Cardiac rehabilitation has been shown to reduce cardiac mortality, improve quality of life, and reduce hospitalizations. Cardiac rehabilitation programs are usually performed over a 12-week period. Studies have shown that similar benefits could be achieved with shorter programs. Abnormal heart rate recovery after exercise has been associated with an increased risk of cardiovascular events and mortality. The main aim of this study was to compare the effect of a 6-week phase 2 cardiac rehabilitation program on heart rate recovery to a 12-week one in patients who had recovered from an anterior wall ST segment elevation myocardial infarction.This prospective study included 60 patients enrolled in cardiac rehabilitation programs randomized into two equal groups: a 6-week and a 12-week program. Baseline patient demographics, lipid profile, and left ventricular ejection fraction (LVEF) were assessed. METs achieved, total exercise time, resting heart rate, peak heart rate, and heart rate recovery at 1 min were examined. These were re-assessed at the end of each program.p = 0.051).Results showed no difference between both groups at the end of each program regarding lipid profile and LVEF. Patients enrolled in the 12-week cardiac rehabilitation program were able to achieve more METs, had a longer exercise time, a higher peak heart rate, and had a lower resting heart rate at the end of the program. Heart rate recovery was slightly higher in patients enrolled in the 6-week program 26.5 ± 6.78 versus 23.17 ± 6.12 bpm (On comparing the magnitude of change between both programs, those in the 12-week program had more increase in HDL-C levels, METs achieved, and exercise time. Additionally, they had more reduction of resting heart rate. Heart rate recovery was more increased for those in the 6-week program.Although heart rate recovery increases after completion of each of a 6-week and 12-week cardiac rehabilitation program compared to their baseline, there is no difference on comparing heart rate recovery between both programs at their end.Patients enrolled in a standard 12-week cardiac rehabilitation program achieve more METs, have a longer exercise time, a higher peak HR, and a lower resting HR at the end of the program compared to those in the 6-week program. Ischemic heart disease is the leading cause of death worldwide with more than 9 million deaths each year . It is eCardiac rehabilitation has been defined by multiple organizations and medical societies. It involves several activities and interventions that provide cardiac patients with prescribed exercise training, education, counseling, and risk factor modification with the purpose of limiting the physical, social, and psychological consequences of heart disease; controlling symptoms; and reducing the risk of recurrence of MI to help patients maintain or resume their active place in society –7.Several studies, meta-analyses, and systematic reviews have shown that cardiac rehabilitation reduces cardiac mortality by up to 26%. Additionally, it has been shown to significantly improve the quality of life and reduce hospitalizations –10.Cardiac rehabilitation programs are usually performed over a 12-week period. However, studies have shown that similar benefits could be achieved with shorter programs performed over 4 or 6 weeks with more frequent weekly sessions, so-called high contact frequency programs .Abnormal heart rate recovery after exercise has been associated with an increased risk of cardiovascular events and mortality with the risk increasing significantly when it falls below 10–12 bpm .The primary aim of this study was to compare the effect of a 6-week (high contact frequency) phase 2 cardiac rehabilitation program on heart rate recovery to a 12-week (low contact frequency) one in fully revascularized patients who had recovered from an anterior wall ST segment elevation MI (STEMI) managed by primary PCI. Secondary aims included comparing both programs for changes in lipid profile, left ventricular ejection fraction, and exercise parameters.This was a prospective randomized study performed in the period from April 2017 to April 2018. Informed consents were provided by all participants and institutional ethics committee approval was obtained.The study was conducted on 60 adult ischemic heart disease patients who had recovered from an anterior wall STEMI managed by primary PCI and for whom complete coronary revascularization was performed using coronary angioplasty in the 6 months prior to enrollment. At the time of enrollment, all patients were free of angina or angina-equivalent symptoms and on guideline-directed medical therapy for ischemic heart disease titrated up to the maximal tolerated doses. The study protocol did not allow the type or dosage of lipid-lowering medications, beta-blockers, or other heart rate control medications to be changed for the whole duration of the study. If a change in dosage or type of the previous medications was deemed medically necessary, the patient would be excluded from the study.n = 24).All patients were enrolled in the outpatient phase 2 cardiac rehabilitation program provided at our institution. We randomized the patients using 1:1 allocation into two equal groups (30 each). One group joined the standard 12-week program provided at our institution with twice weekly sessions (low contact frequency). The other team joined a 6-week program with four weekly sessions (high contact frequency). The total number of sessions for all patients was the same  acute coronary syndrome or MI; left ventricular ejection fraction (LVEF) less than 35%; New York Heart Association functional class III or IV ; recent Patient interviews involved through history taking and clinical examination. Baseline patient demographics and risk factors for coronary artery disease were acquired. It was confirmed that patients were on guideline-directed medical therapy including a beta-blocker; an angiotensin-converting enzyme inhibitor or an angiotensin II receptor blocker; aspirin; and a lipid-lowering medication with titratable medications at maximal tolerated doses.A venous blood sample was obtained after a 12-h fasting period to assess each participant’s lipid profile: total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels. The first three were measured using enzymatic methods, while LDL-C was defined using the Friedewald formula: LDL-C = [total cholesterol] − [HDL-C] − (1/5 triglycerides) .Trans-thoracic echocardiography was performed using a Vivid 7 machine  using an M4S matrix-array probe with a frequency range of 1.7–3.4 MHz with the patient in the left lateral decubitus position. Echocardiography was performed by an individual accredited by the European Association of Cardiovascular Imaging. LVEF was calculated using the bi-plane Simpson’s method of discs from the apical 4- and 2-chamber views .Patients were enrolled into one of two 24-sessions cardiac rehabilitation programs. Both programs were managed by a team of cardiologists, nutritionists, physiotherapists, trained nurses, and psychiatrists.This is the standard program offered at our institution which involves twice weekly supervised exercise training sessions over a 12-week period.Each session had 5–10 min of warm up, at least 20 min of aerobic treadmill training, and 5–10 min of cool down. Sessions would last up to 40 min towards the end of the program.Patients’ exercise intensity was gradually increased from 50 to 80% of the heart rate reserve.This program involved four times weekly supervised exercise training sessions over a 6-week period.Each session had 5–10 min of warmup, at least 20 min of aerobic treadmill exercise training, and 5–10 min of cool down. Sessions lasted up to 40 min towards the end of the program.Patients’ exercise intensity gradually increased from 50 to 80% of heart rate reserve according to each patient’s ability.Both programs included group teaching sessions about coronary artery disease and risk factor modification, in addition to, nutritional counseling, and smoking cessation advice. Group and individual psychiatric counseling were provided to patients.Symptom-limited treadmill exercise training was carried out at baseline for all patients following the modified Bruce protocol to estimate each patient’s peak heart rate.Target heart rate was then estimated by the Karvonen formula  with an The Karvonen method for training heart rate estimation is as follows: target heart rate = resting heart rate + (training percentage required × heart rate reserve).Heart rate reserve is calculated as follows: peak heart rate − resting heart rate.During the sessions, patients were monitored using telemetry and were directly supervised by a nurse. An on-site physician supervised three patients in the cardiac rehabilitation unit at a time.Metabolic equivalent of tasks (METs): these were automatically calculated by the treadmill software where one MET is defined as the amount of oxygen consumed while sitting at rest. One MET equals 3.5 ml of oxygen per kilogram of body weight per minute .Total exercise time: defined as the total duration of the symptom-limited exercise test.Resting heart rate: defined as the patient’s baseline heart rate before starting the symptom-limited exercise test.Peak heart rate: defined as the maximum heart rate achieved in the symptom-limited exercise test.Heart rate recovery at 1 min: heart rate recovery is defined as the rate at which heart rate declines in the minutes following cessation of physical exercise. We calculated it by subtracting each patient’s heart rate after 1 min of recovery