addition, the feasibility of implementing the Without Memories escape room in other careers related to health and community should be investigated. Severe mental illness (SMI) refers to different nosological entities with certain common severity criteria, lasting more than 2 years, and is associated with a loss of functionality . FormallTo reduce stigma, different proposals for socioeducational programs have been made, including the dissemination of information, leisure activities such as sports or art, and contact with people with mental disorders. For example, in a meta-analysis study , throughWith the arrival of serious games and educational video games, the possibilities of electronic resources in socioeducational intervention are also beginning to be studied. For example, in the video game Stigma-Stop, which aimed at reducing stigma toward people with mental disorders, its characters had various mental disorders, with whom participants could empathize. The results of 552 students aged between 14 and 18 years showed a statistically significant decrease in the levels of stigma toward people with schizophrenia .Playful strategies, such as video games, have the potential to be widely motivating for students, making them pivotal to the acquisition of social skills among young people with mental disorders  and to tIn recent years, new forms of games have emerged and established themselves as a leisure alternative, including escape rooms . Escape The results of recent studies  show thaUniversity students in health care and social work studies are especially concerned that they are empathic with the population affected by mental disorders because they are one of the vulnerable groups in their professional future that they will have to attend to. The objective of this study was to evaluate the impact of a web-based escape room on raising awareness of SMI among university students in the health care branch during the 2020-2021 academic year in web-based mode. The specific objectives of this study were (1) to determine whether through a web-based escape room, it is possible to modify the stigmatizing attitudes of university students taking a nursing degree regarding SMI; (2) to study the influence of the immersion (flow) experienced during the escape room experience and on the awareness of the participants toward stigma; and (3) to determine whether the degree of stigmatizing attitudes is different between students with family members with severe mental disorders and students without family members with SMI and to analyze whether the program has the same effects on these people.All the participants were first-semester nursing students. The participants were chosen based on the purpose of the research, consideration of the resources available when performing the intervention with web-based escape rooms, and the willingness of the university teachers to carry out the intervention in their class schedule.Both the escape room of the control group and the escape room of the experimental group were used during class time in subjects that had some content related to mental health. Students enrolled in the first semester of their nursing degree could participate in the research if they gave their written consent. The escape room did not score on the average of the course, and no financial reward or any other type of incentive was offered .A total of 306 nursing students participated in the study, with a mean age of 23.34 (SD 8.37) years, comprising 57 (18.6%) men and 249 (81.4%) women. They were selected through cluster sampling, where a particular batch of students were selected as participants, dividing them by participating classrooms. They were randomly allocated between the control group and the experimental group. The experimental group consisted of 197 students with a mean age of 22.93 (SD 7.93) years, comprising 37 (18.8%) men and 160 (81.2%) women; the control group consisted of 109 students with a mean age of 24.1 (SD 9.10) years, comprising 20 (18.3%) men and 89 (81.7%) women.To fulfill the third objective of this study—to determine whether the degree of stigmatizing attitudes is different between students with family members with severe mental disorders and students without family members with SMI and to analyze whether the program has the same effects on these people—demographic questions were asked of the students before beginning the intervention about whether they had relatives who had SMI. The data showed that 67 students had relatives with SMI, with a mean age of 22.78 (SD 4.27) years. Of these 67 students, 23 (34.3%) belonged to the control group, 19 (82.6%) women and 4 (17.4%) men, with a mean age of 23 (SD 4.62) years, and 44 (65.7%) belonged to the experimental group, 38 (86.4%) women and 6 (13.6%) men, with a mean age of 22.7 (SD 4.07) years.This study was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent. Ethics approval was obtained from the Research Ethics Committee of the University of Almería (Ref. UALBIO 2021/01).The 14-item abbreviated version of public stigma in mental health (Spanish) was used, adapted from the Attributional Questionnaire (AQ) 27 , with 14Dangerousness—measures whether people with SMI pose a threat or create feelings of fear. An example question is, “Do you feel that people with SMI are dangerous?”Solidarity—measures the willingness to help a person with SMI; for example, “If I owned an apartment, I would rent it to people with SMI.”Coercion—people with SMI are required to participate in treatment. An example of an item for this factor is “I think it is in the best interest of the community where a person with SMI lives to be placed in a psychiatric facility.”Avoidance—indicates the willingness to live or work near someone with SMI. For example, “I will share my car every day with a person with SMI.”The Motivation Questionnaire for Cooperative Playful Learning Strategies  was usedWithout Memories escape room. The narrative is that the main character must remember his or her identity and leave the apartment where he or she is to be on time for an appointment. In the escape room, the player wakes up with no memory of who or where he or she is and, through solving tests and exploration, discovers that he or she is a young man with a mental disorder. Through different elements, such as newspapers, mobile phones, social networks, and computers, a normalized life is gradually presented together with the different barriers that people with SMI face because of the stigma and discrimination they face; for example, difficulties in health services or difficulty finding work.For this study, an escape room has been designed based on SMI, namely, the Genialy digital platform, designed as a graphic adventure (Click and Point). The difficulty of the escape room is low because it is designed for a nonexpert audience in these activities, and its duration is approximately 1 hour.The escape room features linear mechanics: one test allows the next to be solved and so on until the final task, which requires the resolution of 2 tasks to obtain the code that ultimately allows the player to exit. The escape room is web-based and is designed within the To investigate whether web-based escape rooms could modify stigmatizing attitudes toward SMI, 2 web-based escape rooms were designed and evaluated according to the intervention group .Genially digital platform, allowing the creation of Click and Point spaces, which is why it favors creating an interactive experience that can favor student immersion. To create the scenarios proposed in the escape room, images and illustrations from the FreePik resource bank were used, owing to a premium license for free use , the class began, and the game master explained in a general way what an escape room was, how the Genially platform was used, and that this escape room was cooperative. Once any questions among the students had been resolved, the escape rooms were distributed to the students in teams of 4 people.Each team was housed in a web-based workroom so that they could share ideas and opinions about solving the escape room. They had 2 options: either one of them shared a screen and they solved it together through that screen or each person solved the escape room simultaneously on their computer, maintaining the relationship between their partners in both options.The game master was in the general room of the Blackboard platform during the entire escape room implementation time, in case a team contacted to request clues. There was no limit to the number of clues, as it was important that they felt they could receive feedback and were able to get out of the escape room even if they had never played before.Once the team passed the last test and managed to leave the escape room, they completed the posttest questionnaire individually.Without Memories, interviews were conducted with people with SMI and 2 professionals from a local mental health association with the aim of understanding the difficulties people with SMI deal with daily and the reality they face as told from their own experiences. The game design for an escape room has a linear structure. Solving one clue will provide the object necessary to solve the next clue, and so on and so forth until the students escape. Tasks in a linear room must be solved in a particular order. The experiment was conducted on the web and synchronously. The average duration of the escape room was 60 minutes.To design the escape room The escape room narrative involves waking up in a room without any memories of who or where you are. You must overcome different challenges until you know that your name is Enrique, you have an SMI, and you have a normal life.The different tasks of the escape room are presented in the following formats inside the game:Computer: in this game, there are elements hidden around the room (click and point) that allow the student to know the password of a computer. On the computer, Enrique’s browsing history can then be accessed, where there are 2 videos of awareness campaigns on mental health and mental disorders, and Enrique’s social networks can be accessed, where 2 conversations with friends and a conversation with his family physician are observed, in which he mentions difficulties related to medication, such as the fear of not being able to have erections and to relate intimately with the woman he likes. Between these conversations, the key for the next test is observed.Diary and mobile phone: more conversations with Enrique’s family and friends are reflected on the mobile phone. These conversations reflect Enrique’s concerns and limited relationships. Some responses to his messages can be interpreted as condescension, childishness, and other behaviors that people with SMI reported in previous interviews. It is also narrated how these people may have a perception of the world that is different from the norm and may display different concerns and hobbies.Safe and door: the safe is opened by means of a phrase that is encoded in the computer. To achieve this, it is necessary to find the decoding key located in one of the rooms. This coded information allows the safe to be opened using information contained in the mobile phone and in the bedroom and allows you to remember that you are Enrique, you are an adult, you have an SMI, and you have met the girl you like for a coffee. The box finally gives you the key to open the door. When you click on the door again, there is a concluding message. This message provides statistical information about SMI and the possibility that the player may have an SMI or meet someone close with an SMI in the future.Locked In was prepared. This escape room is a copy of the previous one in which all the elements that had been prepared to raise awareness, such