Graduate teaching assistants (GTAs) play important roles in engineering education at the undergraduate level. Since there are lots of technological content knowledge (TCK) in engineering curriculums, the improvements of GTAs’ teaching skills on TCK will help the teaching effectiveness of the curriculums. As the instructor’s knowledge about technology-infused instruction for TCK is the core of the teaching skill, Technological Pedagogical Content Knowledge (TPACK) is taken as a framework to measure the extent to which instructor can teach with technology. In this study, an online questionnaires survey covering GTAs’ program coordinator, teacher, graduate student and undergraduate student has done to explore the factors of enhancing GTAs’ TPACK performance. The quantitative analyses through a structural equation modeling approach indicates that the roles of the GTAs should be clearly recognized by the teacher, program coordinators and GTAs themselves. An evaluating procedure for GTAs should be established; The attitude and self-efficacy of GTAs should be improved through the training courses and the field trips while the promising expectation from the undergraduate student on the roles of GTAs can improve the performance of GTAs’ program. Our results will be helpful not only for engineering curriculum, but also for other curriculums. Engineers have been valued for their technical expertise for centuries . This exIn order to reduce the burden of teachers, Graduate teaching assistants (GTAs) are often adopted to lead laboratories and recitation including practice the new versions of technologies and field trip, especially at large, research-intensive universities , 36. GTAThe main goal of GTAs’ program for engineering curriculum is how to facilitate TCK to be understood and mastered by undergraduate students through the technology with pedagogical issues. There have been increasing applications of TPACK to build instruments to assess preservice teacher’s technology integration skills , 31, 32,There are many ways of measuring the TPACK, such as self-assessment surveys , classroIn the process of teaching a core professional curriculum in engineering major, there are always four types of people involving in GTAs’ program. The first one is the GTAs’ program coordinator who oversees the implementation of the program including the selection and assessment of GTAs. They connect the instructors (teacher and GTAs) and the undergraduate students and often monitor the program. The second one is the teacher who works together with GTAs. The third one is the graduate student who participates in the program. The teacher and graduate student are instructors for the curriculum, and they should have TK, PK and CK. The fourth one is the undergraduate student who is the receiver from the program. The relationships between them can be illustrated in Fig. There have been 229 participants taken part in our survey. They are from the school of water resources and hydropower engineering, Wuhan university, China. This school is the biggest engineering school in terms of number of graduate students in the university. There are seven GTAs’ program coordinators including current and former staff. They have done the graduate affairs for many years and know the whole situations of the program and the procedure relating to teacher, graduate student and undergraduate students very well. Their experiences on the GTAs’ program are rich, showing that their answers to the survey reflect effects of the program. 47 teachers have participated in our survey. All of them have a lot of teaching experience for undergraduate students and they know their teaching curriculum very well. As only Ph.D. students have right to do teaching assistant, 93 Ph.D. graduate students  have taken part in the survey. Half of them have done the teaching assistant while the left will do this work in the coming semesters because the qualified teaching assistant work is one of the necessary requirements for their graduation. The purpose of the GTAs’ program is to improve the quality of engineering teaching for undergraduate students and also is to prepare the next generation of teacher. As the performer of the program, the conception and expectation of the graduate student on the program are very important for their assisting activities. Thus, their responses to our survey will be vital to find the factors that influence the program. Furthermore, there are 82 undergraduate students that have participated in the survey and all of them have already experienced in the GTAs’ program. The percentages of participants are shown in the Table Different amounts of experience as GTAs’ program participants have been involved in the survey to increase the ability of the results discussed in this study to be transferrable to other settings. Our comprehensive account of the study also lends itself to the trustworthiness of our conclusions. There might be bias of this study due to inadequate comprehension of our stakeholder, but a lot of feedbacks from the pioneer and experienced professors can assure the accuracy of the results.In order to facilitate answering the questions for the survey, online questionnaires for all kinds of participants were developed to establish basic demographic information about each participant, capture how they teach or learn the technological knowledge content, and figure out what their opinion on this program are. For every item in the survey, participants were asked to identify their level of agreement with the following choice: “strongly agree” as 5 score, “agree” as 4 score, “somewhat agree” as 3 score, “disagree” as 2 score, “strongly disagree” as 1 score.The suggestions of the program from every participant were also collected. Open-ended question was formulated in semi-structured interviews for the qualitative analysis. This survey and interview had been done during the first semester of 2020–2021. To further explore the relations between their perception, expectation, attitudes and self-esteemed to the GTAs’ TPACK program on core professional curriculum for undergraduate engineering major students, a structural equation modeling approach is chosen  for corrIn order to explore the factors of impacting the performance of the GTAs’ TPACK program for curriculum teaching in engineering major, the quantitative results with respect to the relation between the contextual factors or latent variables  from the coordinator, teacher, graduate student and undergraduate student have been presented and discussed as well as the qualitative analysis regarding to the connections between the graduate student and the other three partners. The relationships between contextual factors or latent variables are investigated via the structural equation models. The recommendations on enhancing the performance of the program are also provided according to the quantitative and qualitative results.Cronbach’s α values were calculated to confirm the internal reliability of defined latent variables. A value of more than 0.7 shows an acceptable level of reliability of a factor or latent variable , 41. The1(2): λ = 0.97, q1(3): λ = 0.97 and q1(4): λ = 0.98), which can be found by the percentages of the Five score chosen in Fig. 1(7) for the coordinator perception on teacher, while the factors q1(12), q1(10) and q1(11) for the perceptions on graduate student should be paid attention to improve the performance of the GTAs’ TPACK. The roles of GTAs are very positive in TCK learning for undergraduate student in coordinator perception. TK for a teacher is the dominator factor in the coordinator’s perception on teacher’s TPACK. According to the face-to-face interview for quality analysis, the procedure of selection and assessment of GTAs is not regular and should be invested more money and people to improve the GTAs’ teaching quality. There are only seven program coordinators participate our interview, but their poles plaid in this program shown in Fig. Figure 2(2) (the highest) and q2(7) (the lowest) are very well, which reflect the high TK levels of the teacher. There are two parcels or latent variables that are teachers’ self-efficacy on teaching and their perception on GTAs’ TPACK program as shown in Fig. 2(2)), CK (q2(3)), PK(q2(5) and q2(6)) in the self-efficacy parcel while the TK (q2(12)) and contribution (q2(13)) of GTAs are the two high factors in teacher’s perception parcel. However, the PK of GTAs is not the high factor q2(15), which means its importance has not been recognized by teachers. As the correlations between q2(4), q2(9), q2(10) and themselves esteemed on the teaching are not high , the goal and the way of TCK in a curriculum are not clear by every teacher, which can directly impact their perception on GTAs. Thus, the correlations in their perception on GTAs parcel are relative lower than those in themselves self-efficacy parcel. And the percentages of the five scores in their perception are also lower.Teacher plays principal role in the teaching process of a curriculum, but TCK of the curriculum is not only taught at classroom as a form of a lecture, but also should be guided to be practiced through computer software or field trip. A teacher majored in an engineering should be good at solving technical problems for teaching even for the first online teaching during the COVID-19 pandemic in 2020, thus the scores of q3(4) and q3(2). The computer programming capability as a TCK is important factor to do GTA. The updated TCKs for the curriculum are also higher correlated to the self-efficacy on TCK (shown in q3(4): λ = 0.81), but it has not been cared by graduate student : λ = 0.73), its lower mean score in Fig. 3(9): λ = 0.84) and TK (q3(7): λ = 0.82), thus training works on PK should be done for GTAs even the score of q3(6) referring to the PK is relatively high. The high factors for their perception on the program are the ways of selecting and evaluating for GTAs : λ = 0.95 and q3(11): λ = 0.93) while there are high mean scores in Fig. The graduate student is the performer of GTAs program and directly determines its effectives of the program. It is interesting to find that the percentages of the four score and the three score are highest in Fig. c while tore of q3 referrin4(2) in Fig. 4(4) and q4(5), which can further explain why the undergraduate student like to communicate with the GTAs. It has also proven that the importance of the GTAs for the teaching. The high factors in expectation parcel are q4(8) and q4(9) which are the expectations of the undergraduate student for GTAs and of their learning for the programming or software operation curriculum, especially the new requirements for GTAs during the corvid pandemic period (q4(9)). The scores of these two factors are also the highest in the expectation parcel  and q4, which c4) and q4, which cAccording to the findings discussed above, all the participates complain that there is no complete rule for choosing GTAs and evaluating their performances. The importance of GTAs has still not been recognized by the program coordinator and the teacher. Some teachers even don’t know this program. If the roles of the GTAs played in a curriculum are not assigned by teacher and the GTAs themselves cannot do anything. And of course, the undergraduate student as a receiver don’t have good experiences with GTAs even they have high expectation on them. Beside the passive results of the assessment on the current situation of the program, there are also positive findings. The participating teacher or the GTAs indeed hold good TKs including the teaching tools and the professional software, even though they may lack PK while the attitudes and expectations from the undergraduate students will force the coordinator, teacher and GTAs to know the importance of the program and the advantages of the GTAs. Especially, the roles of GTAs are expected to be friends rather than only instructors, which can attract them to the curriculum or the major and can help them to relieve psychological pressure as well. Another important point from the findings is the high self-efficacies on PK, CK and TK for GTA. They can assist teaching very well, if they have been trained by the necessary pedagogical knowledge including the ways of teaching TCK.Our findings provided insights into the development of research tools for probing GTAs performance. Objective evaluations of performances of the GTAs’ program may be an immediate indicator of success or failure of the program for the teaching in engineering curriculum. Our findings in this research may inform not only the teacher and the program coordinators to establish a system of selecting and evaluating but also the GTAs themselves with a way to know the roles and importance of GTAs.Psychological research has addressed the influence of self-efficacy over psychometric features about teaching, but the scores of the self-efficacy of the graduate students are low, which indicates the relatively poor performance for their teaching. The self-efficacy of the teacher is good and also has led to successful teaching esteemed by themselves. And the successful mastery teaching experience is deemed as the most important source contributing to the self-efficacy in accomplishing a specific teaching activity. Furthermore, such experience can affect graduate student’s beliefs and attitudes on GTAs’ program, so the coordinator and teacher should pay more attention to the ways of acquiring mastery experience for graduate students regarding their individual characteristics. Eventually, the performances of GTAs’ program will be improved to enhance quality of the engineering education.This paper examined the GTAs’ TPACK for engineering curriculum teaching. Based on their responses through the survey, it can be concluded that more efforts should be done for improving their performances. First, their perceptions on the importance of GTAs have not been recognized by the teacher and even the coordinators as there are no rational procedures in respect of selecting and assessment of GTAs and planning program for the future. Second, the attitude and self-efficacy of GTAs should be improved through the successful teaching. The training course and field trip for their PK for the TCK might be an effective way of obtaining the success. Third, the expectation from the undergraduate student on the roles of GTAs is very promising for the implementation of the GTAs’ program. Therefore, our findings have not only found the promising future of the GTAs’ program, but also provided insights for development of GTAs’ program. As the GTAs’ program is still implementing, more researches should be done, including not only quantitative but also qualitative research, to improve curricula.