from their peak heart rate .The following exercise parameters were recorded for each patient:At the end of each of the 6- and 12-week programs, lipid profile, LVEF, and exercise parameters were re-assessed using the same methods described before.t test. Significance level  was set at less than 0.05.Data were collected, revised, coded, and inputted into the IBM statistical package for social science (SPSS) version 24. Data were tested for passing normality. The study power was more than 80. Categorical data were presented as number and percentages; continuous data were presented as mean and standard deviations. Categorical data were compared by using Fisher’s exact test. Continuous data were compared using student’s paired There were no differences between patients enrolled in the 6-week and 12-week programs regarding mean age, gender distribution, smoking status, and presence of hypertension or diabetes mellitus , had a longer exercise time of 16.2 ± 2.31 versus 14.91 ± 2.5 min (p = 0.041), had a higher peak heart rate of 154.03 ± 25.08 versus 136.63 ± 14.31 bpm (p = 0.002), and had a lower resting heart rate at the end of the program of 63.83 ± 6.75 versus 68.8 ± 9.08 bpm (p = 0.019).Patients enrolled in the 12-week cardiac rehabilitation program were able to achieve more METs 14.77 ± 2.01 versus 12.67 ± 2.57 , LDL-C (p < 0.001), and triglyceride levels (p = 0.012) compared to baseline. However, there was no change in HDL-C levels.At the end of the 6-week program, there was a significant reduction in total cholesterol (p < 0.001), LDL-C (p < 0.001), HDL-C (p < 0.001), and triglyceride levels (p < 0.001) compared to baseline.On the other hand, at the end of the 12-week program, there was a significant reduction in total cholesterol (p < 0.001). There was no difference on comparing the magnitude of change for total cholesterol, LDL-C, and triglyceride levels . However, there was no difference in comparing the magnitude of change in LVEF between both programs , exercise time (p = 0.001), and heart rate recovery compared to baseline (p < 0.001). There was no change in peak heart rate. Resting heart rate was reduced by 7.80 ± 8.31 bpm compared to baseline; however, it did not quite reach statistical significance (p = 0.055).At the end of the 6-week program, there was a significant increase in METs achieved (p < 0.001), exercise time (p < 0.001), peak heart rate (p < 0.001), and heart rate recovery (p < 0.001) compared to baseline, as well as a significant reduction in resting heart rate (p < 0.001).On the other hand, at the end of the 12-week program, there was a significant increase in METs achieved (p = 0.003). Exercise time was more prolonged for those in the 12-week program 2.66 ± 0.73 versus 1.13 ± 1.62 min (p < 0.001). Resting heart rate was more reduced for those in the 12-week program − 12.17 ± 5.79 versus − 7.80 ± 8.31 bpm (p = 0.021). However, heart rate recovery was more increased for those in the 6-week program 11.57 ± 5.16 versus 10.10 ± 4.87 bpm (p = 0.026). There was no difference on comparing the magnitude of change for peak heart rate  cardiac rehabilitation program to a standard 12-week (low contact frequency) program on heart rate recovery and other exercise parameters.n = 24) and exercise intensity as the standard 12-week program to try, in theory, to preserve the benefits of a cardiac rehabilitation program.The 6-week program was designed in a manner to have the same number of sessions  There was no difference between both programs regarding patients’ heart rate recovery at the end of the program; (2) Patients in the 12-week program had a lower resting heart rate at the end of the program; (3) Patients in the 12-week program were able to exercise for a longer time, achieve more METs, and achieve a higher peak heart rate on symptom-limited exercise testing at the end of the program.Secondary findings of this study come from comparing both programs for the magnitude of change in each program at its end compared to its baseline: (1) Patients in the 6-week program had more increase in heart rate recovery; (2) Patients in the 12-week program had more increase of HDL-C levels; (3) Patients in the 12-week program had more increase in METs achieved and exercise time duration on symptom-limited exercise testing; (4) Patients in the 12-week program had more reduction of resting heart rate.It can be concluded from these findings that a 6-week program has similar effects on heart rate recovery and changes in lipid profile compared to the 12-week program. However, benefits in exercise parameters are in favor of the 12-week program. This suggests that the benefits of cardiac rehabilitation on exercise parameters are not just dependent on the number of sessions attended but rather on the number of sessions and the duration of the exercise program.Short-term cardiac rehabilitation was previously examined and showed safety and efficacy. A study was performed on 60 patients recovering from an MI managed by primary PCI. Patients were enrolled in a 3-week cardiac rehabilitation program with either daily cycling or walking for 45 min at an intensity of 70–80% of the maximal heart rate. At the end of the program, exercise tolerance, peak heart rate, and heart rate recovery after 1 min all improved. The authors concluded that such a program was safe, improves exercise capacity, improves test duration, and improves heart rate response .An attenuated heart rate recovery has been shown to be associated with an increased risk of cardiovascular and all-cause mortality. A reduction of heart rate by 15 to 20 beats per minute in the first minute of recovery is typical for healthy individuals , 26.p < 0.001). However, on comparing both programs at their end, heart rate recovery was slightly more in the 6-week program 26.5 ± 6.78 versus 23.17 ± 6.12 bpm although it did not quite reach statistical significance (p = 0.051).In our study, heart rate recovery improved significantly in each individual program compared to its baseline (A study examined the effect of a 12-week (three times weekly) cardiac rehabilitation program on heart rate recovery after 1 min in patients who had undergone coronary artery bypass grafting. Fifteen patients were enrolled in the program and compared to 15 controls who had undergone CABG but did not participate in cardiac rehabilitation. Patients enrolled in the program had a significantly lower resting heart rate and higher heart rate recovery at the end of the program compared to controls as well as compared to their own baseline values .Another cohort study performed on 285 who completed a range of 5 to 24 training sessions of cardiac rehabilitation found that all patients showed an increase in heart rate recovery regardless of the number of sessions completed, with a significant correlation between heart rate recovery increase and the number of completed sessions [Similar results were seen in other studies comparing the effect of 12-week cardiac rehabilitation programs on heart rate recovery in 1 min for ischemic heart disease patients recovering from acute myocardial infraction all showing a significant increase in heart rate recovery with cardiac rehabilitation , 29.To the best of our knowledge, this was the first study to compare the effect of a 6-week and 12-week cardiac rehabilitation program on heart rate recovery.In our study, all exercise parameters  improved in both programs with more significant improvement favoring the 12-week program.In a study on 59 patients who participated in a standard cardiac rehabilitation program and were re-assessed 12 months after completion of the program, it was shown that exercise tolerance time and METs achieved were significantly improved .A study was performed on 961 low-risk cardiac patients who self-selected either a 12-week (high contact frequency) or a 4-month (low contact frequency) cardiac rehabilitation program. Patients in both programs achieved equivalent results. Both groups of patients had similar improvements in METs achieved. The authors concluded that the low contact frequency program can be used as an alternative to widen patient access and participation .There was no difference on comparing the lipid profile status between patients in both programs at the end of both programs. However, on comparing patients in each program compared to their baseline, participating in both the 6-week and 12-week programs led to a significant reduction in total cholesterol, LDL-C, and triglyceride levels compared to baseline with HDL-C levels increasing only for those participating in the 12-week program.Similar changes in lipid profile for participating in a 12-week program were reported in several studies , 32.Limitations of the current study are that it was performed in a single medical center with a relatively small number of patients. Patient compliance to lifestyle modifications was not assessed. When considering results of this study, the possible confounding effects of concurrent medications should be considered although no change happened during the study period for doses of lipid-lowering and heart rate control medications.Although heart rate recovery increases after completion of each of a 6-week and 12-week cardiac rehabilitation program compared to their baseline, there is no difference on comparing heart rate recovery between both programs at their end.Patients enrolled in a standard 12-week cardiac rehabilitation program achieve more METs and have a longer exercise time, a higher peak HR, and a lower resting HR at the end of the program compared to those in the 6-week program.