as narrative texts and correspondence on social networks, were removed, replacing them with others without any burden of awareness, as they are logical tests, puzzles, and numerical locks. The narrative of this escape room assumes that you wake up in bed with no memories and must leave the apartment you are in. The steps for resolution are the same as those carried out by the experimental group; that is, they follow a linear logic in which one test leads to another until the final resolution is reached.For the control group, an escape room called d>0.20). Using these data, the minimum sample size needed for ANOVA testing was 277 students. Therefore, the sample size for this study was considered sufficient.Initially, a sample size calculation was performed using the G*Power 3.1 program . The parBefore starting the analyses to answer the research questions, the direct scores of the administered tests were calculated using the corresponding descriptive statistics.To answer the first research question, an ANOVA (2×2) with repeated measures was performed for each factor of the AQ14, as well as another test for the total result of the battery. The Bonferroni correction was used as the adjustment method for the comparison of post hoc tests.To determine the relationship between flow and the change in stigmatizing attitudes, a linear regression was performed, in which the differential scores were calculated. In other words, pretest scores were subtracted from posttest measurements. With the differential scores and the score obtained in the flow, the linear regression was performed with the score of the flow as an independent variable.To address the last research objective, a 3-way ANOVA was carried out for each of the factors and the total score of the AQ14.The analyses were carried out using the R programing language with the R Studio development environment. The libraries used were ggplot2, tidyverse, emmeans, and rstatix.The means and SDs are presented in 2gη>0.14), except in coercion, where the size is medium, and avoidance, where the size is small (2gη>0.01).Regarding the first research question, ANOVAs (2×2) were performed with repeated measurements. The results of the tests between the groups are reported in 2r=−0.845) and a moderate slope of the curve ; that is, the higher the score obtained for flow, the more the stigma displayed was reduced.As for the second research question, as already mentioned, a linear regression was carried out between the flow variables , such as the independent variable, and the differential scores , such as the dependent variable. The results obtained in the analysis indicated that the predictor variables explained 84.5% of the total variance , with statistically significant differences in favor of people who have a close relative. In other words, people with a close relative with SMI display a lower level of stigma than those without a close relative with SMI.The results of the ANOVA indicated that having a close relative influences the level of stigma of the participants in all study variables (dangerousness: With the intention of knowing how the program affects the fact of having or not having family members with SMI, the 3-way ANOVA was continued, and the results of this are reported for the posttest measures. The ANOVA results are presented in As can be seen, statistically significant differences were found between the control and experimental groups, especially in the case of not having a family member affected by SMI. In this case, statistically significant differences were observed in all study variables. However, when someone had a family member affected by SMI, no statistically significant changes were found for the variables solidarity, coercion, and avoidance, whereas for the variables dangerousness and total battery, there were statistically significant changes between the people in the control and experimental groups who had family members affected by SMI.This study sought to provide new methodologies to raise awareness among the young adult population, especially university students who in their professional future could work with people with severe mental disorders. There are very few studies that have used a web-based methodology , and at However, as stated earlier, escape rooms are an increasingly used tool in the educational field ,24 becauIn this study, we investigated the effectiveness of an escape room as an awareness tool based on empathizing or putting oneself in the role of a leading character with an SMI. The intervention significantly reduced the stigmatizing attitudes of university students in the experimental group compared with the control group. It is worth highlighting the size of the large effect observed in the factors of danger, perceived fear, and in solidarity, variables in which the greatest reduction was obtained.Dangerousness is the most common stigma dimension in the general population , and it The implementation of the escape room was carried out with university students from the health sector, which is important considering that some of these professionals will work directly with people affected by SMI, and their expectations and beliefs will affect the recovery of their patients . AnotherIn contrast, it has been proven how the narrative of the story and immersion in the activity encourage the re-elaboration of value judgments that alter the beliefs and attitudes of people with SMI. Escape rooms have already been tested as active methodological tools that promote motivation and student commitment toward learning ,29, obtaAs for other findings, a variable found to influence the results is whether the person has a family member with mental health problems. When a person has a family member with this type of difficulty, we can observe that the stigma is lower and possibly owes to the greater knowledge and direct contact that an individual has around these problems, which is fundamental in the reduction of stigma .This study has several limitations. First, the sample is relatively small, it comes from 2 universities, and it is not representative of the general population. Furthermore, the sample of university students included only individuals from nursing schools. This may undermine the generalizability of the results and limit the interpretation of the effect size. Second, the specific characteristics of the participants and the possible confounding factors that may influence the results, such as their sociocultural origin, their level of knowledge, and their desire to work with patients with SMI, were not evaluated; the only exception was whether they had family members with SMI, in which case they were evaluated. Finally, another limitation was not conducting a reevaluation months after the intervention to determine whether the changes achieved in the experimental group could be perpetuated over time.Mental health awareness is a very important subject for the proper professional development of health care students. It is the responsibility of university teachers to prepare students to learn, understand, and know how to work with their patients. The lack of theoretical knowledge and stigma toward people with SMI can contribute to discrimination toward this group, which, in turn, can influence their self-esteem and quality of life. Therefore, it is necessary to implement new strategies in university education to improve the knowledge and awareness of mental disorders. The integration of playful strategies such as escape rooms can be of great interest because of their immersive and motivating capacity.Without Memories, a web-based escape room where the narrative is about a character with SMI, is an effective tool to promote awareness around mental health because it favors learning by discovery and playful challenges through attractive dynamics and mechanics, which can encourage students to learn about SMI and be more empathetic toward these people.Without Memories is very limited, and although this may have benefits for its replication, there is a need to plan future awareness programs through playful strategies of longer duration to evaluate whether they can achieve a greater impact on motivation, acquisition of knowledge, and awareness.Future studies could use this escape room not only in nursing degree studies but also in other degrees related to health and community education to promote awareness among other professionals who also have direct contact with people with SMI. Furthermore, future research could assess whether the results on the impact of playful strategies for mental health awareness vary because of other sociodemographic factors. Finally, the duration of the escape room


Ischiofemoral impingement syndrome is a neglected cause of posterior hip pain which is derived from narrowing of the space between the lateral aspect of the ischium and the medial aspect of the lesser trochanter. Its diagnosis is challenging and requires the combination of physical tests and imaging studies. In the present narrative review, we found that femoral anteversion predisposes patients to the narrowing of the ischiofemoral space and subsequent quadratus femoris muscle injury. Magnetic resonance imaging serves as the gold-standard diagnostic tool, which facilities the quantification of the ischiofemoral distance and the recognition of edema/fat infiltration/tearing of the quadratus femoris muscle. Ultrasound is useful for scrutinizing the integrity of deep gluteal muscles, and its capability to measure the ischiofemoral space is comparable to that of magnetic resonance. Various injection regimens can be applied to treat ischiofemoral impingement syndrome under ultrasound guidance and they appear to be safe and effective. Finally, more randomized controlled trials are needed to build solid bases of evidence on ultrasound-guided interventions in the management of ischiofemoral impingement syndrome. A population-based survey investigating 2600 adult participants aged between 38 and 77 years reported that the prevalence of self-reported hip disorders was 32% . Its preThe posterior hip is an important region, where pain can ensue due to piriformis syndrome, osteoarthritis and sacroiliac joint disorders. Ischiofemoral impingement syndrome (IIS) is a neglected cause of posterior hip pain which originates from the narrowing of the space between the lateral aspect of the ischium and the medial aspect of the lesser trochanter . The synThe ischiofemoral space is demarcated by the ischial tuberosity medially and the lesser trochanter of the femur laterally . The craDeep in the quadratus femoris is the obturator externus muscle, originating from the obturator membrane and bony boundaries of the obturator foramen and being inserted into the trochanteric fossa of the femur  9]. The. The9]. The configuration of the femoral neck in relation to the femoral shaft plays a role in the alignment of the lesser trochanter against the ischial tuberosity and it also influences the ischiofemoral space. The angle between the femoral neck and shaft is termed the femoral neck anteversion angle, which is associated with the magnitude of femoral torsion . The femThe range of the normal femoral anteversion angle is between 15 and 20 degrees in adults, although it can reach 40 degrees at birth . ParticiIn the toe-in posture, the femur is internally rotated and this tightens the deep gluteal muscles, including the quadratus femoris and obturator externus. It is well known that an overstretched muscle is vulnerable to strain injury . FurtherTwo physical tests have been commonly performed for the diagnosis of IIS . The lonThe ischiofemoral impingement test is conducted using the lateral decubitus position with the affected side facing upward. The examiner presses