Ms 6.9 earthquake in 2022 and discuss the historical seismic activities and corresponding stress adjustment processes in the research region. To analyze and study the seismogenic structure and mechanism of the earthquake, we investigated the spatial and temporal distribution characteristics of the Menyuan earthquake sequence and analyzed the InSAR coseismic deformation field. We obtained the precise locations of the main shock and aftershocks and the coseismic InSAR deformation field of the main shock. It was confirmed that the Ms 6.9 earthquake was a shallow sinistral strike-slip earthquake, which led to the sequential activation of the Tuolaishan and Lenglongling faults. The main seismogenic fault of the mainshock was the northwestern end of the Lenglongling fault, and the earthquake rupture was segmented. It can be inferred that the earthquake was a stress-adjusted event triggered in the Qilian-Haiyuan tectonic belt caused by the northeasterly push of the Qinghai-Tibet Plateau. The risk of moderate to high earthquakes in the region remains high in the future, requiring enhanced seismic observations.Precise earthquake locations and InSAR (Interferometric Synthetic Aperture Radar) deformation observation are the major methods to understand the earthquake occurrence and disaster-causing process. This paper proposes a processing framework for analyzing strong earthquake mechanisms from one-dimensional velocity inversion to precise earthquake locations combined with InSAR deformation observation, and discusses earthquake-generating fault and dynamic mechanisms of tectonic deformation. We analyzed the Menyuan  After the occurrence of strong earthquakes, precise earthquake locations and surface deformation observation are common research methods for understanding the mechanism and process of earthquake occurrence . AccuratMS 6.9 earthquake occurred in Menyuan County, Qinghai Province, China, at 01:13 on 8 January 2022; the initial location of the epicenter was 37.77° N, 101.26° E, and the focal depth was 10 km. This earthquake was the third MS > 6 event after the two earthquakes MS 6.4 in 1986 and MS 6.4 in 2016. According to a field investigation, the surface rupture length caused by this earthquake exceeded 20 km, with a maximum 2.1 m sinistral dislocation. A total of 4052 houses were damaged and nine people were injured in several counties near the epicenter, and the tunnels and facilities of the Haomen-Junmachang section of the Lanzhou-Xinjiang high-speed railway were seriously damaged. Therefore, this earthquake has aroused widespread concern in academia and the public. According to the tomographic imaging results of the upper and middle crust in the source area, the Mengyuan Ms 6.4 (2016) and Ms 6.9 (2022) earthquake occurred in the zone of dramatic changes in P- and S-wave velocities, and this region also corresponds to the transition zone of high and low values of Poisson’s ratio and saturation [Ms 5.2 aftershock that occurred on 12 January was close to the 2016 earthquake, which suggests that this seismic activity led to a full release of the energy accumulated by the Lenglongling rupture, and that a larger magnitude earthquake is unlikely to occur in the near future [According to the China Earthquake Networks Center (CENC) report, an turation ,22. In ar future . Howeverr future . Our purStrong earthquakes occur frequently in Menyuan area, and the structural characteristics are complex. It is worth noting that the 1986 Menyuan earthquake was mainly a normal fault type, while the 2016 Menyuan earthquake was a thrust type ,25,26. Bhttp://10.5.202.37:8080/JOPENSCat, (accessed on 18 January 2022)). The distribution of earthquake epicenters in Menyuan and surrounding areas is shown in Ms 6.6 mainshock occurred at the western boundary of the Lenglongling fault near the boundary with the Tuolaishan fault. Owing to the regional stress adjustment caused by the mainshock, the following two aftershocks with magnitudes greater than Ms 5.0 occurred on the eastern edge of the Tuolaishan fault and in the middle of the Lenglongling fault.We downloaded the earthquake catalog from 2009 to 2022 from the CENC  was also dominated by compressional thrust. Since the late Quaternary it has changed to be dominated by sinistral thrust. Moreover, the fault was continuously active in the Late Pleistocene and has been active in the Holocene (Q3 and Q4) [The two creasing . The 202f 1–3 km ,38. He ef 1–3 km  found th and Q4) ,41.Ms ≧ 3.0, and two aftershocks exceeded Ms 5.0, i.e., Ms 5.1 at 2:09 on 8 January and Ms 5.2 at 18:20 on 12 January 2022. To ensure the reliability of the earthquake’s epicenter, we selected the seismic events (508 times) recorded by more than four stations for analysis. First, the VELEST location method was used to relocate the downloaded seismic events and optimize the velocity model; 500 km was selected as the threshold for the epicenter distance range, and the screened results of the seismic phase travel-time are shown in According to the CENC monitoring records, from 8 to 15 January 2022, a total of 2357 active events of the Menyuan earthquake were recorded. Among them, 1648 earthquake events were recorded by a single station, and 508 earthquake events were recorded by more than four stations; 13 earthquakes had The accuracy of the seismic location mainly depends on the selection accuracy of the seismic phase arrival and the velocity model used for location inversion calculations. An accurate one-dimensional velocity model can provide more accurate source information. At the same time, the minimum one-dimensional velocity model is reliably used in seismic location, which can minimize the root mean square (RMS) of travel time residual . Velest To further improve the location accuracy of seismic events, we continued to use the double-difference location method to perform precise location processing based on the relocated earthquake catalog. The double-difference location method (HypoDD) proposed by Waldhauser and Ellsworth in 2000 has been widely used in microseismic and natural earthquake locations ,48,49. TWe selected 50 km as the upper threshold of the earthquake event pairing distance in precision location using the double-difference method. If the distance of earthquake event pairing exceeded the threshold, it was not considered. In addition, the number of earthquake events paired with the same earthquake was set to be no more than 10, and we used a total of 64,236 P-wave phases and 55,034 S-wave phases for the double-difference method for precise seismic relocation. Considering the different degrees of errors introduced in the manual selection process of the initial seismic phases, the seismic phase weight of the P wave was set to 1.0, and the seismic phase weight of the S wave was set to 0.6 in the calculation process of the precise location in this study . The preMs 0.5–6.9 earthquakes were obtained after precise seismic location using the double-difference method. The spatial location of the main earthquake (Ms 6.9) was 37.767° N, 101.276° E, and the depth was 12.3 km. Its horizontal location was at the intersection of the Lenglongling fault and Tuolaishan fault. The earthquake location results of this paper are consistent with those of other researchers [Ms 5.1 aftershock at 2:9 on 8 January was 13.3 km, which indicated that the fault slip continued to extend and develop at deeper depths after the mainshock. After relocation, the earthquake distribution was more concentrated compared with the origin catalog, and the Ms 5.2 aftershock on 12 January occurred on the Lenglongling fault. This indicates that Lenglongling fault was activated after certain stress accumulation. A comparison of the focal mechanism inversion results of major earthquakes . The corresponding spatial distribution characteristics of the aftershocks that occurred near the Lenglongling Fault (LLLF) and the Tuolaishan Fault (TLSF) also verified the inversion results of the focal mechanism of the Ms 5.1 and Ms 5.2 aftershocks.In total, acceptable location results of 384 earchers ,52,53. Tthquakes  indicateMs 5.2 aftershock on the Lenglongling fault was about 75°. Under the same tectonic stress, the two fault zones had similar fault tendencies. It is worth mentioning that the AA’ profile was about 4 km away from the surface of the Tuolaishan Fault in horizontal projection. Regardless, according to the fault dip analysis, Tuolaishan Fault may have a specific inclination in this section. Hence, the seismic activity in this section mainly occurred within the main fault of Tuolaishan.The BB’ profile near