Dementia is becoming increasingly prevalent in Latin America, contrasting with stable or declining rates in North America and Europe. This scenario places unprecedented clinical, social, and economic burden upon patients, families, and health systems. The challenges prove particularly pressing for conditions with highly specific diagnostic and management demands, such as frontotemporal dementia. Here we introduce a research and networking initiative designed to tackle these ensuing hurdles, the Multi-partner consortium to expand dementia research in Latin America (ReDLat). First, we present ReDLat's regional research framework, aimed at identifying the unique genetic, social, and economic factors driving the presentation of frontotemporal dementia and Alzheimer's disease in Latin America relative to the US. We describe ongoing ReDLat studies in various fields and ongoing research extensions. Then, we introduce actions coordinated by ReDLat and the Latin America and Caribbean Consortium on Dementia (LAC-CD) to develop culturally appropriate diagnostic tools, regional visibility and capacity building, diplomatic coordination in local priority areas, and a knowledge-to-action framework toward a regional action plan. Together, these research and networking initiatives will help to establish strong cross-national bonds, support the implementation of regional dementia plans, enhance health systems' infrastructure, and increase translational research collaborations across the continent. The prevalence and incidence of dementia appears to be stable or declining in the US and other high income countries (HIC) –3, whereSES , education , health and health care , occupation , and social and community context .Environmental factors seem to be critical for dementia presentation in the region. SDH may selectively impact dementia in LAC , 12 by mAt the same time, genetic factors also seem to drive dementia presentation in LAC, with apparent stronger familial aggregation of dementia compared with HIC. The region hosts some of the largest populations of familial dementing disorders, and some populations may harbor unique genetic influences conferring increased risk of dementia , 18–20. The ways in which the combination of genetic and SDH-related risks interact in the dementia presentation across LAC is not well-understood. New studies in this region are needed to identify novel genetic and gene-environment interactions  leading to dementia, and novel pathways applicable to regional therapies. LAC face a dearth of innovative, harmonized, and cross-regional studies on AD and FTD, and establishing multi-center LAC initiatives is critical for global discovery and research harmonization in these underrepresented populations. Yet, region-specific determinants remain uncharted and, due to different factors , 55 the The Latin America and the Caribbean Consortium on Dementia [LAC-CD ], in assOur first specific aim is to establish genetic contributions to AD and FTD in diverse LAC cohorts. First, by elucidating the genetic substructure and familial contributions to AD and FTD in LAC relative to the US, we will be able to identify proper populations for replication of our genetic findings. We anticipate that, relative to the US, LAC will have a higher frequency of familial forms of AD and FTD. Discovery of new families with multiple affected individuals will advance efforts to treat AD and FTD in patients with rare mutations. Second, by assembling this large cohort, we will also be well-positioned to establish a preliminary LAC-specific polygenic risk score (PRS) for predicting AD and FTD risk in future samples. We expect that PRS will work best at discriminating patients from controls in the European predominant subpopulation  than in the African and Indigenous-majority admixed cohorts .In our second specific aim, we will elucidate the impact of SDH on clinical, cognitive, and brain imaging signatures in LAC and the US. To compare patients across regions, we establish standardized neurocognitive measures and harmonization protocols to understand how SDH impacts the manifestations of dementia in LAC. First, we will evaluate how SDH moderates the relationship between age at onset and disease severity in AD and FTD. We anticipate that AD and FTD will emerge at an earlier age in low-SDH vs. high-SDH (dichotomized) patients, and measures of disease severity, including cognitive performance, and multimodal neuroimaging, will be worse in the low-SDH group even after accounting for age. We expect that difference in disease severity ratings, cognition, and multimodal neuroimaging that reflect low vs. high SDH disparities will be greater in LAC patients compared to US patients. Latin America constitute the region with the largest inequalities in the world . MoreoveOur last specific aim seeks to determine whether genetic risk and SDH yield better discrimination between LAC and US patients as compared with other cognitive, neuroimaging, and clinical variables. To our knowledge, no study has sought to establish which potential predictors prove more sensitive to discriminate between LAC and US patients. In particular, although genetic risk and SDH have the potential to robustly differentiate between such samples, no study has explored their combined role, let alone as compared to other multimodal factors. To address this issue, we will apply data-driven machine-learning pipelines to determine top factors that best discriminate patients in LAC from those in the US . MultimoReDLat will establish a large LAC cohort of harmonized, well-characterized AD and FTD patients and controls. We anticipate the development of a better understanding of genetic and environmental contributions to neurocognitive manifestations of dementia and the identification of novel targets for risk reduction and disease prevention in LAC. Our large multimodal, cross-sectional study will enable clinical assessment of understudied patient groups, extend and harmonize existing data sets, prompt the development of novel measures, and inform future work on the clinical value of combined multimodal profiles to predict disease presentation and progression in longitudinal studies of diverse populations.On January 27, 2020, a kickoff meeting  involvedSubstantial effort has been devoted to developing strategies for harmonization of participant enrollment across sites. We created a detailed study-wide protocol see  and siteWe have worked with the National Alzheimer's Coordinating Center to obtain permission for adaptation of the Uniform Data Set Cognitive Assessment to Spanish and Portuguese. Based on feedback from investigators, we adapted the language for some tasks to optimize cultural appropriateness at each site. We developed a new instrument to systematically assess SES and SDH. This UCSF-ReDLat questionnaire has been culturally adapted with input from each enrolling site, based on previous reports as well as national censuses from Colombia, Brazil, Chile, and Perú and following cross-cultural implementation recommendations –64. The PSEN1, PSEN2, TARDBP, GRN, TREM2, MAPT, EPO4, and C9orf72  and/or DNA per country. All countries have data from participants that belong to AD, FTD and healthy controls. RedLat is also aimed to develop implementation science and capacity building, and several actions has been performed via regional networking, training, and development of educational projects. This is the main goal of the Latin America and the Caribbean Consortium on Dementia [LAC-CD ], the reNowadays, the consortium is promoted by the Alzheimer's Association and the Global Brain Health Institute, and holds more than 240 regional members. LAC-CD involves national representatives working in specific priority areas including dementia biomarkers, genetics and epidemiology, a dementia data platform, a clinical trial program, non-pharmacological interventions, and translational research networks. LAC-CD initiatives include (a) empowering local groups, (b) boosting coordinated efforts across the region, and (c) developing a Knowledge-to-Action Framework to develop a regional action plan.A key ambition of our consortium is to create harmonized approaches to dementia diagnosis in order to allow multi-country comparisons. First, we developed diagnostic recommendations , for diagnosis of AD and FTD across LAC even where there are no available resources required for classification of dementias . Then, shttp://lac-cd.org/, in English, Spanish, and Portuguese) providing information on projects, membership, news, opportunities, dissemination products, press releases, and social media. With the Alzheimer's Association, we have launched a LAC-CD – ISTAART webinar including an annual meeting, and periodic webinars focused on different topics.Several initiatives are being created to expand the visibility and dissemination of the consortium activities, including a