the buttock slightly lateral to the ischium after passively extending the hip. If pain is provoked at the neutral/adducted (but not abducted) femur, IIS is likely . Using MRecently, a two-step provocation test has been proposed . In the There are also some nonspecific physical tests for the diagnosis of IIS which might be helpful for differentiating the origin of gluteal pain. For instance, during the FADIR  test , the patPatients with IIS typically have pain over the posterior buttock, aggravated by a long duration of weight bearing or hip extension/adduction . SnappinThere are miscellaneous disorders mimicking IIS, e.g., lumbosacral radiculopathy, sacroiliac joint dysfunction, sciatic nerve entrapment, posterior hip joint arthropathy and ischiofemoral ligament sprain. Considering the overlapping clinical presentations and anatomy related to IIS, deep gluteal and hamstring syndromes seem to be the two scenarios that should be differentiated.Deep gluteal syndrome can be defined as non-discogenic posterior buttock pain with possible irritation of the sciatic nerve . It is lOn the other hand, hamstring syndrome is manifested by pain on or adjacent to the ischial tuberosity, radiating to the posterior thigh . It is fMRI is the imaging tool most commonly used for the assessment of IIS. The width of the ischiofemoral space and the thickness of the quadratus femoris muscle can be estimated using axial views. Compared with T2 weighted imaging, T1 sequences are better for these measurements owing to their superiority in delineating regional anatomy. The smallest distance between the lateral cortex of the ischial tuberosity and the medial cortex of the lesser trochanter is used to represent the ischiofemoral space . On the In addition to the quantitative evaluation of the ischiofemoral space, MRI is useful in the qualitative assessment of the quadratus femoris muscle . At the Before the assessment of structures inside the ischiofemoral space, the examiner must be aware of the explicit evaluation of the hip external rotators . The curMore distally, the obturator internus can be seen originating from the inner surface of the obturator foramen, wrapping around the ischium and attaching to the trochanteric fossa. The presence of an intra-muscular tendon and a ‘waterfall moving pattern’ upon external/internal femoral rotation indicates the obturator internus. Relocating the transducer on top of the posterior hip joint, the inferior gemellus appears over the ischiofemoral ligament. Lastly, the quadratus femoris arises from the deeper portion of the ischium and the inferior edge of the semimembranosus and is inserted on the intertrochanteric crest. The muscle situated underneath the quadratus femoris is the obturator externus .Another clinical issue is whether the ischiofemoral space can be easily/reliably measured by using US . In 2019r = 0.781, p < 0.001).In 2017, the capability of US imaging for measuring the ischiofemoral space was validated via MRI on 10 asymptomatic participants . The exar = 0.575, p = 0.01) in both patient and control groups. Using the cut-off value of 2.14 cm, US exhibited a sensitivity of 92.0% and a specificity of 68.4% for the detection of IIS.In 2019, another validation study was conducted on 16 patients with hip pain and 19 controls . The iscUS is helpful in delineating the configuration of the sciatic nerve, which is strongly implicated in IIS. In 2018, sonographic examinations were conducted on six fresh cadavers and 31 healthy volunteers . The magA stepwise algorithm was published in 2017 for the management of IIS . In sympIn 2013, Volokhina et al. demonstrated a case with posterior hip pain and quadratus femoris muscle edema on axial T1 and proton-density fat-suppressed MRI . A combiIn light of our literature search, we observed that the current evidence regarding US-guided injections for IIS was limited, i.e., comprising case reports/series. Further randomized controlled trials comparing US-guided interventions with other non-operative approaches  are indisputably needed.IIS is an overlooked entity within the spectrum of posterior hip pain. It usually presents with symptoms that are not distinguished from deep gluteal and hamstring syndromes. Femoral anteversion predisposes patients to narrowing of the ischiofemoral space and subsequent quadratus femoris muscle injury. MRI serves as the gold-standard diagnostic tool, which facilitates the quantification of the ischiofemoral distance and the recognition of edema, fat infiltration and tearing of the quadratus femoris muscle. US is useful for scrutinizing the integrity of deep gluteal muscles and its capability to measure the ischiofemoral space is notably comparable to that of MRI. Various injectates have been applied to treat IIS under US guidance and they appear to be safe/effective. Finally, future randomized controlled trials are needed to solidify the evidence regarding US-guided interventions in the management of IIS.


In the multivariate logistic regression analysis, IOP fluctuation (p = 0.037) and systolic blood pressure (p = 0.016) were all independently associated with positive TNF-α level. In normal-tension glaucoma (NTG) patients, presence of central scotoma (p = 0.029) was significantly associated with positive TNF-α level. In conclusion, positive TNF-α level in OAG patients was associated with greater IOP fluctuation and higher systolic blood pressure. In NTG patients, positive TNF-α level was associated with the presence of central scotoma. IOP factors and vascular factors, including blood pressure and presence of central scotoma, may indicate glaucoma pathogenesis related to TNF-α elevation in OAG patients.Tumor necrosis factor-alpha (TNF-α) is an important modulator of neuroinflammation, secreted from activated glial cells in response to intraocular stress. The purpose of this study was to investigate the clinical factors associated with elevated TNF-α and its level in aqueous humor of patients with open-angle glaucoma (OAG). Aqueous humor was collected from 73 OAG eyes, and TNF-α level was analyzed using the singleplex bead immunoassay method. Patients were divided into TNF-α-positive and TNF-α-negative groups according to the TNF-α level of 10 pg/mL, and baseline clinical characteristics were compared. The TNF-α-positive group showed higher baseline IOP, greater IOP fluctuation, and higher systolic blood pressure than the TNF-α-negative group ( High intraocular pressure is the most significant risk factor in glaucoma, and current therapies aim to lower intraocular pressure; however, despite successful lowering of intraocular pressure, many patients still experience disease progression . In thisTNF-α is an important modulator of neuroinflammation, synthesized primarily by activated monocytes due to tissue ischemia or damage ,8. TNF-αElevated TNF-α levels have been previously reported in glaucoma. In glaucoma mouse models, TNF-α levels were elevated in the retina with retinal ganglion cell loss . In glauSince TNF-α is secreted from activated glial cells in response to intraocular stress such as high pressure or ischemia ,10,11, gTherefore, in this study, we obtained aqueous humor of patients with OAG during cataract surgery and measured the level of TNF-α. We compared the clinical characteristics between TNF-α-positive and TNF-α-negative groups and analyzed factors associated with elevated levels of TNF-α. Furthermore, we also performed subanalyses in patients with normal-tension glaucoma (NTG).This study was approved by the Institutional Review Board of the Seoul St. Mary’s Hospital (KC11SISI0941) and adhered to all relevant tenets of the Declaration of Helsinki. We prospectively recruited eligible patients who were scheduled for cataract surgery and who were willing to participate. All patients provided written informed consent.Our study consisted of 80 eyes of 80 OAG patients who underwent cataract surgery using phacoemulsification at the Seoul St. Mary’s Hospital by one surgeon between July 2017 and June 2020. Patients with angle-closure or secondary etiology of glaucoma, such as uveitic glaucoma and pseudoexfoliative glaucoma, were excluded from the study. Those with any retinal disease—including diabetic or hypertensive retinopathy, history of eye trauma or previous intraocular surgery, history of refractive surgery, and any optic nerve disease or history of neurological disease that may affect the visual field—were excluded from the study.p-value <5%, or a glaucoma hemifield test result consistently outside normal limits on two preoperative visual field tests as confirmed by two glaucoma specialists (YJ and HP). Patients with normal range IOP at serial measurements at baseline were considered NTG before initiation of hypotensive medication.POAG was defined as the presence of an open angle on gonioscopy, a glaucomatous optic disc , visual field defects consistent with glaucoma , a pattern standard deviation with a p < 5%, one of which was p < 1%, among 12 points on the pattern deviation plot.Those who underwent uneventful cataract surgery were included in the study. Preoperative examination included biometry for intraocular lens calculation and blood testing for standard local surgery. All study subjects’ medical history and medical records were reviewed, including systemic symptoms such as migraine and cold extremities. All patients underwent a comprehensive ophthalmic examination, including measurement of visual acuity, intraocular pressure, slit lamp examination, measurement of central corneal thickness by ultrasound pachymetry , measurement of axial length with ocular biometry , dilated stereoscopic disc photo , retinal nerve fiber layer (RNFL) optical coherence tomography , standard automated perimetry using Swedish interactive threshold algorithm 24-2 , and blood pressure. All patients were followed up with every 1–3 months for IOP measurement and optic disc evaluation. Stereoscopic disc photography, OCT, and visual field examinations were performed every year. Baseline IOP was measurement before initiation of glaucoma medication. Mean follow-up IOP was calculated as the average of all IOP measurements, and IOP fluctuation was calculated as the difference between the lowest and highest IOP during the entire follow-up period. All disc hemorrhages (DHs) detected on color disc and fundus photography during follow-up were recorded. A DH was defined as an isolated flame-shaped or splinter-like hemorrhage on the optic disc or in the parapapillary area, extending to the disc border. Alternative causes of hemorrhage  were diagnostically excluded. Eyes with a glaucomatous visual field defect in either or both hemifields within 24 points of a central 10° of fixation, and with no visual field abnormality in the nasal periphery outside 10° of fixation, were considered to have isolated central scotoma. The criteria for central scotoma were the presence of three or more points with Aqueous humor sample was collected using a standard sterilization procedure. The study protocol was approved by the institutional review board of the Seoul St. Mary’s Hospital. Prior to the beginning of cataract surgery, 50–100 μL of aqueous humor sample was collected from each eye using a 30 gauge syringe. Caution was taken to avoid inadvertent touching of intraocular tissues to prevent contamination of aqueous sample with blood. Aqueous samples were then immediately stored at −80 °C.Samples were not diluted and processed for TNF-α analysis using singleplex bead immunoassay . Standards were reconstituted and serially diluted, following the manufacturer’s instructions. 