the mainshock was fitted with an 80° dip angle of the fault plane, while the fitted fault plane dip angle of the DD’ profile of the https://dwtkns.com/srtm30m/ (accessed on 18 January 2022)). After the phase unwrapping of the phase differential interference results by the branch-cut method [InSAR technology uses the reflection of microwaves emitted by a satellite on the ground to calculate the phase difference, and then performs an inverse calculation to obtain the surface micro-deformation . After tt method , an addit method . For thet method  to estimBased on this earthquake’s InSAR coseismic deformation field, the two interferograms from the ascending and descending orbits indicated relatively obvious deformation information a,b, consThere is a linear relationship between the slip (strike-slip and dip-slip components) on the fault plane and surface deformation. According to the InSAR deformation field , we furtMW 6.55–6.78, which is similar to the magnitude in The slip distribution obtained by the inversion is shown in Ms 6.9 occurred at the intersection of the Lenglongling fault and Tuolaishan fault, the Ms 5.1 aftershock that occurred on the same day was located to the west of the Tuolaishan fault, and the Ms 5.2 aftershock that occurred on 12 January was located on Lenglongling fault to the east of the main earthquake. The InSAR coseismic deformation showed a butterfly-like deformation pattern. The similar apparent deformation size and opposite amplitude polarity characteristics of the two walls of the fault also indicated that the earthquake activity was mainly strike-slip.The surrounding terrain of the Menyuan earthquake is complex, and the altitude varies greatly, which corresponds to the complex tectonic stress field in the Menyuan area and the unstable direction of block movement. The earthquake occurred at the intersection of Lenglongling fault and Tuolaishan fault, both of which have sinistral strike-slip characteristics. Our precise earthquake location results showed that the Menyuan earthquake sequence was distributed in the range of about 50 km from east to west and about 30 km from north to south. The main earthquake of MS 6.9 earthquake is Lenglongling fault. The earthquake caused the SW hanging wall of Lenglongling fault to rupture westward toward Tolaishan fault and the NE footwall to rupture northwest toward the end of Sunan-Qilian fault. The rupture length of Tuolaishan fault caused by the earthquake was about 10 km, and that of Lenglongling fault approached 20 km. The slip distribution of this earthquake on the fault plane was mainly concentrated in the depth range of 0–13 km downward along the dip, with a maximum slip of 4.5 m. After the main shock, the aftershocks moved together in Tolaishan fault and Lenglongling fault.Based on the precise location and InSAR results of the earthquake, we believe that the main seismogenic fault of the Menyuan MS 6.4 earthquakes. However, unlike the 1986 event, which was a normal fault earthquake, and the 2016 event, which was a thrust earthquake, this event was a strike-slip earthquake. These findings indicate the complexity of the seismogenic environment in this region. The present crustal motion field shows that the Qilian-Haiyuan tectonic belt where Menyuan is located is experiencing continuous and strong lateral escape, south-north crustal shortening and strong vertical uplift due to the northeasterly push of the Qinghai-Tibet block and the Alashan block in the northeast, which indicates that the dynamic background of this area is mainly crustal shortening and sinistral shearing.This earthquake was the third strongest earthquake in Menyuan after the 1986 and 2016 Ms 5.1 earthquake on the Tuolaishan fault tens of minutes after the mainshock. The occurrence of these two strong earthquakes and other aftershocks released the stress accumulation state on the western side of the Lenglongling fault zone to a certain extent, but also caused the imbalance of the stress state on the eastern and western sides of the Lenglongling fault zone, and finally contributed to the occurrence of the Ms 5.2 earthquake on 12 January. The CC’ profile in Based on this, we believe that the occurrence of this main shock was the result of a sinistral strike-slip dislocation at the intersection of the Lenglongling fault and the Tuolaishan fault when the Qinghai-Tibet block was pushed in a northeasterly direction, resulting in a critical state. The undischarged energy triggered the MS 8.0 earthquake in 1927, Minqin MS 7.0 earthquake in 1954, Menyuan MS 6.4 earthquake in 1986, Menyuan MS 6.4 earthquake in 2016 and this MS 6.9 earthquake. Among them, both the 1986 Menyuan earthquake and the 2016 Menyuan earthquake occurred at the two end of branch fault on the northern side of Lenglongling fault [The Qilian-Haiyuan tectonic belt, where Lenglongling fault is located, is an important boundary fault for adjusting tectonic stress along the northeastern margin of the Qinghai-Tibet Plateau, where there have been many strong earthquakes in the past one hundred years, including the Gulang ng fault ,59,60. Tng fault . These fIt is well known that the occurrence of moderate and strong earthquakes is caused by the overall unstable sliding of a fault, and a decoupling layer is often required to provide the overall dislocation conditions for the fault during the unstable sliding process ,63. The Ms 6.9 earthquake and its aftershock sequence, we analyzed the precise locations of the Menyuan earthquake sequence and InSAR coseismic deformation field and finally drew the following conclusions:(1)Ms 6.9 main earthquake  occurred at the intersection of the Lenglongling fault and the adjacent Tuolaishan fault, while the following two Ms 5.1 and Ms 5.2 aftershocks occurred in the Tuolaishan and Lenglongling fault zone, respectively.According to the relocations of the earthquake sequence, the (2)Ms 6.9 mainshock changed the local stress field and activated the Tuolaishan fault zone. Subsequently, many aftershocks gradually transitioned from the Tuolaishan fault to the Lenglongling fault. The Ms 5.2 aftershock on 12 January was triggered during the eastward transmission of the stress effect.The focal mechanism solution of the earthquake and the spatial geometric distribution of the aftershock sequence exhibited prominent east-west distribution characteristics. The energy released by the (3)The earthquake reflects the calibration of accumulated tectonic stresses inside the Qilian-Haiyuan tectonic belt, where the Lenglongling fault lies under the action of northeasterly compressive stress on the northeastern margin of the Qinghai-Tibet Plateau. The higher recurrence rate of moderate-to-high earthquakes in this region also indicates that the future seismic hazard remains high, and earthquake observation and early warning in this region should be strengthened.To analyze the seismogenic process and structural characteristics of the Qinghai Menyuan


Funded by the Centers for Disease Control and Prevention’s Building Our Largest Dementia Public Health Program through U.S. Congressional appropriations for the BOLD Infrastructure for Alzheimer’s Act, the Los Angeles County BOLD initiative (LA BOLD) was established in 2021 with the goal of addressing the silent but burgeoning epidemic of Alzheimer's disease and related dementias (ADRD) in Los Angeles County (LAC). In 2019, approximately 166,857 LAC residents were living with Alzheimer’s disease; this number is expected to increase 150% by 2040. The initiative brings together a diverse group of multi-sector stakeholders, including two Area Agencies on Aging, two major academic centers, the largest Medicaid health plan in the region, the county’s safety net health agency , and other key community partners. The primary objectives of LA BOLD are: (i) establish a countywide coalition to address ADRD locally, and (ii) develop a strategic plan to guide the coalition’s efforts. The present study describes the first year of this initiative, highlighting the formation of the coalition’s steering group, identifying the three focus areas for strategic planning – risk reduction, early detection, advance care planning – and sharing lessons learned from navigating the contextual realities of ADRD care and prevention in LAC’s diverse communities. Data and program accomplishments for this early phase of LA BOLD are discussed within the context of medical advancement, the financial realities of ADRD programming and services, and the geo-political factors that are being considered as part of the strategic planning and implementation processes.