LAC-CD website  and its modulation by different factors including expertise-related information (knowledge of public policies), individual differences , and location across LACs  toward regional action plan for dementia . InitialThe KtAF comprises five workgroups, each responsible for specific tasks . A Non-pmultidisciplinary innovation, ReDLat focuses on the largely ignored convergence of genetic and SDH risks, especially considering multimodal  effects and innovative machine learning techniques. Regarding translational impact, our project is research-based, but geared to capacity building and implementation science , favoring regional commitment. We also aim to empower local ideas in a global networking landscape, as our initiative merges bottom-up LAC proposals into a single landscape. We promote win-win HIC-LMIC collaborations facing local needs from a local-global perspective. We also focus on barriers, by tackling HIC-LMIC cultural-communicative differences between researchers, assessing underrepresented populations, pushing changes regarding lack of trust among teams based on an equitable platform for collective decision making, and bringing collective support to minimize emergent leaders' disregards. We believe these actions promoting brain health and dementia across LACs and globally will help to truly transform challenges into opportunities.Our recently launched consortium grasps relevant features for upcoming progress and expansion. Regarding AI designed the proposal under supervision of BM. AI wrote the drafts, discussed contributions from all co-authors. All authors searched the literature, participated in discussing the contents of the paper, contributed to editing, and approved the final version of the article.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.


Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptⅡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene (nptⅡ) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptⅡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement. Eukaryotic filamentous yellow-green algae from the  Furthe change ,4,5. TheTribonema, which are widespread in freshwater and salt-water ecosystems, have gained substantial attention [T. minus strain has the ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids [T. minus is an industrial strain of microalgae that is used for biodiesel production under photoautotrophic conditions because of its high lipid content (up to 60.2% of its biomass) and easy harvesting characteristics due to its unbranched filaments. On the other hand, T. minus cells could be made economically feasible for the production of functional fatty acids with high value via heterotrophic/mixotrophic cultivation. In addition, a recent study by Wang et al. [T. minus under photoautotrophic and heterotrophic conditions. Moreover, the recent genome sequencing of T. minus makes the species an attractive model for understanding the molecular nature of cell formation and lipid synthesis in the cells (the manuscript is undergoing preparation for publication). However, information on transgene expression and genetic engineering for understanding molecular mechanisms and strain development is very limited in the Tribonema genus. The filamentous yellow-green algae ttention . The T. ty acids ,8. On ong et al.  revealedChlamydomonas reinhardtii [Nannochloropsis sp. [Chlorella [Phaedodactylum tricornutum [Tribonema genus.The availability of facile and efficient transformation techniques for transgene expression and metabolic engineering is important for biotechnological application. Specifically, species-specific molecular operations in microalgae will allow for the engineering of improved performances to drive down manufacturing costs and overcome the economic threshold for the production of commercially competitive products. The identification and genetic manipulation in this organism are crucial steps for the improvement of lipid productivity and/or the growth rate in microalgae, and the establishment of a genetic transformation method is the first step towards this goal. To date, suitable transformation techniques have been established only in several limited unicellular models of eukaryotic microalgae, such as nhardtii ,11, Nannpsis sp. ,13, Chlohlorella , and Phacornutum . UnfortuNannochloropsis sp. and other green algae, electroporation is a useful tool to introduce exogenous DNA into cells partially deprived of their cell walls [T. minus, we first attempted to use electroporation to introduce the plasmid into the protoplasts of this alga. However, although we tried to degrade the T. minus cell wall using several different enzymes that weaken the cell walls of other microalgal cells [In ll walls ,17. Theral cells ,19, we hal cells .T. minus, a natively robust and oleaginous algae, was investigated using a particle bombardment method. The efficient transformation conditions and some operational details in the transformation were determined by expressing the green fluorescent protein (eGFP) gene and neomycin phosphotransferase Ⅱ-encoding G418-resistant (nptⅡ) gene. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvements. This is the first report of a genetic engineering system in eukaryotic filamentous microalgae. Here, in this study, the genetic transformation of T. minus were first verified for herbicide and antibiotic sensitivity in liquid cultures. Five different antibiotics and one herbicide were assessed at various concentrations. The results showed that in liquid culture, T. minus is very sensitive to some of the antibiotics tested, particularly streptomycin and G418, causing 43.88% and 59.49% reductions in growth at concentrations of 10 and 20 μg mL−1, respectively, in 5 days  caused considerable inhibition to the biomass concentration of T. minus. On the other hand, kanamycin had almost no effect on the growth of T. minus  under the control of a promoter . In orden 5 days A. T. minkaryotes ,23, exhiT. minus A.T. minus to G418 for genetic selection was examined on BG11-based agar plates with different concentrations  of G418. After 3 days, the cells cultured on agar plates without G418 had a good growth status, while those cultured with different concentrations of G418 did not show obvious growth. With prolongation of the cultivation, the results indicated that G418 at concentrations of 10 μg mL−1 and higher could effectively prevent the growth of T. minus, as the alga could still be grown on the plates at 10 days, giving a final concentration of 5 μg mL−1 (nptⅡ) was used as a selection marker gene [−1 were chosen for use in agar and liquid media, respectively. Our tests in liquid medium allowed us to analyze the effects of antibiotics in a quantitative, repetitive, and reliable way. However, in order to provide further information about the concentrations of antibiotics, especially G418, that are appropriate for the selection of transformants, we also assessed the effects of G418 on agar plates. Subsequently, the sensitivity of  μg mL−1 B. Therefker gene  in this T. minus, and thus, we used the upstream and downstream regions of the tub gene to provide promoter and terminator functions, respectively. We cloned and sequenced the tub gene (annotation number of MN481481 in Genbank) and its flanking sequences by the gene walking method, as described in the Methods section, and finally, we found that the nucleotide sequences comprised a promoter region (1373bp) and a terminator region (574bp) from the tub gene in T. minus  and eGFP genes (411bp) were detected in transformants. Moreover, 18S rDNA was detectable in all samples except for the vector control, indicating the successful transformation of T. minus (data not shown). In order to check the presence of the plasmid DNA in transformants, both the predicted transgenic cells that grew well in BG11 medium supplemented with 20 μg mLtub-nptⅡ transgenic cells under 450 and 900 psi were picked randomly and transferred into fresh BG11 medium for a subculture until the third generation. Moreover, according to the sequence of pEASY-tub-nptⅡ, we chose a middle fragment with a size of 803 bp including part of the nptⅡ gene and part of the tub promoter at random and designed the primers to have sequences of T-PK-F (5’-gcaatttgattggccagcgcaac-3’) and T-PK-R (5’-gatggatactttctcggcaggag-3’). We amplified this fragment in the genomic DNA extracted in the third-generation transgenic cells, and the wild type was regarded as the control. Not surprisingly, In addition, three pEASY-nptⅡ genes in T. minus transformants. T. minus bombarded with pEASY-tub-nptⅡ under 450 and 900 psi, respectively. The endogenous tub gene was utilized as a reference gene, and the samples from wild-type T. minus were used as the control. Normalized fold changes of the nptⅡ gene in these transformants to the wild-type cells were calculated with the tub gene as a balance based on the