25 μL of bead and wash solutions were added to each well of a 96-well microtiter plate and incubated for 30 s. Afterwards, supernatant was aspirated with a vacuum manifold. Then 50 μL of incubation buffer was added to each well, followed by 50 μL of assay diluent, and 50 μL of the aqueous humor sample. Standards were added to the designated wells and the plate was incubated for 2 h at room temperature on an orbital shaker at 250–500 rpm. After incubation period, the liquid was removed, and the excess beads were washed off. Then, detector antibody (100 μL) was added to each well and incubated on an orbital shaker for 1 h at room temperature.After removing excess detector antibodies, substrate complex  was added, and the fluorescence was measured using the Luminex 100TM IS fluoroanalyzer  after 30 min of incubation at room temperature. For each reaction, both positive and negative controls were included on the microtiter plate. As a positive control, a known concentration of human recombinant TNF-α was included in each run to ensure a proper assay.Using standard concentration against standard fluorescence, a graphical plot was constructed. The curve fitting linear regression algorithm was performed with GraphPad InStat data analysis software . The concentration of TNF-α level was extracted from the standard curve and expressed as pg/mL. Patients with a TNF-α level of less than 10 pg/mL were considered as the negative group which was the limit of detection. Patients with TNF-α level greater than 10 pg/mL were classified as the TNF-α-positive group based on a previous report that intra-assay coefficient of variation increases below that range .t-test for continuous variables and chi-square test for categorical variables. Data are presented as mean ± standard deviation for continuous variables and number (%) for categorical variables. Univariate and multivariate logistic regression analyses were used to identify factors associated with the presence of TNF-α in aqueous humor of glaucoma patients. In addition, univariate and multivariate linear regression analyses were used to identify factors associated with the level of TNF-α. Independent variables were age, sex, history of migraine, history of cold extremities, axial length, central corneal thickness, baseline IOP, mean follow-up IOP, IOP fluctuation, baseline average peripapillary RNFL (pRNFL) thickness, baseline average macular ganglion cell-inner plexiform layer (mGCIPL) thickness, baseline mean deviation (MD), and pattern standard deviation (PSD) on standard automated perimetry, presence of DH, presence of central scotoma, and systolic and diastolic blood pressure. A subanalysis including only those with NTG was also performed.The baseline characteristics of the study subjects were compared using the Student’s p-value of <0.05 was considered statistically significant. All statistical analyses were performed with SPSS for Windows .A p = 0.007). IOP fluctuation was greater in TNF-α-positive group (8.38 ± 6.29 mmHg) than TNF-α-negative group . Systolic blood pressure was significantly higher in TNF-α-positive group (136.55 ± 15.30 mmHg) compared to TNF-α-negative group .A total of 80 eyes of 80 OAG patients undergoing phacoemulsification were enrolled in the study. Of 80 eyes, 7 (8.75%) were excluded because of small sample amount. Among the remaining 73 OAG eyes, 31 eyes were TNF-α positive, whereas 42 eyes were TNF-α negative. Baseline clinical characteristics are listed in p = 0.037), and systolic blood pressure  were all independently associated with positive TNF-α levels.We used logistic regression analysis to identify factors associated with positive TNF-α levels in aqueous humor . BaselinFurthermore, to identify factors associated with TNF-α level in aqueous humor, we used linear regression analysis. TNF-α level was significantly associated with baseline IOP, mean follow-up IOP, and IOP fluctuation in univariate analysis . In the p = 0.005). The TNF-α-positive group included significantly greater percentage of patients with DH (p = 0.002) and central scotoma  and presence of central scotoma  were associated with positive TNF-α level in univariate analyses. In multivariate analysis, the presence of central scotoma  was significantly associated with positive TNF-α level in the aqueous humor of NTG patients.A subanalysis of those with NTG was performed. In NTG patients, baseline IOP was significantly greater in the TNF-α-positive group (15.63 ± 2.41 mmHg) compared with TNF-α-negative group  compared to cataract patients (5.0%) % compare.In our study, we aimed to find out associated factors to TNF-α elevation in glaucoma patients. This may reveal glaucoma patients who may have neuroinflammation and TNF-α related cell deaths as a pathogenesis of glaucomatous damage. TNF-α-positive was associated with high IOP, high IOP fluctuation, and high systolic blood pressure. These factors are well known risk factors for glaucoma progression ,18,19,20Of note, in our subanalysis including only NTG patients, DH and central scotomas were associated with positive TNF-α level. Only central scotoma was statistically significant in the multivariate analysis. Central scotoma has been associated with vascular instability and increased risk of progression especially in NTG patients ,26,27,28Glaucoma patients with vascular features present with central scotoma and progression in the central visual field have been linked to vascular features, including DH, autonomic dysfunction, migraines, orthostatic hypotension, and Raynaud’s phenomenon ,30. TherIn conclusion, this study demonstrated that POAG patients with positive TNF-α levels showed higher baseline IOP, greater IOP fluctuation, and higher systolic blood pressure. In the subanalysis including only NTG patients, those with positive TNF-α levels were associated with the presence of central scotoma. TNF-α levels are associated with IOP and vascular factors, indicating the potential role of TNF-α in glaucoma.


Background: Pacemaker implantation combined with atrioventricular node ablation (AVNA) could be a practical choice for atrial fibrillation (AF) patients with heart failure (HF). Left bundle branch area pacing (LBBaP) has been widely reported. Objectives: To explore the safety and efficacy of LBBaP combined with AVNA in AF patients with HF. Methods and results: Fifty-six AF patients with HF attempted LBBaP and AVNA from January 2019 to December 2020. Standard LBBaP was achieved in forty-six patients, and another ten received left ventricular septal pacing (LVSP). The cardiac function indexes and pacemaker parameters were evaluated at baseline, and we conducted a 1-month and 1-year follow-up. Result: At the time of implantation and 1-month and 1-year follow-up, QRS duration of LVSP group was longer than that of LBBaP group. The pacemaker parameters remained stable in both the LBBaP and LVSP groups. At 1-month and 1-year follow-up after LBBaP and AVNA, left ventricular ejection fraction, left ventricular end-diastolic diameter, and NYHA classification continued to improve. Baseline left ventricular ejection fraction and QRS duration change at implantation can predict the magnitude of improvement of left ventricular ejection fraction at 1-year after LBBaP. Baseline right atrial left-right diameter, the degree of tricuspid regurgitation, and interventricular septum thickness may be the factors affecting the success of LBBaP. Conclusion: LBBaP combined with AVNA is safe and effective for patients with AF and HF. Baseline right atrial left-right diameter, the degree of tricuspid regurgitation, and interventricular septum thickness may be the factors affecting the success of LBBaP. Atrial fibrillation (AF) and heart failure (HF) frequently coexist and can promote each other. In patients with AF who are intolerant or unresponsive to intensive rate and rhythm control therapy, atrioventricular node ablation (AVNA) combined with pacemaker implantation for rate control can be used as an approach of last resort .Long-term right ventricular apical pacing (RVAP) leads to electrical and mechanical dyssynchrony, associated with an increased incidence rate of AF, HF, and pacemaker-induced cardiomyopathy ,3. CardiHis-Purkinje system pacing has been used as a physiological pacing strategy. Research shows that AVNA and His bundle pacing (HBP) can improve cardiac function and reduce diuretics use in patients with AF and HF. Nevertheless, HBP has some limitations, including higher pacing thresholds, lower R wave amplitude, and the difficulty of lead implantation, which limited the clinical applications of HBP ,7,8. LefThe present study was a single-center retrospective study, and included fifty-six consecutive AF patients with symptomatic HF who attempted LBBaP and AVNA from January 2019 to December 2020. Studies and data collection were performed according to protocols approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University. All patients provided written informed consent. Echocardiogram reports and Pacemaker parameters were available for all patients.Inclusion criteria were as follows: (1) all patients had New York Heart Association (NYHA) classification class II to IV HF symptoms despite optimal medical therapy; (2) age > 60; (3) brain natriuretic peptide (BNP) increased; (4) transthoracic echocardiography demonstrated abnormalities in cardiac structure and/or function. (5) long-lasting persistent or permanent AF, and heart rate was uncontrolled by medical therapy; or (6) AF that failed several catheter ablations, or intolerance to ablation.Exclusion criteria were as follows: (1) patients with poor general condition, unable to tolerate surgery; (2) patients with severe heart , liver , kidney , and other severe dysfunction ; (3) patients with serious coagulation disorders; (4) patients with a life expectancy period < 1-year; (5) patients with contrast agent allergy.LBBaP was performed using the Select Secure lead . In the right anterior 30° oblique projection, the lead was delivered through the fixed curve sheath . The sheath and lead tip were first advanced to the anterior lower site of the His-bundle position and subsequently rotated in a counterclockwise fashion to place the lead tip in a perpendicular orientation to the IVS. At this location, the paced QRS morphology usually displayed a “W” pattern with a midnotch in lead V1 .Then the tip of lead was screwed clockwise until it penetrated deep into the interventricular septum. The QRS changed from left bundle branch block (LBBB) pattern to right bundle branch block (RBBB) pattern ,14.The evidence of LBB capture: during unipolar-tip pacing, right bundle branch configuration was observed in addition to one or more of the following findings: 1. recording of a LBB potential, 2. transition from nonselective to selective LBB capture, 3. Stim-LVAT that shortens abruptly with increasing output and remains shortest and constant at high- and low-output pacing ,14. SuccDuring the procedure, the paced 12-lead electrocardiogram (ECG), intracavitary electrogram (IECG), pacemaker parameters, pacing stimulus to left ventricular activation time , and the paced QRS duration (the duration from the pacing artifact to the QRS end during unipolar pacing) were monitored.AVNA was performed under fluoroscopic guidance. After LBBaP was established, an 8.5 F sheath was inserted through the femoral vein to the right atrium, and a 4 mm irrigated ablation catheter was delivered through the sheath and positioned in the region of the compact atrioventricular node. When the tip of the catheter recorded a near-field Hisian potential, ablation energy was released with a flow rate of 15–30 mL/min, preset power of 35W, and temperature of 43 °C. Desired success of AVNA was evidenced with complete atrioventricular block (AVB) without change in LBBaP parameters  6]..6].Clinical and procedural data were obtained from medical records. Baseline patient