While its basic principles and processes have been planned and implemented, how ViP works, for whom and in what circumstances are not yet well understood. Therefore, we propose for a realist evaluation (RE) – a theory-based evaluation, to address the current knowledge gaps. Evaluation findings may guide, support further development and broader uptake of ViP.Violet Program (ViP) was developed This study will be conducted in three phases: 1. development of initial program theory (IPT), 2. testing of programme theory, and 3. refinement of IPT. First, IPT will be elicited through review of programme documents, scoping review of reviews and in-depth interviews with stakeholders involved in the conceptualization of ViP. Then, a convergent mixed method study will be conducted to assess contexts (C), mechanisms (M) and outcomes (O) to test the IPT through interviews with stakeholders, surveys and analysis of program and administrative databases. Based on findings gathered and through consultation with respective stakeholders, IPT will be refined to highlight what works (outcomes), how (mechanisms) and for whom under what conditions (contexts). Historically, palliative care (PC) focused largely on patients with cancer. However, as the number of deaths attributed to non-cancer causes exceed cancer-related deaths  with pat2PC provided to terminally ill non-cancer patients has been found to be effective in reducing acute care utilization  and impr3Similar to other developed countries, there is a growing need for PC for a range of life limiting illnesses in Singapore . Disprop1012Traditionally, in Singapore, PC is provided to patients suffering  from cancers in hospitals, hospices and at home 15 and si16Contrasting needs, goals and care experiences among those with advanced illnesses nearing the end of life limits generalizability of existing cancer-based PC models . This suThere are compelling reasons for a new model of home PC to cater to the unmet yet growing needs of those with terminal non-cancer diseases. The World Health Organization (WHO) and National Strategy for Palliative Care 20 recommThe Violet Program (ViP) was developed as a collaboration between Changi General Hospital (CGH) and St. Andrew’s Community Hospital (SACH) to provide home-based PC to non-cancer patients residing in the Eastern region of Singapore. VIP aims to (i) reduce unnecessary acute hospital utilization (ii) improve health outcomes (iii) reduce caregiver burden through caregiver support (iv) honour patient preferences better through integrated care while (v) keeping healthcare cost affordable.nd line support. Advice over the phone or urgent home visits are done depending on assessment of the situation by the on-call staff. Patients who have stable PC needs over a period of time have their care de-escalated to the generalist team till their care needs next peak.CGH is a 1,000-bedder acute care hospital with a consult-based PC team who also runs an outpatient PC clinic. SACH is a step-down care rehab and subacute facility that also houses a 24-bedder inpatient palliative ward and home care services. ViP is an integrated program involving specialists from CGH PC team and SACH home palliative supporting the SACH generalist home medical and home nursing services. The specialist team is a multidisciplinary team comprising of doctors, nurses, medical social workers and pastoral care trained in PC with input from other allied health professionals  while the generalist team are doctors and nurses trained in care of chronic illnesses with exposure to end of life care issues. Using a common electronic platform, the specialist team provides care for patient and families with more complex care needs, e.g. symptom control requiring frequent medication titration, subcutaneous medication infusion, unstable symptoms or high psychological needs requiring frequent touch points. The generalist team provides basic PC by engaging patient and family in advance care planning (ACP), education on the dying trajectory, managing non-complex symptoms, providing caregiving training and identifying patients with escalating PC needs requiring specialist intervention. The generalists have lesser touch points of between 3–6 months unless a regular nursing procedure is needed, e.g. change of nasogastric tube, indwelling urine catheter, wound dressing. As such, the specialists also provide ad-hoc rapid response service for patients managed by generalists when acute events occur. The program provides a 24 hours service. An on-call phone number is given on enrolment and patient/Next-of-Kin is briefed on the use of on-call service. On-call number is manned by a PC nurse or doctor with a PC consultant as 22, not for dialysis) with prognosis of <12 months. Other forms of frailty, e.g. stroke or neurodegenerative illnesses, with prognosis <6 months are also accepted. This admission criterion is based on guidance laid down by Ministry of Health and Central Provident Fund Board who oversees the use of Medisave, a national medical savings scheme. Patients who do not fall within these criteria but assessed to have PC needs are discussed on an ad-hoc basis with ViP. Referrals are vetted by ViP doctors and suitable referrals are contacted and an assessment visit date is scheduled. Patients are enrolled into the program once the service agreement is signed. As of May 2022, majority of referrals came from CGH and SACH, with other organizations contributing only 7.5% of total referrals. Patients are identified in the inpatient, specialist clinic and home setting based on a pre-determined selection criterion. Pre-assessment by the PC team of the organizations is not required. In brief, the referral criterion includes (i) dementia (at least FAST 7), (ii) Chronic Obstructive Pulmonary Disease GOLD D, irreversible chronic lung diseases, (iii) heart failure (NYHA III or IV), or (iv) renal failure  assessment . PatientViP recruited its first patient in December 2020 and is in its infancy. While the general program design, processes and guiding principles were drafted by the ViP team with inputs from policy makers, it is not well understood “for whom” the planned program will be suitable and “how” ViP will work and be sustainable in the local context. To the best of our knowledge, ViP is one of the first integrated home PC model for non-cancer patients in Singapore involving an acute hospital, home medical and nursing teams and an inpatient hospice hence there are limited relevant context-specific insights from which ViP can learn from. As such, a rigorous and practical evaluation strategy to address the existing knowledge gaps is warranted. Findings from such evaluation will provide insights to support the implementation and further development of ViP and similar programs in Singapore and beyond.Commonly used evaluation design, Randomised Controlled trial (RCT) design, which focuses solely on outcomes evaluation is rigid when evaluating a complex intervention. It is unsuitable for evolving interventions and rarely adequately or even explicitly address the context-specific drivers behind any outcomes and their relationship to the underlying program theory . This maRealist evaluation (RE) is a more suitable approach for the evaluation of ViP. RE seeks to clarify implementation processes by providing a more explicit and in-depth understanding of the influence of existing and evolving contextual factors and mechanisms driving an intervention 25. FurthThe realist lens purports that an intervention is made of active theories that can only achieve successful outcomes if appropriate ideas are applied to the right context with appropriate social and cultural conditions . The con24Beyond answering if the program “works”, we propose to answer the following evaluation questions:To what extent is ViP successfully implemented?To what extent is ViP effective in achieving its intended outcomes?What are the essential conditions for achieving program intended outcomes?Whom (which population) does the program (not) work for and why?How does ViP work to achieve its intended outcomes?According to the framework for RE outlined by Pawson and Tilley , evaluatProgram related documents will be reviewed and a scoping review of reviews will be conducted. The JBI methodology for scoping review and PRISMA-ScR reporting guideline will be used . The scoConcurrently, semi-structured interviews with key stakeholders will be conducted to assess initial program theories, perceived roles of ACP, caregiver support, capacity building of generalists and formation of integrated PC team. A purposive sampling of approximately 10 key stakeholders who were involved in the conceptualization of the program including program director, key medical providers and planning team will be interviewed.Interviews will be recorded, transcribed verbatim and each transcript will be checked against recording for accuracy. Then, interview transcripts will be coded and analyzed by 2 evaluators using NVivo as suggested by Gilmore et al . TranscrA convergent parallel mixed methods study based on the triangulation design will be undertaken. Using the triangulation strategy, both quantitative and qualitative data will be collected concurrently. Data streams will be given equal weight and two datasets will be analyzed, compared and merged through iterative cycles of validation and confirmation of findings. Two distinct groups of study participants – healthcare providers/managers and healthcare users will be recruited into this study. Healthcare providers/managers who are involved in the implementation of ViP will be invited to participate in the interviews through emails. Healthcare users will be recruited through ViP team/physicians in-charge and/or home care nurses.Context, mechanisms and relationships between context and outcomes, as defined in phase 1, will be tested through interviews with healthcare providers and users. As suggested by Manzano , questioIn ensuring the quality of data collected, an interview guide will be used and interviewers will routinely reflect on interview experience and discuss data as they are being collected. Based on emerging understanding and identification of key knowledge gaps throughout the process of interviewing, interview questions will be revised accordingly.Healthcare services utilization, health outcomes, healthcare cost and caregiver burden data will be collected and analyzed over time to evaluate intervention outcomes of ViP. A quasi-experimental design will be adopted by comparing the outcomes of the intervention group against a comparator group for healthcare utilization, outcomes and cost. Due to limited data available, a pre-post comparison will be made for caregiver burden. A quasi-experimental design is selected as it more truly mimics real-world constraints and provide a balance between internal and external validity . PragmatIn identifying retrospective comparator group, relevant databases will initially be filtered