ΔΔCt method. The signals of nptⅡ genes identified in the transformant genomes were calculated to be 235–394 times higher than those in the wild-type cDNA. All these results confirm that the plasmids were successfully integrated into the genome and expressed efficiently in T. minus transformants.Quantitative real-time PCR was performed to quantify the efficiency of genomic integration and transcription of the introduced Tribonema minus, which is classified within the family Xanthophyceae, has recently become an attractive organism for use in biotechnology. This alga has the ability to grow under autotrophic, mixotrophic, and heterotrophic conditions, and it can synthesize and accumulate a significant amount of fatty acid intracellularly [T. minus would increase the potential value of this alga in biotechnological applications, and it could serve as a molecular model for Xanthophyceae. llularly . EstablinptⅡ) [shble) [sat) [T. minus is very sensitive to G418 (neomycin); therefore, the resistance gene nptⅡ was used in this study as the selection maker. Active promoters and terminators are important constitutions of efficient transformation and expression vectors. Researchers once chose heterologous eukaryotic vectors, such as SV 40 [tub) is a principal component of microtubules that forms part of the cytoskeleton in eukaryotic cells [tub gene has a constitutive and high level of expression in T. minus. Thus, the upstream and downstream regions of endogenous tub could be considered to provide active promoter and terminator functions. In this work, the complete sequence of the tub gene, including its exons, introns, and 5’ and 3’ flanking sequences were successfully identified in T. minus. Subsequently, a fusion PCR method was performed to fuse the genes of eGFP and nptⅡ with the tub promoter (1373 bp) and terminator (574bp) of T. minus, respectively. Finally, two vectors that were controlled by the endogenous tub promoter and terminator with sizes of 6124 and 6580 bp were successfully constructed and named pSimple-tub-eGFP and pEASY-tub-nptⅡ, respectively. Transformability is usually tested using a selection marker; thus, in order to develop a system for the efficient selection of transformants, knowledge about the sensitivity of a strain to various antibiotics is essential. Selectable marker genes, including neomycin phosphotransferaseⅡ (nptⅡ) , phleomy [shble) , and stre) [sat) , have beas SV 40  or CaMV as SV 40 , to drivas SV 40 . Generalas SV 40 . Alpha tic cells , and itsic cells . TranscrH. pluvialis cells was used for bombardment, no successful transformation was achieved. However, the transient expression of a gene could be served in a logarithmic culture with cells after bombardment [T. minus cells were easily shattered as the tungsten powder appeared on the filter membrane under higher pressure conditions. Hence, we layered a piece of filter membrane on the upper part of the cells prepared for transformation under a pressure of 900 psi and higher. Transformation was achieved successfully under only two lower pressure conditions of 450 and 900 psi. The effective pressures were lower than those used in the genetic transformation of a unicellular green microalgae Pseudochoricystis ellipsoidea, the bombardment of which can achieve success from 900 to 1550 psi [During biolistic transformation manipulation, many manipulation details influenced the success rate . The culbardment ,28. In t1550 psi . This miT. minus was carried out on agar plates of G418-containing medium followed by further resistant treatment in liquid medium. For unicellular microalgae, the transformant colonies could be easily observed on the agar plates [T. minus transformants instead of a flake or a speck of green cells on the agar plates  in liquid BG11 medium [2. The Escherichia coli strain DH5α was used for the construction, propagation, and maintenance of plasmid. It was cultured in Luria–Bertani (LB) medium at 37 ℃. The yellow-green microalgae 1 medium  that wasT. minus in liquid BG11 medium were monitored. Aliquots of BG11 medium supplemented with varying concentrations of basta or each antibiotic  were inoculated with freshly grown T. minus to an initial biomass concentration of 0.2 g L−1 (OD680 and density may not be accurate because of the filaments). The effects of the herbicide basta (Sigma) and the antibiotics hygromycin, chloramphenicol, geneticin (G418), kanamycin, and streptomycin (Sigma) on T. minus to G418 on solid BG11 medium, aliquots of 200 μL suspension (0.2 g L−1) were inoculated on agar plates containing different concentrations of G418 . The flasks and agar plates were incubated under continuous light at an irradiance level of 60 μmol m−2s−1 and 23 ± 1 °C, and the growth of T. minus in the liquid and the solid medium was monitored at daily intervals for 10 days and 2 weeks, respectively. The sensitivity of T. minus to the antibiotics and the herbicide was repeated at least three times. To further determine the sensitivity of T. minus to obtain tub genes. Through PCR with designed primers (tubulin (tub) gene was obtained and the nucleotide sequences comprised a promoter region and a terminator region from a tub gene in T. minus. Genome walking [tub gene. Three specific synthetic primers, tub-sp1, tub-sp2, and tub-sp3  gene [nptⅡ) were flanked by the T. minus-derived tub promoter (Ptub) and terminator (Ttub), respectively. The A fusion PCR method was performed to fuse the nptⅡ and eGFP genes with the tub promoter and terminator of T. minus, as described in a previous study [nptⅡ cassette as an example, three target fragments, Ptub, nptⅡ, and Ttub, were obtained by regular PCR with the primer pairs of Ptub-F/Ptub(nptⅡ)-R, nptⅡ-F/ nptⅡ-R, and Ttub(nptⅡ)-F/Ttub-R, respectively  gene  and the us study . Taking ectively . The priT. minus cultures at the logarithmic growth phase were pelleted by filtration and suspended in BG11 medium to a biomass concentration of 2–3 g L−1. Then, a 2 mL aliquot of cell suspension was layered directly onto the central area of an MF-Millipore membrane filter , which was placed on a BG11 solid plate. After growth for 2 days, the samples were used as the recipient cells for transformation.For transformation, −1 in H2O) with 5 μL of a DNA solution (>1 μg μL−1), 50 μL of 2.5 M CaCl2, and 20 μL of 0.1M spermidine base. This was followed by 10 min incubation on ice and two centrifugal washes with 70% and then 100% ethanol before final resuspension in about 50 μl ethanol. A total of 10 μL of prepared DNA-coated particle solution was layered on a microcarrier for one trial. The bombardment was performed with a PDS-1000/He Biolistic Particle Delivery system . Plates were bombarded from a distance of 6 cm and bombarded under vacuum pressure at 125 mm Hg using helium pressures of 450, 900, 1100, and 1350 psi to accelerate particles. DNA coated particles were prepared by mixing 50 μL of 17 tungsten particle solution  was used to examine eGFP expression using excitation at 488 nm. Detection was in the range of 495–556 nm for eGFP and 685–735 nm for chloroplast autofluorescence.pSimple-T. minus cells were disrupted by grinding in liquid nitrogen with a mortar and pestle and transferred to a 1.5 mL microcentrifuge tube. The cells were suspended in TEN buffer . After phenol/chloroform/isoamylalcohol extraction and 2-propanol precipitation, samples were treated with RNase A, and the genomic DNA was recovered by ethanol precipitation following phenol/chloroform/isoamyl alcohol and chloroform treatments. The G418-resistant Genomic PCR was performed with isolated 0.1 μg genomic DNA as a template and 5 μL of Go Taq Master Mix. Amplifications were performed by incubating reaction mixtures at 95 ℃ followed by 20 s at 52.5 ℃ and 60 s at 72 ℃. Assays with no template were examined for every experiment as a negative control.T. minus transformants were harvested via filtration. After being washed with distilled water twice, the cells were transferred into fresh BG11 medium for subculture. The extracted genomic DNA and PCR analysis of the resistant gene from transformants, which was cultured to the third generation, were examined and analyzed as described above.The G418-resistant Total RNA was prepared by the ConcertTM Plant RNA Reagent  according to the manufacturer’s instructions and purified with an RNeasy Mini Kit column . The purified RNA (1.5 μg) was transcribed into cDNA by employing the Transcriptor High Fidelity cDNA Synthesis Kit , as described by the manufacturer. PCR was performed as for the genomic PCR analysis described above with slight modifications. The conditions of the PCR were as follows: 95 ℃ for 2 min prior to 25 cycles of 15 s at 95 ℃, followed by 20 s at 52.5 ℃ and 60 s at 72 ℃. Assays with samples that were prepared without reverse transcriptase in the cDNA synthesis reaction and with no template were examined for every experiment as a negative control.