characteristics, including the demographic characteristics, patient history, medication use, and NYHA classification, were recorded. The baseline mean heart rate was measured using dynamic electrocardiogram (Holter). Furthermore, 12-lead ECG and echocardiography (Echo) were routinely performed before the procedure. Echo parameters, including left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDd), left atrial anterior-posterior diameter (LAAPD), right atrial left-right diameter (RALRD), right atrial superior-inferior diameter (RASID), right ventricular left-right diameter (RVLRD), and interventricular septum thickness (IVST) were obtained. The severity of mitral regurgitation (MR) and tricuspid regurgitation (TR) was assessed according to current guidelines, defined as follows: 0-None; 1-Mild; 2-Moderate; and 3-Severe .All patients were followed up in the outpatient department. Routine Echo and pacemaker data were recorded at 1-month and 1-year follow-up. Procedure-related complications included capture threshold increase of 1 V, loss of capture, lead septal perforation, lead dislodgement, and infection. Clinical outcomes included mortality and HF hospitalization.p-value < 0.05 was considered statistically significant.Continuous variables were described as mean ± standard deviations (SD) or median with interquartile range . Student’s t-tests were used for parameters that were normally distributed . Wilcoxon signed-rank tests were used for within-group comparisons for parameters with skewed distribution, and Mann–Whitney U-tests were used for between-group comparisons. Data comparison at different time-points was conducted by repeated-measures analysis of variance. Pairwise comparisons were performed post hoc to identify which groups were different. Categorical data were expressed as numbers (%). Qualitative data were compared using Chi-squared or Fisher’s exact tests. Multiple linear regression models were constructed to predict the prognosis of operation. Statistical analyses were conducted using SPSS version 23.0 . All tests were two-sided. A p = 0.003).Fifty-six consecutive AF patients with symptomatic HF attempted LBBaP and AVNA from January 2019 to December 2020. Forty-six patients were successfully treated with permanent LBBaP as LBBaP group, and ten patients failed at five attempts of lead positioning and finally received permanent LVSP as LVSP group. Baseline characteristics of the study population are shown in p = 0.743, p = 1.000, and p = 0.092). At the time of implantation and 1-month and 1-year follow-up, QRSd of the two groups were longer than that before operation . QRSd and stim-LVAT of LVSP group were longer than that of LBBaP group during the same period   .p < 0.001) . No patient demonstrated an increase in the pacing threshold above 1V (compared to baseline). The median ventricular pacing rates were 99.0% and 99.8%, respectively  mm, p < 0.05 for all pairwise comparisons). Decreases or improvements were also observed in the LAAPD, RALRD, and the degree of MR at 1-year follow-up ((47.8 ± 7.3 vs. 45.8 ± 3.8) mm, p = 0.001 and (45.1 ± 6.6 vs. 43.6 ± 6.9) mm, p = 0.031 and p = 0.025). There was no significant change in the degree of TR, RVLRD, and IVST . However, the changes of Echo parameters did not reach statistical differences in the LVSP group (p > 0.05), while greater numerical improvements were observed in LVEF, LVEDd, and LAAPD at the LBBaP group (A series of Echo parameters largely improved after LBBaP and AVNA: 1) the improvements in LVEF and LVEDd were present at 1-month of LBBaP and more improvements were observed at 1-year follow-up ((53.0 ± 11.9 vs. 56.7 ± 11.1 vs. 60.4 ± 8.8)%, SP group . There waP group .p < 0.001 for all pairwise comparisons). In the LVSP group, the improvement of NYHA classification reached statistical difference at 1-year after operation (p = 0.025) (p = 0.021)  . A more = 0.021) .p-values < 0.1 on univariate analysis were included in the multivariable model. The final variables included baseline LVEF, stim-LVAT, postoperative change in ventricular rate (record as ΔHR), and QRSd change at implantation (record as ΔQRSd). The established regression model was statistically significant, F = 12.293, p < 0.001, and adjusted R2 = 0.501. Among the four variables included, baseline LVEF and ΔQRSd were statistically significant (p < 0.001 and p = 0.019). The multiple linear regression equation is “ΔLVEF = 44.295-0.463 * baseline LVEF-0.182 * ΔQRSd”, indicating that the lower the baseline LVEF, the smaller the QRSd broadening, or the more obvious the QRSd shortening at implantation, the more significant the improvement of LVEF at 1-year follow-up (The magnitude of improvement of LVEF at 1-year after LBBaP and AVNA (recorded as ΔLVEF) was taken as the prognostic indicator. Multiple linear regression models were constructed to predict the prognosis of operation. Baseline variables that were considered clinically relevant and candidate variables with ollow-up A.p = 0.010; 1.0  vs. 2.0, p = 0.028; 10.0  vs. 14.5  mm, p < 0.001)  mm, < 0.001) B, suggesp < 0.001). However, there were no significant differences in the numbers of patients taking angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB), angiotensin receptor neprilysin inhibitor (ARNI), and β-blockers (p = 0.482 and p = 0.388)  .All patients had at least one hospitalization for HF within 1 year before LBBaP, but only two patients (4.3%) had HF-related hospitalization within 12 months of LBBaP. Both were induced by severe infection. In the LVSP group, one patient with cardiomyopathy was rehospitalization for HF during the 1-year follow-up period. There were no records of patient deaths during the follow-up period in both LBBaP and LVSP groups.p < 0.001). The pacemaker parameters remained stable in both the LBBaP and LVSP groups, and no severe complications occurred at implantation or during the follow-up period. (2) At 1-month and 1-year follow-up after LBBaP and AVNA, the LVEF, LVEDd, and NYHA classifications continued to improve . Greater numerical improvements were observed in LVEF, LVEDd, and LAAPD at LBBaP group. Additionally, a more significant improvement in NYHA classification was observed in LBBaP group at 1-year follow-up when compared with the LVSP group (p = 0.021). (3) Baseline LVEF and QRSd change at implantation can predict the magnitude of improvement of LVEF at 1-year after LBBaP and AVNA. The lower the baseline LVEF, the smaller the QRSd broadening, or the more obvious the QRSd shortening at implantation, the more significant the improvement of LVEF at 1-year follow-up. (4) Baseline RALRD, the degree of TR, and IVST may be the factors affecting the success of LBBaP. (5) Compared with baseline values, the number of patients taking diuretic agents and digoxin significantly decreased after LBBaP and AVNA . The main findings of this retrospective, observational study are as follows: (1) At the time of implantation and 1-month and 1-year follow-up, QRSd were longer than that of the LBBaP group during the same period  can restore the intrinsic electromechanical activation sequence of the heart, avoiding ventricular dyssynchrony and improving cardiac function. Previous studies confirmed the safety, efficacy, and clinical applications of HBP. However, it must be mentioned that HBP also has its insufficiencies, such as both undersensing and oversensing issues, having a higher pacing threshold owing to the fibrous structure (less myocardium), and having an increased battery drain because of inherently higher pacing thresholds ,11,12,13The applications of LBBaP in symptomatic bradycardia, HF and AF have achieved good results preliminarily, while there are still few studies on permanent LBBaP combine with AVNA for treating AF patients with symptomatic HF. The latest research shows that AVNA in the presence of an LBBaP lead is associated with a higher success rate and fewer acute and chronic lead-related complications; LBBaP preserves left ventricular systolic function in patients with refractory AF post AVNA . For patHowever, in our study, there are still ten patients who failed in five attempts of lead positioning and finally received permanent LVSP. We further explored the factors affecting the success of LBBaP. An analysis on the baseline heart structure of the two groups found that baseline IVST, the degree of TR, and RALRD may be the factors related to the success of LBBaP. The LBBaP procedure rotated the 3830 lead into the interventricular septum and allowed it to reach the left ventricular surface LBB area, directly pacing the conduction system and rapidly agitating the entire ventricle to achieve electro-mechanical synchronization. (1) When the interventricular septum was thickened, it was difficult to rotate the 3830 lead deep into the interventricular septum to reach the left ventricular endocardial surface and capture the left bundle branch conduction system. Additionally, septal fibrosis existed in some patients with thickened interventricular septum and further increased the difficulty of screwing the 3830 lead into the deep ventricular septum. (2) Large right atrium might lead to cardiac rotation and insufficient sheath support. C315His sheath floating in the large right atrium increased the difficulty of crossing the tricuspid annulus. Even if the C315His sheath crossed the tricuspid annulus successfully, it was difficult for the sheath to contact the ventricular myocardium due to the insufficient length of the sheath entering in the ventricle . (3) SevAdditionally, we found that the pacing QRSd and stim-LVAT of LBBaP group were significantly shorter than those of LVSP group. Pacing QRSd can reflect the synchronization of left and right ventricular contraction to a certain extent. Stim-LVAT is a suitable subrogate indicator of evaluating left ventricular activation. LBBAP is a physiological pacing approach characterized as direct capture of the main left bundle or each of the fascicle branches. After pacing the LBB area, there was a narrow QRSd and stim-LVAT, indirectly indicating that the left and right ventricular synchronization was good. LVSP activated the ventricular myocardium and not the specialized conduction system; therefore, it does not achieve complete electrical resynchronization. The pacing QRSd and Stim-LVAT were longer. Long-term alteration of ventricular activation causes persistent electrical remodeling via a mechano-electrical feedback mechanism. This further induces significant changes in ventricular structure and function, leading to ventricular remodeling and HF. In addition, previous studies found that the selection of an optimal pacing site during operation mainly depends on imaging. Therefore, the pacing lead of some patients may not be located on the left side of the septum, which may further decrease the benefit of LVSP .(1) There is still a lack of long-term, large-sample, and multiple-centered randomized controlled prospective trials to confirm our observation further. (2) The sample size in our study was relatively small, which might have led to statistical bias. (3) We still lack a high level of evidence supporting whether LVSP combined with AVNA can benefit AF patients with HF and whether LBBaP is superior to LVSP. (4) This study did not investigate whether beneficial clinical outcomes by LBBaP combined with AVN ablation in patients with AF and HF would be better than those from other pacing strategies (such as RVAP or BiVP-CRT).LBBaP combined with AVNA is safe and effective for AF patients with HF. The cardiac function was significantly improved, and the pacemaker parameters remained stable. No serious adverse events occurred during the follow-up period. Baseline RALRD, the degree of TR, and IVSP may be the factors affecting the success of LBBaP.


Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp64 open reading frame, which encodes the major envelope protein of AcMNPV. After comfirming the site-specific targeting of gp64 leading to reduced budded virus (BV) release, the gag gene of human immunodeficiency virus type 1 was expressed to produce Gag VLPs. This approach was effective for producing VLPs using the BEVS whilst simultaneously obstructing BV release.The manufacture and downstream processing of virus-like particles (VLPs) using the baculovirus expression vector system (BEVS) is complicated by the presence of large concentrations of baculovirus particles, which are similar in size and density to VLPs, and consequently are difficult to separate. To reduce the burden of downstream processing, CRISPR-Cas9 technology was used to introduce insertion-deletion (indel) mutations within the  Virus-like particles (VLPs) are an emerging class of biotherapeutic modality for delivery of therapeutic cargo such as chemotherapy, protein, and nucleic acid-based drugs, and as antigens for vaccination ,2. VLPs The Baculovirus Expression Vector System (BEVS) has many features that make it an attractive platform for VLP production, including ease of manipulation and large capacity for foreign gene insertion that allows simultaneous expression of multiple proteins from the same recombinant BEV (rBEV) . As suchtrans-complementing cell line, in which the deleted gene is constitutively expressed, is required. The mutant rBEV is then used to infect parental cells  for production of the recombinant protein/therapeutic. This approach has been used with the AcMNPV vp80 and gp64 genes to produce enhanced green fluorescent protein (EGFP) and HIV-1 Gag VLPs, respectively [To reduce the burden of baculovirus contamination on downstream processing, strategies have been devised wherein a gene encoding a baculovirus structural protein required for viral genome packaging, nucleocapsid assembly, or release of budded viruses (BV) is deleted from its genome. To enable initial production of infectious virus seed stocks, a ectively ,8. Both ectively , whereasectively . Althougectively ,8.gp64 gene for disruption. After confirming that targeting the gp64 open reading frame (ORF) resulted in decreased GP64 abundance in infected cells, expression of the green fluorescent protein (GFP) reporter gene was assessed. Consistent with previous reports, disruption of gp64 reduced progeny virus release but did not affect expression of GFP. Next, production of HIV-1 Gag VLPs was demonstrated with this approach (targeting gp64 and vp80). The yield of Gag VLPs was similar for all rBEVs in Sf9-Cas9 cells and Sf9 cells, further indicating that CRISPR-mediated disruption of structural genes may be an effective strategy for reducing BV release while maintaining high expression of foreign genes.Here, a recently developed approach for generating rBEV KOVs using CRISPR-Cas9  was usedcas9 gene.Development of the Sf9-Cas9 cells was described previously . Sf9 andAll plasmids used in this study were constructed using the NEBuilder HiFi DNA Assembly Master Mix  according to manufacturer’s directions. Primers used for construction of all plasmids were synthesized by Integrated DNA Technologies  and are given in gp64 or vp80 ORF [gag gene, the gfp ORF was replaced with the gag gene from the plasmid pAdCMV5-gagGFP [The plasmid p6.9GFP-sgRNA, which encodes the p6.9GFP reporter cassette and SfU6-sgRNA for targeting Cas9, has been described previously . Brieflyvp80 ORF . To gene5-gagGFP  using PC5-gagGFP .50 as described previously [Transfer plasmids for rBEV generation were co-transfected with flashBACGOLD™  genomic DNA to Sf9 cells using Escort IV transfection reagent (Sigma-Aldrich) according to manufacturer’s directions. Supernatant from each transfection was harvested 4–5 days post transfection and used to infect suspension Sf9 cultures .Fluorescent cells were acquired using a BD Accuri™ C6 Plus flow cytometer  equipped with 488 nm and 640 nm lasers. Samples were run at the low flow setting and 10,000 events were collected and analyzed using FlowJoSample preparation for analysis via flow cytometry was described previously . BrieflyThe supernatants of Sf9 and Sf9-Cas9 cells infected with Gag-expressing rBEVs were harvested by centrifugation at 1000gp64 gene were target-specific and resulted in the disruption of progeny virus release. Accordingly, the abundance of GP64 protein was analyzed by western blot and immunofluorescence staining in the cell membrane. Analysis of cell lysates from infected cells revealed that GP64 present in Sf9-Cas9 cells infected with rBEVs targeting the gp64 ORF was reduced to gp64 ORF result in decreased abundance of GP64 protein.Initial experiments were conducted to confirm that sgRNAs designed to target the  control . Detecti control . Taken tgfp reporter gene transcribed from the late p6.9 gene promoter and sgRNAs targeting the gp64 gene resulted in significant reduction of infectious viral titer (IVT) at 48 hpi compared to the untargeted control. Specifically, the mean IVT for control rBEVs in Sf9-Cas9 cells was gp64 KOV was gp64-targeting sgRNAs, respectively) and similar to the untargeted control rBEV in Sf9-Cas9 cells  ,7,8. ThiV genome . This syAcMNPV gp64 gene for disruption. To this end, the abundance of GP64 in infected cell lysates and in the membrane of infected cells was measured. Our results indicated gp64 was indistinguishable from control infections, indicating that disruption of GP64 expression was the result of CRISPR-mediated targeting of the gp64 ORF.To assess this strategy for its utility as an effective production platform for VLP production with concomitant reduced BV release, we again targeted the gp64 on late gene expression and progeny BV release was measured. Disruption of GP64 resulted in >98% and gp64 gene truncations [gp64 KOV via western blot, however direct quantification of BV in the supernatant was not conducted in that report [gfp reporter gene was not significantly affected by gp64 disruption. Although the median fluorescence intensity was slightly lower for gp64-targeting rBEVs compared to the control, this difference in expression was similar for both Sf9-Cas9 and Sf9 cell lines. This data could indicate that variability between individual virus stocks may have accounted for these differences as opposed to decreased late gene expression as a result of CRISPR-mediated targeting. Nevertheless, these differences were not statistically significant. This is an important result, as previous reports indicated that high MOIs were required for similar EGFP yields between vp80 KOV and the control virus [gp64 KOV and the control [Next, the effect of targeting ncations . Similarol virus , whereasgag ORF encodes a 55 kDa polyprotein (Pr55 or Gag) that is processed into several proteins, including the 17 kDa matrix protein (p17 or MA), the 24 kDa capsid protein (p24 or CA), and the 7 kDa nucleocapsid protein (p7 or NC) [gp64, rBEVs with sgRNAs targeting the vp80 ORF were prepared in order to compare VLP production using both of these strategies. Similar to previous results, targeting the gp64 ORF resulted in significant reduction of GP64 abundance in the plasma membrane of infected cells and IVT. The IVT of vp80-disrupted rBEVs was also significantly reduced compared to control infections in Sf9 cells. Unexpectedly, immunofluorescence staining of GP64 in the plasma membrane of infected cells was observed to be lower in Sf9-Cas9 cells compared to Sf9 cells, suggesting that disruption of VP80 expression may impact GP64 production. Reduced GP64 was not observed by western blot analysis of cell lysates infected with a vp80 KOV previously [gp64 KOV [vp80 ORF showed similar results (data not shown). Similarly, there were insignificant differences between GP64 measurements in the cell membranes infected with control or vp80/gp64-targeted rBEVs (data not shown). As such, this observation appears to be the result of a potential and as yet unreported interaction between vp80 disruption and GP64 expression, and may require further scrutiny to assess this relationship. Nevertheless, both of these strategies were successful for producing Gag VLPs with concomitant reduction in rBEV contamination. Importantly, although the estimated yield of VLPs by p24 ELISA was lower compared to a control , yields of VLPs were similar in Sf9-Cas9 and Sf9 cells for all of the rBEVs, suggesting that these results might be due to variance among virus seed stocks as opposed to the strategy itself.Finally, we assessed the production of HIV-1 Gag VLPs with concomitant reduced BV contamination. The HIV-1 7 or NC) . Express7 or NC) ,19,20,21gp64 gene was assessed. After confirming that this strategy resulted in target specific obstruction of GP64 and reduced BV release, production of HIV-1 Gag VLPs was assessed and compared with a similar strategy in which the vp80 ORF was targeted for disruption. Both strategies resulted in high level production of VLPs along with reduced rBEV contamination in culture supernatants. This strategy may be impactful for simplifying the purification of recombinant proteins and other complex biologics such as VLPs, and may be an improvement over previously reported strategies in which initial virus seed production was impaired and overall yield may be impacted.In this report, CRISPR-mediated disruption of the