for individuals who were referred to home hospice services between 2016–2019, have completed their advance care planning (ACP) and have died as of December 2020. Death status will be obtained from National Registry of Diseases Office records. Then, using a propensity matching strategy, a comparator group matched to intervention group in terms of age, gender, ethnicity, functional status, clinical status and re-admission characteristics will be identified. A ratio of 1:1 of intervention to comparator group will be used for matching.In the assessment of healthcare utilization and health outcomes, date of death will be used as reference point for comparison. Acute hospital utilization (emergency department (ED) attendances, inpatient admissions, intensive care unit (ICU) admissions and length of stay (LOS)) will be extracted backwards from the date of death for 1 month (30 days), 3 months (90 days) and 6 months (180 days). Concordance of actualized place of death with preferences discussed as part of ACP will be determined after death as described by Tan et al .We take a healthcare system perspective in examining the effect of ViP on healthcare cost. Development costs, program implementation costs and healthcare utilization costs will be measured. Development costs include costs incurred during the development of ViP. The program implementation costs consider costs related to the implementation of the program including the manpower, travel, equipment, materials used for patient and caregiver training and costs associated with multidisciplinary team meetings. The development and implementation costs will be systematically collected using the WHO’s CostIt instrument  and willData for both intervention and comparator group will be extracted from SingHealth-iHiS Electronic Health Intelligence System (eHints), a common database of patient level data across all SingHealth institutions. Development and implementation cost will be extracted from ViP program database and potentially finance department. For comprehensiveness, we may also merge dataset with data held by the Ministry of Health to obtain nationwide acute hospital utilization  using National Registration Identification Card (NRIC) of subjects included in the study.Primary informal caregivers of ViP patients will be invited to complete a Zarit Burden Interview (ZBI) – 12 items to assess caregiver burden at enrolment and every 3 months after enrolment until patients’ death. The ZBI is a widely used tool for assessing caregiver burden among those who provide care for patients with various illnesses, including those at the end-of-life 35. A higInterviews will be transcribed verbatim and each transcript will be checked against recordings for accuracy. Interview transcripts and observation notes will be coded using NVivo. Interviews conducted in languages other than English will be translated and transcribed in English. Like phase 1, interview transcripts will be coded and analyzed by 2 evaluators using NVivo as suggested by Gilmore et al . To ensuQuantitative data analysis will be conducted using STATA. First, univariate analysis will be conducted using chi-square for categorical variables and t-test/ANOVA for continuous variables. Then, to estimate the effect of the program on the outcome of interest, multivariable regression analysis  will be used for count data and generalized linear models will be used for continuous data. For dichotomous outcome variables, logistic regression will be used. Subgroup analysis may be conducted by patients’ primary conditions to allow for the detection of differential effects of ViP on various disease groups. To ensure robustness of the analysis, sensitivity analysis will also be performed.After both quantitative and qualitative data have been analyzed separately, findings will be integrated through a triangulation process at interpretation stage. The triangulation protocol  will be A few potential CMO configurations will be proposed. Stakeholder participants of phase 2 will be invited back for focus group discussions (FGD) to validate and ensure program evolution has been accounted for. The IPTs will be refined to highlight how to improve ViP by detailing what works (outcome), as well as how (mechanisms) and under what conditions (contexts).This protocol describes a RE of a home-based PC program for non-cancer patients in Singapore. RE is adopted to provide an in-depth understanding about whether the program works, for whom and in what circumstances. This study carries significant implications for various stakeholders. For the program, the study will generate new insights about the implementation, effectiveness of the program and explicit context-specific recommendations. By understanding what works and does not work, relevant stakeholders can refine program components and focus resources to ensure effectiveness of the program. Information on whom ViP works will help to refine selection criteria. Meanwhile, insights on mechanisms and contextual influence on ViP may be useful in informing required adaptations in the program strategies.For policy makers, findings from the study will inform the potential of generalist involvement in home-based PC and policy-relevant insights for future enhancement of PC to support the aging population. Globally, most prior evaluations of home PC programs 39404142 394041The RE approach adopted in this study provides a useful guiding framework for a comprehensive evaluation of ViP. It is well fitted as RE seeks to answer relevant questions of interest, is flexible and able to accommodate changes surrounding interventions implemented in real-world setting. Moreover, the use of mixed methods allow us to draw on the strengths of both qualitative and quantitative methods, enhancing the credibility of the evaluation findings and allows for generation of in-depth insights.With this evaluation approach, evaluators work closely with the key stakeholders  throughout the evaluation journey to co-define evaluation objectives, IPT, identify constraints to the feasibility of implementation of the evaluation itself, collect data, interpret findings and refine program theories. This provides confidence on the relevance of the evaluation efforts and likelihood for adoption of recommendations to improve relevant components of ViP and/or define future PC models.Nevertheless, RE approach and mixed methods design are relatively new in comparison to the positivist approach commonly used in the evaluation of medical interventions. This may draw criticism for not being as rigorous as the typical RCT due to the lack of knowledge on the utility of this niche approach. Furthermore, we acknowledge that the use of mixed methods design also increases complexity. We strive to reach optimal integration of data at multiple levels – study design, methods, interpretation and reporting – using the convergent parallel mixed methods approach which connects and merges methods and findings .Furthermore, as participation in the evaluation efforts is voluntary, we also recognize that selection bias may be introduced in this study. To account for selection bias, demographic information including age, gender, ethnicity and role  are collected and will be compared between study participants and those who declined to participate. Given the sensitivity around the topic of PC and prospective data collection methods, difficulties in recruitment and follow-up with respondents are also anticipated. In order to maximize recruitment numbers, evaluators will work closely with main care providers to obtain “buy-in”. If necessary, the recruitment period will also be extended to allow more people to participate. To minimize the numbers lost to follow-up, research appointments are scheduled at participants’ time and place of convenience in collaboration with participants’ healthcare provider. Where loss to follow-up inevitable due to death, survival analysis will be conducted to account for the missing data.Using the proposed methodology, the planned evaluation will highlight what works (outcomes), how (mechanisms) and for whom under what conditions (contexts) related to ViP. Findings gathered from this study will shed insights to inform decisions about implementation and further development of ViP. Furthermore, application of realist methodology for evaluation of complex interventions will also be illustrated.


While losartan and proton therapy were associated with reduced lymphatic ECs, these therapies also correlated with an increase in reactive EndMT. Thus, the development and inclusion of EndMT-inhibiting drugs  to a neoadjuvant chemoradiotherapy regimen featuring losartan and/or proton therapy may be most effective in depleting both lymphatic and reactive EndMT populations and potentially improving patient outcomes.Pancreatic ductal adenocarcinoma (PDAC) is one of the most treatment refractory and lethal malignancies. The diversity of endothelial cell (EC) lineages in the tumor microenvironment (TME) impacts the efficacy of antineoplastic therapies, which in turn remodel EC states and distributions. Here, we present a single-cell resolution framework of diverse EC lineages in the PDAC TME in the context of neoadjuvant chemotherapy, radiotherapy, and losartan. We analyzed a custom single-nucleus RNA-seq dataset derived from 37 primary PDAC specimens . A single-nucleus transcriptome analysis of 15,185 EC profiles revealed two state programs , four lineage programs , and one program that did not overlap significantly with prior signatures but was enriched in pathways involved in vasculogenesis, stem-like state, response to wounding and hypoxia, and endothelial-to-mesenchymal transition (reactive EndMT). A bulk transcriptome analysis of two independent cohorts ( Pancreatic ductal adenocarcinoma (PDAC) is a treatment refractory and lethal malignancy that is projected to be the second leading cause of cancer mortality in the United States by 2030 . The tumn = 18), with neoadjuvant FOLFIRINOX + chemoradiation (n = 14), or with neoadjuvant FOLFIRINOX + chemoradiation + losartan (n = 5) as previously described  implemented in sklearn to decompose the gene expression matrix into two matrices, one of which embeds the endothelial gene expression programs. Because the result of NMF optimization can vary between runs based on random seeding, we repeated the NMF 50 times and computed a set of consensus programs by aggregating results from all 50 runs and determining the stability and reconstruction error. This consensus NMF was performed by making custom updates to the cNMF Python package. To determine the optimal number of programs k, we struck a balance between maximizing stability and minimizing error of the cNMF solution, while ensuring that the resulting programs were as biologically coherent and parsimonious as possible.We extracted single-nucleus gene expression data from high-quality ECs using a custom subset of primary PDAC patients (DUOS dataset ID 000139) who