Neurodegenerative diseases are characterized by the accumulation of disease-related misfolded proteins. It is now widely understood that the characteristic self-amplifying  capacity once only attributed to the prions of transmissible spongiform encephalopathy diseases is a feature of other misfolded proteins of neurodegenerative diseases, including tau, Aβ, and αSynuclein (αSyn). Ultrasensitive diagnostic assays, known as real-time quaking-induced conversion (RT-QuIC) assays, exploit these seeding capabilities in order to exponentially amplify protein seeds from various biospecimens. To date, RT-QuIC assays have been developed for the detection of protein seeds related to known prion diseases of mammals, the αSyn aggregates of Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy, and the tau aggregates of Alzheimer’s disease, chronic traumatic encephalopathy, and other tauopathies including progressive supranuclear palsy. Application of these assays to premortem human biospecimens shows promise for diagnosis of neurodegenerative disease and is an area of active investigation. RT-QuIC assays are also powerful experimental tools that can be used to dissect seeding networks within and between tissues and to evaluate how protein seed distribution and quantity correlate to disease-related outcomes in a host. As well, RT-QuIC application may help characterize molecular pathways influencing protein seed accumulation, transmission, and clearance. In this review we discuss the application of RT-QuIC assays as diagnostic, experimental, and structural tools for detection and discrimination of PrP prions, tau, and αSyn protein seeds. Neurodegenerative diseases are complex, multifactorial, heterogenous diseases identified by the accumulation of misfolded proteins in the brain. Alzheimer’s disease (AD) is characterized by tau and β-amyloid (Aβ) deposits, Parkinson’s disease (PD) by αSynuclein (αSyn) aggregates, and prion diseases by misfolded prion protein (PrP). Variability in disease progression, clinical presentation, and overlapping disease symptoms complicate diagnosis. Further, neurodegenerative diseases frequently have misfolded protein co-pathologies that have been suggested may contribute to the spectrum of disease heterogeneity. For example, AD can have misfolded αSyn in addition to tau and Aβ aggregates, and Lewy body dementias, such as dementia with Lewy bodies (DLB) and Parkinson’s disease dementia (PDD) can have comorbid tau deposits ,2. TargeThe misfolded proteins of neurodegeneration are capable of faithfully self-amplifying their misfolded structures via seeded polymerization mechanisms. The propagation of structurally matched, highly ordered protein assemblies is initiated by the misfolded “protein seed”. The capacity of misfolded proteins to seed further self-propagation was originally defined in prion diseases and is now a broadly recognized feature of the proteins underlying neurodegenerative diseases including AD and PD. Amplification of protein seeds correlates with progressive disease processes, with protein seeding events occurring before the onset of clinical signs. By definition, “protein seeds” describe the misfolded protein conformers capable of protein-based self-propagation, with the prototypical protein seed being that of prions. PrP prions occur when the largely α-helical conformation of native PrP is converted into a predominant β-sheet structure. Continued, prion-seeded conversion events result in propagation, accumulation, and spread of prions within a host. While prion transmission certainly depends on multiple factors such as prion type, titer, and route of exposure, PrP prions can be naturally and readily transmitted between hosts, as is the case with the readily transmissible prions of chronic wasting disease (CWD) in cervids. However, prions come in a plethora of shapes, sizes, and ability to induce pathological and disease-related events (reviewed in ). PrP seSeed amplification assays include both cell-based and cell-free platforms. Cell-based assays measure the amplification of seeding activity initiated by biospecimens using cells that express tau or αSyn protein fragments fused to fluorescent protein tags. Fluorescence resonance energy transfer (FRET) readouts are used to assess seeded aggregate formation. Cell-based assays are ultrasensitive, able to quantify fM quantities of seeds and typically demonstrate a 3-log dynamic working range . ProvidiBoth the protein misfolding cyclic amplification (PMCA) and the real-time quaking-induced conversion (RT-QuIC) assays are cell-free, in vitro seed amplification assays. PMCA assays use protein substrates derived from brain homogenates  or recom50) with Spearman Kärber analysis [RT-QuIC assays use recombinant protein substrates with shaking cycles and incubation to measure seeding activity in a high throughput (96- or 384- multiwell) format. Using the amyloid-specific dye ThT to measure seeded fibril formation, RT-QuICs can detect fg–ag quantities of protein seeds . Biospecanalysis  as a meaanalysis ,45,46,47analysis ,49, and There is considerable interest in developing biomarkers to classify neurodegenerative disease entities, including those that specifically target the characteristic aggregated proteins. In addition to improving our mechanistic understanding, such markers will certainly be useful to identify and stratify patient cohorts in the development of targeted therapeutics. Disease-specific biomarkers can help identify appropriate patients for clinical trials, as well as to follow the efficacy of therapeutics over time. In addition to specificity, the ideal biomarker is highly sensitive to support early diagnosis, demonstrates a sufficient dynamic range to evaluate clinically relevant quantitative changes with longitudinal studies, and can be measured in easily accessible biofluid or tissue specimens. Prion RT-QuIC assays have been successfully implemented for the diagnosis of prion diseases, with RT-QuIC evaluation of cerebrospinal fluid (CSF) currently being used as part of the diagnostic work-up for sporadic Creutzfeldt-Jakob disease (sCJD) ,52,53,54MAPT gene. Alternative splicing gives rise to six tau isoforms in the human brain, which are characterized by 0, 1, or 2 N-terminal sequence inserts and either 3 or 4 microtubule binding repeats resulting in classifications as 3-repeat (3R) or 4-repeat (4R) tau, respectively [Tau is a microtubule-associated protein encoded by the ectively ,62,63. TRecent near-atomic cryo electron microscopy (EM) structures have further defined the distinct structural conformers that encode the 3R/4R paired helical and straight tau filaments of AD , the 3R/The original tau RT-QuIC assay was developed for the 3R tau conformers of PiD . Using aSubsequent tau assay developments led to the first 3R/4R tau RT-QuIC assay for the detection of tau filaments of AD and CTE from brain tissue, known as the AD RT-QuIC . The AD Additional assays have been developed for the detection of 4R tau aggregates , as wellSynucleinopathies are characterized by the accumulation of αSyn aggregates and include PD, DLB, and MSA. Outside of prion RT-QuIC assays, αSyn RT-QuIC is thus far the most developed RT-QuIC seed amplification platform to aid premortem diagnosis, largely using CSF specimens. n = 76), 100% for DLB (n = 10), and 80% for MSA (n = 10) [n = 65) was 96.9%, whereas that for unrelated and neurodegenerative disease controls was 94% [n = 105) and control cases (n = 79) [The application of RT-QuIC for the detection of αSyn seeds was first reported by Alison Green and colleagues. Using CSF, they detected seeding activity from DLB and PD cases with a sensitivity of 92% and 95%, respectively, with 