Additionally, the V2 and the Azure both showed high agreement with each other on the ML outcomes but not against a ground truth. Therefore, the ML models may need to be retrained ideally with the Azure, as it was found to be a more reliable and robust sensor for tracking wheelchair transfers in comparison to the V2.Wheelchair users must use proper technique when performing sitting-pivot-transfers (SPTs) to prevent upper extremity pain and discomfort. Current methods to analyze the quality of SPTs include the TransKinect, a combination of machine learning (ML) models, and the Transfer Assessment Instrument (TAI), to automatically score the quality of a transfer using Microsoft Kinect V2. With the discontinuation of the V2, there is a necessity to determine the compatibility of other commercial sensors. The Intel RealSense D435 and the Microsoft Kinect Azure were compared against the V2 for inter- and intra-sensor reliability. A secondary analysis with the Azure was also performed to analyze its performance with the existing ML models used to predict transfer quality. The intra- and inter-sensor reliability was higher for the Azure and V2 ( In 2016 there were approximately 282,000 individuals in the United States living with a spinal cord injury (SCI), with around 12,500 new cases occurring each year ,4. This Proper sitting-pivot-technique (SPT) for independent transfers in MWUs is vital to avoid predisposition to upper extremity pain and discomfort; therefore, proper training, practice, and management of SPTs are integral to the quality of life and opportunities for MWUs ,7. MethoA large number of movement recognition and auto-technique classification applications have been described in the literature. These applications use a wide variety of sensors to capture the spatial and temporal features of the motions of interest. One group of applications uses inertial measurement units (IMUs) for capturing the biomechanical features of interest, which are used to classify the techniques ,13,14. AA third group of applications entails the use of depth sensors which can provide full-body three-dimensional (3D) motion capture with facial, hand/finger, and/or body joint tracking capabilities while permitting total freedom of movement without holding or wearing any sensors or markers. These sensors are also inexpensive (<USD 500), portable, and easy to set up and use, making them an attractive option to consider for technical and clinical applications. Ensuring the simplicity of system use and flexibility of system setup is crucial to fit the needs of busy clinical professionals and to support the integration of an auto-scoring system into the existing facilities and treatment processes. The Microsoft Kinect V2 camera is a popular depth sensor that has been used in numerous studies to successfully automate the scoring of various clinical tools using 3D body tracking  data ,20,21.Building upon the success of these prior studies, we developed TransKinect—a software application used to make TAI scoring easier and less subjective, using a Microsoft Kinect V2 depth camera to watch and auto-score the TAI. This is accomplished through trained machine learning (ML) models that use biomechanical features to determine if the SPT was conducted correctly or incorrectly in accordance with TAI principles . The TraOur research team has demonstrated that the Kinect V2 has the ability to recognize at-risk body motions during independent sitting pivot transfers ,22,23. TFor the continued development of TransKinect and successful translation into clinical practice, a new sensor must be identified. While there has been some research in comparing the performance of various depth sensors in their ability to track biomechanical variables ,25,26,27The overall goal of this study was to determine the potential for the RealSense and Azure sensors to act as surrogate sensors for the TransKinect application. Specifically, we aimed to determine the intra-rater (within sensor) reliability, inter-rater (between sensor) reliability, and agreement of select key features between the Intel RealSense and V2 and the Azure and V2. We hypothesized that the reliability within each sensor and between each sensor pairing would be high (interclass correlation coefficients (ICC > 0.80) and that the reliability would be similar between the sensors (ICC differences < 0.10). We further expected high agreement between the V2 and RealSense and V2 and Azure features  limits of agreement). A secondary goal of the study was to determine the agreement between TAI scores auto-generated with the V2 to those auto-generated with the Azure for proper and improper SPTs. We hoped to find that the Azure TAI scores would have high agreement with the V2 TAI scores (>70%) and that the Azure TAI scores would also have high accuracy with the ground truth scores (>70%).Both phases of the study followed the proposed flowchart shown in The first phase of the study comparing the RealSense to V2 sensors was conducted pre-COVID-19 pandemic at the 2019 Annual National Veterans Wheelchair Games (NVWG) in Louisville, Kentucky. The second phase comparing the Azure and V2 sensors was conducted during the COVID-19 pandemic in the Human Engineering Research Laboratories.The inclusion criteria for Phase 1  participants were: (1) have a discernable neurological impairment affecting both lower extremities or persons with transfemoral or transtibial amputation of both lower extremities and who do not use prostheses during transfers, (2) at least one-year post-injury or diagnosis, (3) able to independently transfer to/from a wheelchair without human assistance or assistive devices, (4) use a wheelchair for the majority of mobility (over 40 h/week), and (5) over the age of 18 years. Participants were excluded if they had (1) current or recent history of pressure ulcers in the last year, (2) history of seizures or angina, or (3) were able to stand unsupported.The Phase 2 (Azure vs. V2) participants were able-bodied research staff without any upper or lower limb impairment who were knowledgeable on proper and improper wheelchair transfer techniques.n = 1), traumatic brain injury (n = 1), and poliomyelitis (n = 1).For Phase 1 RealSense vs. V2 reliability and agreement analysis, 26 men and 4 women with an average age of 56.6 years (standard deviation (STD) = 11.8) contributed a total of 150 transfer trials for the analysis. The group regularly performed, on average, 13 transfers  per day. Participants had 16.8 years (STD = 5.8) experience in using wheelchairs and used their wheelchairs for 13.2 h (STD = 5.8) per day. Nine (30%) were African Americans, twelve (40%) were Caucasian, three (10%) were Hispanic, two were Asian, one denoted mixed race, and three did not answer the question. Twenty-three participants (77%) used a manual wheelchair. Nineteen (63%) had a spinal cord injury, six (20%) had an amputation, two (7%) had multiple sclerosis, and others included Guillain barre  with an average age of 29.4 years participated for a total of 70 transfers. Four individuals were Caucasian, two were Asian, and one was Hispanic.The analysis comparing the ML predicted TAI scores between the Azure and V2 involved three participants (1 man and 2 women) with an average age of 32.7 years. They conducted 10 transfers for each of the five different transfer types . One participant was Asian, and the other two were Caucasian.In both phases of the study, participants transferred from a wheelchair to a height-adjustable firm surface (transfer bench). Custom software programs were developed that utilized existing SDKs for each sensor, allowing for visualization and recording of body tracking data. For the V2, a graphical user interface (GUI) was programmed in C#, Visual Studio 2012, .NET Framework 4.0 and used the Windows Kinect SDK for obtaining the joint center coordinates. The RealSense D435 utilized a third-party SDK by Nuitrack, released by 3DiVi Inc., to obtain the coordinates. A custom Python GUI was created to facilitate data collection from the Kinect Azure, utilizing the pyKinectAzure library. Each individual sensor was connected to its own laptop computer for the data collection. The joint center data from all sensors were collected at 30 Hz and saved in *csv  files. The data were synchronized via timestamps. Specifications for each sensor are shown in Each depth sensor was positioned in front of the transfer surface and the wheelchair at a distance specified by the manufacturer .Participants were asked to position their wheelchair to the right of a level-height bench (70 cm × 55 cm) based on their transfer preferences  left. DaParticipants were asked to position themselves to the right of the level-height bench (same one as Phase 1) and prepare to perform a transfer  right. Tx-axis position of the spine base/waist/pelvis joint center. The raw positional data was filtered with a digital lowpass Butterworth filter with a cutoff frequency of 15 Hz, and the start and end points of the lift phases were determined. The start of the lift phase was determined by the end of the first plateau region, as shown in The specific joint centers identified by each sensor type are shown in x-axis, calculated by taking the final position and subtracting the initial position. This value is given in millimeters and converted to centimeters.DSWP—the displacement of the spine base/waist/pelvis joint center along the LPOE—the average plane of elevation angle on the leading side shoulder (the left side). This angle is calculated between two vectors: a normal vector to the chest and the upper arm (left shoulder to elbow), projected onto the transverse plane. The normal vector is calculated by taking the cross product of the trunk vector  and the shoulder across vector (left to right shoulder markers). This value is given in degrees.LE—the average elevation angle on the leading side shoulder (the left side). This angle is calculated between two vectors: the trunk vector and the upper arm vector. This value is given in degrees.y-axis. This value is given in degrees.TF—the average flexion angle of the trunk calculated as the angle between the trunk vector and the vertical A secondary goal of the study was to determine the agreement between TAI scores auto-generated with the V2 to those auto-generated with the Azure for proper and improper SPTs. Specific details concerning the development of the ML models used to predict (auto-generate) TAI scores can be found elsewhere . These mThe features used in the ML models  were then computed using the identified and approximated Azure marker locations and