underwent surgery without neoadjuvant therapy  and comparison with previously characterized endothelial signatures. Based on these annotations, each program was additionally classified as either a state or lineage program. To measure the similarity between PDAC-derived gene expression lineage programs and prior lung-derived single-cell endothelial lineage signatures  as an offset. The mixed effects model was implemented using the glmer R package.n = 19) into subgroups with  and without losartan  and separately into subgroups receiving low-dose radiotherapy  versus those receiving high-dose photon radiotherapy . We further stratified the subset of low-dose radiotherapy-treated patients into subgroups with low-dose photon radiotherapy (n = 7) versus those with low-dose proton radiotherapy (n = 4). No patients in our cohort received high-dose proton radiotherapy. Two-sided Mann–Whitney U test with Benjamini–Hochberg correction (FDR = 0.1) was used to compare treatment status with the proportion of ECs classified as a given program as well as the average expression of a given gene across patients.We stratified neoadjuvant-treated patients  and previously described  and non-irradiated (n = 24) conditions. To compute endothelial cNMF lineage program scores, we averaged the targeted transcriptome expression of genes overlapping with the top 200 weighted genes for each lineage program. Two-sided Wilcoxon signed-rank test with Bonferroni correction was used to compare lineage program expression scores across treatment conditions.Targeted transcriptome data from cultured primary human umbilical vein endothelial cells (HUVECs) were obtained from Morilla et al . HUVECs n = 140; PanCuRx (n = 168). Patients with metastases were excluded from this analysis. Gene expression levels from RNA-seq data were estimated using RSEM. We then deconvolved the cell-type proportions in each tumor using the following marker genes: endothelial , epithelial/acinar , myeloid , lymphoid , cancer-associated fibroblast , pericyte , Schwann , endocrine , intra-pancreatic neurons , and adipocytes . To compute cNMF endothelial program scores, we summed the endothelial compartment expression of the top 200 weighted genes for each program, and the z-score normalized the expression scores within the TCGA , and overall survival (OS) were available for 269 patients, of whom 154 had progression events and 167 died during follow-up. Multivariable Cox regression analysis was performed for TTP and OS with age, sex, grade, stage, and EC lineage program z-scores as covariates. Separately, the same analysis was performed with EC state program z-scores, instead of EC lineage program z-scores, as covariates.+, n = 210) and without  lymph node metastasis. Two-sided Mann–Whitney U test was used to compare N staging with z-score-normalized lineage program expression scores.Histopathologically annotated lymph node (N) staging was available for 279 patients . Patientn = 19) or without neoadjuvant treatment (n = 18) (n = 5) or without  losartan . We selected the number of programs (k = 9) based on optimizing stability and error  and general capillary-aerocyte (p = 4.20 × 10-9)], Lineage Program 2  [Schupp arterial (p = 2.37 × 10-18) and arterial-venous (p = 2.32 × 10-2)], Lineage Program 3 (venous) , and Lineage Program 4 (lymphatic) [Schupp lymphatic (p = 1.69 × 10-18)] .To learn recurrent nd error , yieldinnd error  overlapp). Treatment was associated with a higher proportion of Lineage Program 5 endothelial nuclei  and a lower proportion of Lineage Program 1 (capillary) endothelial nuclei    versus a high-dose photon radiotherapy subgroup (n = 8) with a BED of 55–59 Gy and separately into low-dose photon (n = 7) versus low-dose proton (n = 4) subgroups , consistent with prior observations that radiation-induced endothelial damage is concentrated in the microvasculature  and low-dose proton therapy  were both associated with a higher proportion of Lineage Program 5 endothelial nuclei  for vascular effects, in which high-dose radiation is typically defined as greater than 10 GyE (Radiation has dose-dependent effects on the vasculature . Proton culature  rather tn 10 GyE  so both ). We observed that Lineage Program 5 endothelial nuclei were enriched in leukocyte adhesion molecules , which when combined with enrichment in HALLMARK_TNFA_SIGNALING_VIA_NFKB pathway genes suggests an inflammatory role for Lineage Program 5  and enrichment for mesenchymal markers   analysis of Lineage Program 5 ECs versus all other EC subtypes  . In the n = 24) versus irradiated (n = 24) primary human umbilical vein endothelial cells (HUVECs) with targeted transcriptome data  (in vitro (p = 6.15 × 10-4) , mesenchymal , and neuroendocrine-like  program expression, respectively, compared with those of patients in the bottom quartile of Lineage Program 5 EC expression , we observed that the ratios of CD8+ T cells , dendritic cells (p = 2.16 × 10-2), macrophages (p = 2.17 × 10-2), natural killer cells (p = 2.76 × 10-2), neutrophils (p = 1.85 × 10-3), and Tregs (p = 1.40 × 10-2) to endothelial cells were enriched in patients with a higher expression of Lineage Program 5 ECs . The high expression of macrophage chemoattractants by Lineage Program 5 endothelial cells such as CSM1 and CCL2 , CD4+ T cells (p = 3.00 × 10-3), natural killer cells (p = 4.30 × 10-3), Tregs (p = 5.67 × 10-3), macrophages (p = 2.17 × 10-2), dendritic cells (p = 3.40 × 10-2), mast cells (p = 3.45 × 10-2), and pericytes (p = 3.41 × 10-2) to endothelial cells were enriched in tumors that had a higher expression of Lineage Program 1 (capillary) ECs . We note, however, that both of these analyses were conducted at an aggregated patient level since we lack spatial information in the snRNA-seq dataset  and State Program 2 (cycling) do not have significant associations with treatment (n = 135) and PanCuRx (n = 134) (Methods). We deconvolved the cell-type proportions for each tumor and computed the z-score-normalized program scores for the endothelial compartment. We then performed a multivariable Cox regression analysis of the overall survival (OS) and time to progression (TTP) endpoints with age, sex, stage, grade, and the z-score-normalized endothelial program expression as covariates. While age, sex, stage, and grade were not prognostic for TTP, age and stage were prognostic for OS   and Lineage Program 3 (venous)  trended toward a positive association for both OS and TTP but did not reach significance  or not . Node-positive patients had significantly higher Lineage Program 4 (lymphatic) EC expression in the primary tumor , suggesting that lymph node involvement can potentially be predicted from the prevalence of Lineage Program 4 (lymphatic) ECs in the primary tumor  and/or proton therapy  was associated or borderline associated with lower Lineage Program 4 (lymphatic) EC expression  or without neoadjuvant treatment (n = 18). Performing consensus non-negative matrix factorization (cNMF), we learned seven endothelial expression programs: two cell state programs , four cell lineage programs , and one program (Lineage Program 5) that did not significantly overlap with prior endothelial subtype signatures  and Lineage Program 5 (reactive EndMT) EC expression portended a poor prognosis in patients with untreated primary resected PDAC –11. Whiln vitro . Taken tn vitro   have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession numbers GSE202051.This study was reviewed and approved by Massachusetts General Hospital Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.CS, JS, JG, KJ, and WH developed the study concept and designed the computational analyses. CS analyzed the data with guidance from KJ and WH, scientific insights from JS, JG, and WH, and clinical insights from TH and JW. CS generated the figures. WH supervised the research. CS and WH wrote the manuscript, and all authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by NCI K08CA270417 (WH), Burroughs Wellcome Fund Career Award for Medical Scientists (WH), and Hopper Belmont Foundation Inspiration Award (WH). This study was also conducted with support of the Ontario Institute for Cancer Research  through funding provided by the Government of Ontario, the Wallace McCain Centre for Pancreatic Cancer supported by the Princess Margaret Cancer Foundation, the Terry Fox Research Institute, the Canadian Cancer Society Research Institute, and the Pancreatic Cancer Canada Foundation.We are grateful to our patients and their families, Aviv Regev, Tyler Jacks, Hannah I. Hoffman, Payman Yadollahpour, Cristina R. Ferrone, Motaz Qadan, Rakesh K. Jain, Ramnik Xavier, Andrew J. Aguirre, Mari Mino-Kenudson, Carlos Fernandez-del Castillo, Andrew S. Liss, David T. Ting, and many others for their contributions to the single-nucleus RNA-seq dataset.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Consumer and community engagement (CCE) in the implementation of genomics into health services and associated research is needed to ensure that changes benefit the affected patients. Queensland Genomics was a program to implement genomics into a public health service. We describe its Community Advisory Group’s (CAG) structure and function and provide recommendations based on the CAG members’ perspectives. The CAG provided advice to the Queensland Genomics program and its projects in an advisory capacity. The CAG was also resourced to develop and lead community-focused activities. Key enablers for CAG included; diversity of CAG members’ skills and experience, adequate resourcing, and the CAG’s ability to self-determine their direction. The CAG experienced limitations due to a lack of mechanisms to implement CCE in the Program’s projects. Here, we provide insights and commentary on this CAG, which will be useful for other initiatives seeking to undertake CCE in genomic research and health care. Consumer and community engagement (CCE) in research and health care is working in partnership with end-users to improve processes and outcomes  and addrIn general, a Community Advisory Group (CAG) consists of community members working with organisation representatives to provide community and consumer perspectives . In healHere we describe a CAG established to provide CCE within a program implementing genomics across the whole health system. We detail the CAG’s structure, which had the dual role of an advisory committee and developing community-led activities and research within the health implementation program. Finally, we provide recommendations for implementers based on CAG members’ experience as they move beyond an advisory capacity to activity-based leadership.Queensland Genomics (“the Program”) was a 5-years program (2016–2021) that aimed to implement genomics into Queensland’s health care system via a series of health implementation initiatives and research projects (‘the Program’s projects’) that focused on whole of system capacity and capability building .The CAG was established as an