100% specificity . PD case(n = 10) . Specifi was 94% . A compa(n = 79) . A compa(n = 79)  versus t(n = 79) . Cases e(n = 79) ,76.Important aspects of diagnostic biomarkers include their ability to distinguish clinical syndromes, as well as to be used as potential prognostic indicators. This becomes particularly helpful in the cases where symptoms of disease overlap, such as the discrimination of synucleinopathies from dementias including AD and atypical parkinsonisms such as PSP and CBS. αSyn RT-QuIC was used to evaluate αSyn seeding activity in CSF from uncertain cases of parkinsonism and controls . This stThe ideal premortem biomarker is readily measured in easily accessible biospecimens/biofluids. αSyn RT-QuIC demonstrates extraordinary sensitivity for αSyn detection from CSF, however, while CSF can be obtained premortem, such samplings require an invasive lumbar puncture. Thus, for RT-QuIC use in routine preclinical screens, or for longitudinal patient assessment in clinical trials, a more readily accessible peripheral biospecimen may be preferential. αSyn seeding activity can be detected from olfactory mucosa nasal brushings in PD and MSA cases, albeit with incomplete sensitivity (19 of 29 cases) and specificity against PSP and CBD tauopathies . SeedingThe defining characteristics of the seeds detected by αSyn and tau RT-QuIC assays are only beginning to be explored. Seeding activity of 3R/4R tau in AD RT-QuIC is largely sarkosyl insoluble and protease resistant . The selPrion strains are thought to reflect distinct structural conformers, characterized by differences in fibrillar ultrastructures, protease resistance, and seed amplification properties. Ultimately, different prion strains result in strain-specific neuropathology and clinical disease manifestations. Akin to the strains of prion diseases, distinct strains of αSyn and tau aggregates have been suggested based on apparently different seeding properties, including amplification characteristics and resulting clinical and pathological outcomes ,28,29,83RT-QuIC assays collectively represent a platform of selective, seed-optimized amplification assays. As such, multiple assays can be used concurrently to qualitatively distinguish distinct seeds. This approach has been used previously to discriminate prion strains of classic and atypical (L-type and C-type) BSE ,86,87, aWhile utilization of multiple RT-QuIC assays concurrently can help discriminate strains, strain subtyping can be achieved within the same RT-QuIC assay. This has been observed as a feature for some prion, αSyn, and tau RT-QuIC assays. A modified second-generation prion RT-QuIC assay using full-length hamster substrate and CSF biospecimens is capable of identifying sCJD subtypes. An evaluation of CSF samples from 2141 patients indicated that different maximum ThT fluorescent amplitudes and lag times for seeded reactions were measured for MM1 versus MM2 and VV1 versus VV2 sCJD cases . SubtypiIn the 4R tau RT-QuIC, subtypes of 4R tau seeds from PSP, CBD, and FTDP-17 P301L or N279K mutation cases can be distinguished by differences in maximum ThT amplitudes of seeded reactions, as well as by distinct FTIR signatures of the resulting fibrillar RT-QuIC products . 3R tau RT-QuIC assays are powerful experimental tools with the ability to measure minute quantities of protein seeds from different tissues and biofluids in ways that can be applied to follow seed transmission and accumulation in cell culture and rodent models. Often, assessments of seeding events, seed accumulation, and transmission instead relies on evidence of histologically identifiable misfolded protein accumulation, or immunoblot detection of insoluble or protease resistant material. This might preclude measurements of early seeding events below the detection limits of these experimental methods. With the increasing reports indicating divergent pathogenic and transmission properties of protein seeds (reviewed in ), seedinPrion seeding activity has been used to establish time courses of prion propagation and spread, including very early propagation events following infection ,93. RT-Q8 seeding doses), in brain regions without histologically visible PrP deposits or other neuropathological indications of prion disease such as gliosis or spongiform change [Measurements of seeding activity have also been used to identify transmission events of prions that do not induce clinical disease or readily detectable pathological changes ,10,11. Tm change . WithoutThe networks and distributions of tau and αSyn protein seeds in preclinical and diseased states are largely not understood. Histological evaluations of tau and αSyn aggregate distribution suggest progressive, staged occurrence of aggregates in ways that correlate with disease ,97,98. HTo understand how protein seed networks correlate with neurodegenerative disease processes, when and where seeds occur, and with what other disease markers they are associated with must be defined. As neurodegenerative diseases often include the accumulation of multiple misfolded proteins, this includes how distinct protein seed networks may or may not overlap. From a practical perspective, it becomes important to also understand how comorbid protein seeds might influence seed amplification via RT-QuIC. In the AD RT-QuIC, inclusion of sub- and superstoichiometric quantities of synthetic Aβ oligomers does not influence the kinetics or sensitivity of AD tau seed amplification . HoweverFundamentally, RT-QuIC reactions are real-time kinetic readouts of amyloid fibril formation of the reaction substrate, and the seed-dependent acceleration of such formation. Thus, RT-QuIC assays can also be used to understand molecular features of substrates that influence amyloid formation. For example, RT-QuIC assays have been used to determine amino acids that influence PrP amyloid formation. This includes studies examining the influence of specific proline and lysine residues (within amino acids 101–110) on PrP amyloid formation ,100. DisUse of distinct protein substrates with single amino acid changes can confer selectivity, or stability to an RT-QuIC assay. These characteristics can be exploited both for diagnostic and experimental purposes. One recent study compared the ability of αSyn aggregates from DLB brain tissue and A53T mice to convert either wild-type or A53T mutant αSyn recombinant substrates with RT-QuIC . These fPrion RT-QuIC analysis has been applied to a wide variety of culture models. RT-QuIC assays have been used to establish prion propagation in organoid models , to evalSeeding activity has also been used to establish the efficacy of anti-amyloid activity of disinfectants, including hypochlorous acid (HOCl)  and sodiThe advantages of RT-QuIC assays have been discussed at length above, and certainly include their ultrasensitivity, high throughput amenability, as well as their ability to discriminate disease specific protein seeds, and recapitulate strain specific conformational features with recombinant protein substrates. Advantages over cell-based seeding assays include an apparently increased dynamic working range, with RT-QuICs having at least a 9-log working range, compared to the 3-log range of cell-based assays, as well as a more accessible laboratory setup that does not require cell culture facilities or a flow cytometer. While these features make RT-QuIC assays well-poised for diagnostic application and use as experimental tools in clinical and laboratory settings, current limitations of RT-QuIC assays must be taken into account.The ultrasensitive capabilities of RT-QuIC assays allow them to detect protein seeds that occur in even the most minute amounts. Prion RT-QuIC