separately using the equivalent V2 marker locations for each transfer. The features generated from each of the sensors were entered into the ML models to predict the TAI items scores. The TAI contains 15 items that break down the transfer into components that are scored either a ‘1’ for properly executed technique or a ‘0’ for improperly executed technique . Only 11infimum (inf) and supremum (sub) of the agreement were set as:m” is the mean of differences of both sensors and “s” is the standard deviation. Based on the Gaussian hypothesis, if 95% of the data are within the range between the inf and sub, it is valid to affirm that the two methods are interchangeable [All statistical analyses were performed in SPSS 27. Intra-rater reliability and reliability within a sensor were calculated using ICC 3,1 (two-way mixed-effects model with absolute agreement) for all the trials. Inter-rater reliability, reliability between sensors, was calculated using ICC 2,1 (two-way random-effects model with absolute agreement). ICC values for both analyses were characterized as excellent (ICC > 0.8), good (ICC 0.6–0.79), moderate (ICC 0.4–0.59), fair (ICC 0.2–0.39), or poor (ICC < 0.2). Furthermore, agreement between sensor features was evaluated using Bland–Altman plots. The “agreement” in this study is the characteristic that describes how close the two measurements are. The angeable .A percent agreement was computed between the Azure, and V2 ML predicted TAI scores across 150 transfers . A confusion matrix was used to determine the accuracy of the ML-predicted TAI scores for each sensor in comparison to the ground truth scores for each transfer.The V2 showed higher intra-rater reliability (ICC = 0.60–0.82) than the RealSense (ICC = 0.25–0.70) for the four key variables . Both V2The LPOE and TF have moderate inter-rater reliability (ICC = 0.52 and 0.51) , while tThe ICC values for the Azure were higher (0.91–0.92) than those of the V2 (0.82–0.92) for three of the key variables analyzed . The TF The DSWP had excellent between-sensor reliability (ICC = 0.91) and all other variables  had good reliability  . The BlaThe percent agreement between the two sensors for the predicted TAI scores across all the trials and items was 85.9% (STD 12.8%) . The TAIBoth the V2 and Azure sensors showed a high level of accuracy (>77%) for predicting the TAI scores of ‘properly’ executed transfers. Neither of the sensors did well at detecting when improper transfer technique was being used . For exaA recent application was developed to auto-score the quality of wheelchair transfer techniques (TransKinect); however, the sensor that is central to capturing the movement strategies (Microsoft Kinect V2) is no longer being produced. A comparative study was conducted to determine if another sensor could be interchanged with the obsolete one. Overall, the results show that Microsoft’s successor to V2, the Azure, showed even better reliability compared to the V2 and Intel’s RealSense for measuring the key variables important for evaluating the quality of transfer techniques. The Azure also showed high levels of agreement with the V2 in relation to the magnitude values of these variables, and the two closely agreed with each other on the auto-generated scoring of proper and improper transfers. However, when looking at how each of these sensors did in detecting proper from improper technique compared to ground truth, both sensors were good at predicting when ‘proper’ technique was used, but both fell short in predicting when improper technique occurred. These results suggest that the ML models of the TransKinect application may need to be further examined, refined, or retrained to improve their accuracy in predicting improper technique. The results of this study provide important information for the next steps of TransKinect application development.The quality of wheelchair transfer techniques can be evaluated by multiple components related to wheelchair setup, body setup, and flight movements using the latest version of the TAI 4.0 . The kinWe examined the intra-rater reliability of the sensors by analyzing repeated trials performed by the same participant. Some intra-trial variability is expected because the individuals could have varied their technique for each transfer. However, because both sensors are watching the same transfers, the variation from the participants would have been controlled for in the analysis. Thus, the intra-rater reliability from the repeated transfers provides an indication of how reliable the sensors are relative to each other in detecting the motions.The reliability analysis comparing RealSense to the V2 shows that the RealSense would not be a viable substitute for the V2 in the TransKinect application. The RealSense had worse intra-sensor reliability than the V2, with only two of the key variables scoring a good ICC (the other two being fair). On the other hand, the V2 had two excellent and two good ICC values. Additionally, the RealSense had broader 95% confidence intervals than the V2. Furthermore, the inter-sensor reliability analysis showed that the RealSense and V2 have moderate ICC for only two of the key variables (LPOE and TF). The sensors had a very low agreement for DWBW and LE, the former being the variable used to identify the different phases of the transfer. These results indicate that the V2 is much more reliable for repeated data collection than the RealSense, and that the RealSense should not be substituted into the TransKinect application due to its lower intra- and inter-sensor reliability than the V2.The reliability analysis between the V2 and the Azure, however, showed an opposite trend. While all the key variables showed excellent intra-sensor reliability, the ICC values for the Azure were even higher than those of the V2. The 95% confidence intervals for the Azure were also narrower than the V2, indicating greater stability of the calculated features. The inter-sensor reliability between the V2 and the Azure also showed ICC values in the good and excellent range. The Bland–Altman plots also showed that the magnitude differences between the two sensors for the key variables are small and that most of the data lie within the 95% limits of agreement with the exception of a few outliers. These results suggest that the Azure could be a viable substitute for the V2 in the TransKinect application.Before full integration of the Azure into the TransKinect application, a proper validation and compatibility test of the ML models used to predict the TAI item scores is necessary. The results showed that the Azure and V2 had high levels of agreement on most TAI items, indicating that the Azure should be compatible with the existing ML models. All but two items scored an agreement over 75%, Item 7 and 11 (59.3% and 71.3%) (feet position and leading arm-hand grip). It should be noted that the V2 has very low accuracy in body tracking with the lower extremities for individuals in wheelchairs. Often, the sensor would confuse the wheelchair frame or headrest as part of a body and interfere with the data collection. It was specifically seen in this analysis that the pelvis marker on the V2 would consistently lag behind the actual body; additionally, there were times when the V2 lost sight of several segments during the transfer. This problem was not seen in the Azure data collection; all joints were tracked accurately, and there was no data loss for any of the joints. These differences between the actual body position and the skeletons of the two sensors are shown in The accuracy of the ML-predicted TAI scores using both Azure and V2 sensor data tested against the ground truth scores for proper and improper transfer techniques showed high accuracy in detecting proper technique when proper technique was used but tended to assign proper technique when improper technique was used. Thus, the current ML models appear to over predict good quality transfers, regardless of the sensor used. There are several possible reasons this could have occurred. The ML models were developed using 100 unique wheelchair users who each contributed multiple transfer trials (up to five) to the analysis. All the trials were randomized and split into a 70/30 training and testing set. It is possible that some of the same subject’s trials were part of both the training and testing sets. While this is a relatively common practice in ML, this method can lead to overfitting . TherefoThe TransKinect application offers a promising opportunity to automate the evaluation of SPTs and support clinical decision making. The use of commercial hardware, beginning with the low cost, marker-less Kinect V2 sensor, makes the system more accessible to clinics and easier to use. However, using commercial hardware may also lead to the inevitable possibility of discontinued production leading to the need to find an acceptable substitute depth sensor for future use. The Intel RealSense, while an affordable commercially available device, unfortunately, does not demonstrate the necessary reliability and agreement with the V2 to justify it as a viable substitute sensor. On the other hand, the Azure demonstrates sufficiently high inter- and intra- rater reliability when compared to the V2, which suggests it may be an adequate replacement within the TransKinect system. The final, and most clinically relevant, finding from this analysis is the true accuracy of the ML algorithms used to produce TAI scores from the collected data. The results of this analysis suggest that while the Azure and Kinect V2 are tracking body movement throughout the transfer process precisely, the predicted ML TAI scores did not match the ground truth scores for transfers that were performed using improper technique.The future steps for this application are to retrain the ML models with the new Azure sensor, placing an emphasis on identifying incorrect technique, using a more balanced dataset, and performing testing validation on a new batch of subjects.