advisory group that reported to the Program’s Governance Board via the Executive Director, who attended CAG meetings as an ex-officio member . The ProThe selection of CAG members was a deliberate process by the Queensland Genomics business team and existing CAG members. Recruitment occurred through multiple methods; nomination of a person by key stakeholders, identification of suitable people at stakeholder meetings and events, and through expression of interest–circulated on behalf of the Program by health consumer organisations (for consumer recruitment) and health services . Applicants were considered based on targeted criteria, with a specific effort to recruit representatives with a broad range of experience .The selected CAG members had diverse personal and professional backgrounds and skills - contributing specialist knowledge and expertise to the Program. They provided individual and system-level input from the perspective of consumers, carers, health delivery workers, medical researchers, consumer advocates, and community representatives . MembersThe CAG ranged between ten to eleven members throughout the Program, with an annual renewal unless the member chose to withdraw. Most members continued for the Program’s duration, but some did take a leave of absence and return to the CAG to facilitate their health needs or carer responsibilities. Members were eligible to receive an honorarium to help support participation unless they were an investigator on a Program’s project, a Queensland Health employee, or a paid employee of a consumer, patient, carer or health peak body. The honorarium was based on local health advocacy policy  and equaThe minimum participation for CAG members was attending 3-h quarterly meeting with completion of the associated reading. Participation in all other Program events or CAG-led activities was at the discretion of the individual member. CAG members were also remunerated when invited to attend Program events or meetings. However, all CAG members provided significantly more time to the Program than then minimum, based on their own availability and areas interests.The Program established the CAG with a broad Terms of Reference outlining scope and structure while ensuring the CAG had sufficient autonomy to set their direction and agenda. Shortly after establishment, the CAG developed its strategy to inform the group’s direction within the scope outlined by the Terms of Reference. The CAG strategy was developed through a workshop with all members and facilitated by the CAG Chair and iterative feedback from CAG members on the draft. The resulting CAG Strategy outlined the principles underpinning the direction of the CAG’s activities for the Program’s duration and included the CAG vision, objectives, themes of work, and action areas. Themes for the CAG focused on collaboration, equity and access, education, advocacy, foundation and prioritisation, and real-world context .The Program allocated funds and resources for running the CAG. The CAG received administrative, logistical, and operational support from the Program’s Communication and Engagement Manager, who operated as the CAG Project Manager . The ProThroughout the Program, the CAG undertook tasks nominated by the Program, including contribution to the Program Governance Review and the Program’s project selection processes . In addiIn the Program’s early phase, the CAG self-nominated areas of interest and associated activities aligned with the CAG Strategy. These activities came from CAG members’ networks and inquiries directly to the Program, including Federal Government Parliamentary health inquiries, other organisation and research projects, and Queensland Health policy planning for genomics .During the Program’s later phases, the CAG identified, designed and ran research and non-research activities funded by the Program . The expCAG members nominated the CAG activities through a written expression of interest process. Members presented their ideas for nominated activities at a CAG meeting, with other members contributing their perspectives to develop the activities further. All CAG members nominated their preferred activities from the ideas presented and made recommendations to the Program’s Executive Director, who made the final selection. The Program’s selection was predominantly based on each activity’s viability within resource and time constraints.For each CAG activity selected, the CAG formed working groups consisting of self-nominated CAG members . These gCommunity education was achieved through presentations and seminars at Health Consumers Queensland Forums in 2018 and 2019. These presentations centred on integrating genomics into health care (2018) and facilitated a question and answer session to address community questions around genomics (2019). The community perspective at these forums was explored through surveys. These surveys were used to improve our knowledge of Queensland Consumers’ understanding of Genetic Services .Multiple CAG activities focused on equity and access to health services and education. The CAG developed a series of written resources to help patients navigate the Queensland’s health system genomic services (known as the Genomics Information Toolkit). These patient resources were translated into five languages other than English: Arabic, Persian (Farsi), Vietnamese, Japanese and Chinese (simplified) . The relAdvocacy, foundation and prioritisation, and real-world context themes  were addSupporting the CAG to undertake community-led activities was a unique feature of this Program. This positioned CAG members as experts collaborating to identify and provide solutions to areas of unmet need rather than advisors to already established research projects or health service initiatives. This recognition of expertise enabled members to use their collective skills and knowledge to develop real-world outputs for consumers and the community. It provided opportunities for skills and knowledge development, as CAG members gained exposure to insights from other members and the activities of project officers and contractors.The CAG was not established with the capacity to undertake multi-year, community-led activities. The Program’s trust in the CAG to undertake more substantial work came through the CAG’s demonstration of capability and readiness over their first 12-months  by develThe efforts to establish the CAG’s readiness for community-led activities resulted in the secondary effect of improving their advisory capacity within the Program. The Program’s trust and relationship enabled the CAG to develop a voice that had influence. The CAG identified that the Program’s operation had insufficient mechanisms and opportunities for the CAG to provide CCE support to projects. The issue was raised by the CAG members several times throughout the Program. These discussions resulted in changes, including; the CAG being involved in the Program’s project selection process and the inclusion of new CCE requirements around expectations and reporting mechanisms for projects.Despite the Program’s desire to have embedded CCE across projects, the CAG was established concurrently with the Program’s first round of projects. So it was unable to contribute to this initial series of projects. While advocacy by the CAG for greater involvement in the Program and its projects resulted in improved CCE, the initial lack of mechanisms for CCE and the difficulty with the later implementation of CCE measures is evident in the fragmented engagement with the CAG and Program’s projects. Only 2 of the 15 clinical projects and 3 of 28 capability projects took up the CAG offer for consumer input. Some of the Program’s projects did incorporate CCE through direct recruitment of or consultation with disease-specific consumer or consumer organisation representatives rather than engaging with the CAG.This inconsistent engagement stemmed from two areas. Outside of the Program, at the organisational level, there was a lack of support for CAG being involved in Program decision making, for example, being represented in the Program’s Board. The Program also experienced push-back from some project leaders who questioned the need for CCE in their projects. These experiences reflect two known barriers to effective implementation of CCE practices; implementers’ knowledge and experience, and the level of organisational support .An area of need for organisation-wide implementation of CCE going forward, which was not captured by this Program, is the education of the implementers of CCE . CAGs are key knowledge holders and advocates for developing and delivering educational interventions for CCE implementers by presenting examples and conveying their experiences and advice.To have acceptance of CAGs, there needs to be cross-organisational normalisation of consumer and community involvement in health services and research . In thisBased on the CAG member experiences, both positive and negative, a series of recommendations for CAGs and organisations implementing CAG as part of research or healthcare delivery are summarised in CAGs formed by researchers, organisations, or health services often have specific tasks or ask to contribute in ways that reflect the organiser’s intent . While tThe CAG’s self-determination of its activities significantly impacted the Program, the individual CAG members and the group as a collective. Primarily, these CAG activities facilitated meaningful dialogue with the Program on the needs of consumers and the community as a whole. It created awareness of issues and provided a mechanism for the Program to address unmet needs, using the CAG as an expert resource that defined and led activities. The ability to self-determine activities was beneficial to the CAG members’ motivation and engagement in the Program. It provided an opportunity for the CAG to contribute to the Program actively and gave the CAG responsibilities and ownership of self-determined activities.There is debate over the level of experience CAG participants should have when recruited . EngaginIn addition, the diverse mix of CAG members’ experiences created a dynamic within the group, where each member had areas of expertise and inexperience. The resulting combination of people made a CAG with diverse yet complementary skillsets enabling within group learning. Providing education opportunities, often through group training, to develop the skills of CAG members enables effective contribution for CAGs with novice members . In thisCAGs are dependent on the generosity and commitment of the members that volunteer their time and skills. It is recognised that the remuneration of consumer and community volunteers is an integral part of supporting CCE participation . HoweverCAGs are one mechanism of incorporating CCE in health and medical research and health service delivery . The pur


Cirrhinus mrigal  serum to produce polyclonal antibodies is described in the present study. The purified immunoglobulins (IgM) were isolated from the serum of mrigal  by the bovine serum albumin (BSA)-CL affinity column purification method, and the IgM was used to produce a polyclonal rabbit anti-mrigal IgM antiserum. The IgM preparations were employed in the characterization of mrigal serum immunoglobulin. Reduced mrigal IgM on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to consist of two subunits, compatible with heavy and light chains. A single heavy chain at approximately 90 kDa and variant of light chain 30 kDa were found. The dominant form of nonreduced IgM had a MW of approximately 900 kDa, suggesting a tetrameric structure based on estimated molecular weights, the relative protein content, and the reactivity with anti-mrigal IgM antisera, was obtained. The antisera were characterized as to specificity and reactivity by means of the enzyme linked immuno-sorbent assay (ELISA) and western blotting method. The information on the structure and character of immunoglobulin of fishes is essential in health management. The study described here investigates the possibility of using the serological techniques to assess the reactivity of antibody with the anti-mrigal IgM antisera.A method for the isolation of immunoglobulin M (IgM) in Indian major carp  Fish immunoglobulins share various characteristics with mammalian immunoglobulin; however, some differences exist; those partly arise from the process of evolution from common ancestors and partly reflect the effect of fish adaptation to a changed life environment. The fish immune system contains five types of immunoglobulins: IgM , IgD 2––2–4, IgZA. hydrophila have been reported. [Wllson et al.  had repoeported.  NoncommuThis study presents methods for processing and purification of Indian major carp Cirrhinus mrigal immunoglobulin and its reactivity with anti-mrigal IgM antisera. The antisera were characterized by specificity and reactivity by means of the enzyme linked immuno-sorbent assay (ELISA) and western blotting method.μl of phosphate buffered saline  emulsified with an equal volume of Freund's complete adjuvant (FCA). Similar doses of BSA emulsified with an equal volume of Freund's incomplete adjuvant (FIA) were given 15 and 22 days later. Two mrigal fishes injected with PBS (pH 7.4) alone were maintained as control for the collection of unimmunized serum. On the 30th day, fishes were anaesthetized with 10 ppm benzocaine for 3 min followed by the collection of blood by puncturing the caudal vein, which was allowed to clot for 1 h at room temperature and stored overnight at 4°C. Serum was collected after centrifuging at 6000 rpm for 10 min and pooled and stored at −20°C for further use [Five mrigal fishes weighing about 500 g each, maintained in a 25 m [μm syringe filter  and loaded onto the column. The serum was allowed to interact with the BSA for 45 min. Unbound serum protein was washed out with 10 column volumes of elution buffer . Approximately, 10 fractions of 0.55 ml each were collected and neutralized with 0.45 ml of 0.5 M Tris buffer (pH11). Absorbance at 280 nm of each fraction was measured spectrophotometrically. The eluted samples were pooled and subjected to dialysis at 4°C against PBS using a low molecular weight (12 kDa cut off) dialysis membrane . The dialysis membrane was shifted to a tray containing sucrose to concentrate it to 2 ml, and the concentrated IgM was stored at −20°C for further use.Purification of serum was carried out by affinity column chromatography according to Gopalakrishnan et al. , using aμl of the 4x native PAGE sample buffer , glycerol 0.8 ml, and 0.01% bromophenol blue (W/V) was added to 30 μl of the purified IgM. The purified IgM along with a molecular weight marker  and native human IgM  were loaded onto the gel containing 3% acrylamide and 0.5% agarose and subjected to electrophoresis at 25 mA till the dye front reached the bottom of the gel which was strained with coomassie blue  for 8 h and destained with destaining solution , and the molecular weight of the IgM was estimated. Further characterization of the IgM was carried out by 10% SDS-PAGE according to Laemmli [μl of the 4X sample buffer , glycerol 0.8 ml, and 0.01% bromophenol blue (W/V) was added to 30 μl of the purified IgM and boiled at 100°C for 5 mins. For comparison, crude serum from an immunized fish was prepared in a similar way. The samples along with molecular weight markers  were loaded onto 4.5% stacking gel and subjected to electrophoresis at 30 mA in 10% separating gel till the dye front reached the bottom of the gel. The gel was stained with coomassie blue, and the molecular weight of heavy and light chain Ig were determined [Purified IgM was characterized by nonreducing SDS-PAGE, briefly 10  Laemmli . About 1μg in 250 μl PBS) mixed with 1 : 1 with FCA. A similar second dose (ip) was given with the purified IgM in FIA (1 : 1) on the 15th day. On the 27th day, a booster dose of 10 μg purified IgM in 0.05 ml PBS was given intravenously. Blood was withdrawn on the 30th day from the tail vein using a hypodermic syringe and collected blood was allowed to clot for 1 h at room temperature. Later, the serum was obtained by centrifuging the blood clot at 6000 rpm for a period of 10 mins, which was used as a polyclonal antibody.A polyclonal antibody-based ELISA method was adapted for the detection of mrigal antibodies. Ten-week old Swiss albino mice were immunized (ip) with purified mrigal IgM  followed by (1) blocking with 5% skimmed milk powder in PBS with 0.1% Tween 20 (PBS/Tween), (2) incubation with doubling dilutions (1 : 100 to 1 : 12400) of purified Ig, immunized sera and unimmunized sera in PBS/Tween in the two sets, (3) incubation with polyclonal mice anti-mrigal IgM antiserum (1 : 1000 in PBS/Tween), (4) incubation with horseradish peroxidase conjugated rabbit anti-mouse Ig antiserum  (1 : 1000 in PBS/Tween), and (5) incubation for 10 mins with TMB H2O2  (1 : 20 in PBS/Tween). The reaction was stopped by addition of 1M H2SO4 and the colour development was observed at 490 nm (OD490) which was measured with ELISA reader . All incubations were for 1h at room temperature. All the steps between (1) and (5) were separated by three washes with PBS/Tween.Indirect ELISA was performed to analyze the BSA specific antibody activity in immunoaffinity chromatographic fractions pf pooled mrigal sera as described by Towbin et al. . MicrotiPurified mrigal IgM was solubilized in the sample buffer under reduced conditions. After separation in 10% polyacrylamide gel , the pro2 size. Purified IgM was dotted on the paper as positive control and Aeromonas hydrophila and vibrio sp bacteria were dotted as a negative control, and three such dotted membranes were prepared separately to detect antibodies in the affinity purified IgM sample, crude immunized, and unimmunized sera. Free sites on the nitrocellulose membrane were blocked with 05% skimmed milk powder in PBS for 02 hours and washed 03 times with PBS (pH 7.4), and one of the membranes was treated over night with 01 ml of purified IgM and at a concentration of 0.1 mg/ml, and the other with crude immunized fish serum diluted to 10 times with PBS (01 ml). The third membrane was treated overnight with unimmunized fish serum diluted to 10 times with PBS (01 ml). After washing with PBS Tween 20, rabbit anti-mouse IgG horse radish peroxidase  in 3% BSA in PBS  was added and incubated for 90 minutes. After washing with PBS Tween 20 three times, 1 ml of substrate ) was added, and clear purple dot development on the nitrocellulose paper was considered as positive reaction.An immunodot assay was carried out by dotting BSA onto nitrocellulose (N.C.) papers cut into 02 cmFish serum was obtained after immunization with bovine serum albumin (BSA), the sera obtained was purified by affinity column chromatography method eluted fractions and characterized by poly acrylamide gel electrophoresis (PAGE) under reducing and nonreducing conditions Figures  and 2. P3In the present study, we tried to purify the immunoglobulin of mrigal by a single step affinity purification by the BSA-linked column, which was suitable to get adequate amount of immunoglobulin (Ig); moreover, the same column was found to be useful to purify more than once without reduction in the binding capacity of the antibody adapting established immunization protocol and the purification technique, and an average yield of immunoglobulin (Ig) was 1.25 mg/ml of serum. Ig levels in different fishes after immunization vary within the range of 0.25–23.5 mg/ml .A. hydrophila and Vibrio sp. Bacterins) did not show any colour reaction.The immuno dot test clearly showed specific reaction of the antibodies in the affinity-purified IgM and crude antisera with BSA. In the NC membrane treated with affinity-purified IgM  and the Cirrhinus mrigal , Catla catla (Catla), and Lobeo rohita (rohu) with nonreducing SDS-PAGE, different populations of Ig molecules such as tetrameric and dimeric ano-monomeric forms have been reported, but we have observed a single band of protein in nonreducing SDS-PAGE indicating that protein is pure and was eluted in its native form. The molecular weight of C. mrigal IgM was 900 kDa in nonreducing SDS-PAGE, which is similar to the molecular weight of IgM of the fishes such as Turbot (835 kDa African catfish 840 kDa Cod 851 kDa).In the present study, only one form of IgM-like molecule was eluted in the affinity purification. In the earlier studies on SDS-PAGE analysis revealed two clear bands of polypeptides having molecular weights of 90 and 30 kDa which are considered as the heavy and light chains of the purified IgM molecule, respectively. The immunized crude serum also shows the corresponding heavy and light chain bands. The molecular weights of heavy and light chains can be compared with those from other species of fishes like Tilapia (90 and 30 kDa), Cod (811 and 27.5 kDa), Trematomus bernacchii (83.5 and 27.5 kDa), Turbot (78 and 27 kDa), and common carp (70 and 25 kDa) .The present paper describes the attempts made and strategy employed to obtain specific polyclonal antibody against mrigal antiserum to develop a strategy to obtain a specific polyclonal antibody against mrigal antiserum and to develop a sensitive, rapid, and specific ELISA to employ in routine screening in disease management. In the present study, BSA antigens were selected to raise the antibodies in mrigal because of the accessibility of affinity columns for antibody purification. BSA has been used as an immunogen for elucidating antibody production in different fishes by many investigators –18.Eluted fractions of immunoglobulin were analyzed afterwards by the antiserum which was found extremely immunogenic as assessed by the titration of immune sera. The ELISA developed was shown to be very sensitive with titer of 1 : 12400. The antibody reacted more strongly with pure mrigal IgM than with crude mrigal sera as ELISA, immmunodot, and western blot has confirmed the results , 19.