assays can detect ag levels of seeds, which corresponds to 10–40 prion particles . As suchFrom a diagnostic perspective, it remains to be established what the clinical implications of low levels of seeds are, particularly in specimens from non-symptomatic patients. Careful longitudinal analysis with samples collected prior to, and throughout clinical disease course will be important to help establish the quantitative correlates of seeding activity and clinical disease.3Citrate instead of NaCl allowed a million-fold increase in detection sensitivity. Hofmeister effects were further exploited to enhance detection of seeds in plasma, nasal brushings, and ear homogenates. Thus, modulation of ion effects [Toward further development of RT-QuIC assays, recent efforts to explore the influence of ionic environment on seed amplification has revealed that the fidelity and sensitivity of seed detection can be drastically altered by inclusion of more weakly or strongly hydrating ions . The Hof effects , and oth effects  can certOver the last 7 years, efforts have built on premises established for PrP prions to further develop RT-QuIC and related seed amplification assays to amplify Aβ, tau, and αSyn protein aggregates. Just like prion RT-QuIC assays, assays for tau and αSyn have shown extraordinary sensitivity and have further revealed qualitative distinctions in seed detection and faithful propagation of seed characteristics. As the field works to define and understand the clinicopathological heterogeneity of neurodegeneration, the ability of RT-QuIC assays to distinguish protein seeds combined with their ultrasensitive and modulatable detection capabilities underscore the value of RT-QuIC assays as diagnostic and experimental tools.


Literature on earthquake impact on hospital admissions is lacking, particularly in low-resource settings. Our aim was to study the pattern of admissions before and after the 2015 earthquake in a tertiary hospital in Nepal. We used routine hospital data from 9,596 admissions, and defined four periods: pre-earthquake (pre-EQ), acute (EQ1), post-acute (EQ2), and post-earthquake (post-EQ). We compared length of hospital stay (LOS) across the study periods using negative binomial regressions. We used logistic regressions to study changes in probability of admission for diagnostic categories, and Generalized Additive Models to model the difference in number of admissions compared to pre-EQ baseline. LOS was longer in EQ1 than during pre-EQ, in particular for injury-related admissions. In EQ1, the odds of injury admissions increased, while they decreased for the majority of other diagnoses, with the odds of pregnancy-related admissions remaining low until post-EQ. The number of admissions dropped in EQ1 and EQ2, and returned to pre-EQ trends in post-EQ, accumulating 381 admissions lost (CI: 206–556). Our findings suggest that hospital disaster plans must not only foresee injury management after earthquakes, but also ensure accessibility, in particular for pregnant women, and promote a quick return to normality to prevent additional negative health outcomes. While deaths and direct injuries are immediate and expectable consequences of such disasters, drastic changes in the surrounding environment may have longer-term health effects7. These disasters also heavily disturb health systems, which must provide healthcare in a context of sudden increase of health needs, radical change of priority conditions, infrastructural and material damage, and staff shortages8. While most of the victims in need of care during and after a disaster are managed in an outpatient basis at emergency departments, a considerable amount of people will require hospitalization9. However, there is inconsistent evidence on the impact on hospital admissions immediately after and in the months following violent, sudden-onset disasters12.Violent, sudden-onset disasters, such as hurricanes or earthquakes, cause considerable damage in communities, leading to widespread destruction13. Rapid population growth, urbanization, poverty, and geological risks contribute to Asia’s seismic vulnerability.Earthquakes are the most destructive type of natural disaster, having killed nearly 720,000 people globally between 2000 and 2018. About two thirds of all events worldwide occurred in Asia, where earthquakes affect much more people than earthquakes in all the other continentsth 2015, an earthquake with a moment magnitude of 7.8 severely hit the country. The epicenter was in Gorkha district, and heavy infrastructural damage occurred in neighbouring regions, including in the capital city of Kathmandu located 76 km away. There were several aftershocks, the strongest occurring on May 12th. National health services were the only services available to treat the high number of injured victims in the first days, since external assistance arrived with a considerable delay14. The Tribhuvan University Teaching Hospital (TUTH) is a major tertiary hospital in Nepal, built with earthquake resistance standards, and was functioning immediately after the earthquake, having assisted many earthquake victims15. TUTH belonged to the Hospital Preparedness for Emergencies (HOPE) network since 201416, and activated its disaster management plan after the earthquake, reorganizing its services and implementing a mass casualty triage system to categorize earthquake victims.Nepal is located on the boundaries of two colliding tectonic plates. On April 2517, or do not capture the specificities of the initial days after the disaster19. Understanding the pattern of hospitalizations after earthquakes is essential to improve hospital preparedness and surge capacity in future disasters, and facilitates a quick return to regular activities. We studied the pattern of hospital admissions at TUTH from six weeks before to four months after the 2015 earthquake, in terms of length of hospital stay (LOS), diagnostic categories, and number of daily admissions.The literature portraying how earthquakes affect health service outputs, such as hospital admissions, is scarce in resource-poor settings. The available studies in Nepal either fail to show the longer term consequences of the earthquake on hospital admissionsth and August 17th 2015 were collected from centralized hospital registries. Three datasets were available: hospital admissions, hospital discharges, and admitted earthquake victims. The first dataset contained sociodemographic information and served as panel data for daily admissions. We linked it with the remaining datasets to include date of discharge and diagnosis. The victim dataset underwent hand verification in original patient files in case of missing data or inconsistent information. We identified common admissions and merged datasets through deterministic linkages of identification number, name, sex, age, and date of admission. If these did coincide, we considered them the same admission. If not, we only considered the entry from the hospital admission dataset.Data on hospital admissions between March 15th revision (ICD-10), until the third level (letter and two digits), and was entered directly by TUTH’s administrative staff. In the earthquake victim dataset, diagnosis was originally entered as free text, and two independent researchers later codified it into ICD-10. If their coding differed, they would discuss and agree on a final code.Available variables were sex, age (continuous), date of admission, date of discharge, and diagnosis. In the discharge dataset, diagnosis was classified according to the International Classification of Diseases, 10th until April 24th 2015Pre-earthquake (pre-EQ): admissions from March 15th (including) until May 15th 2015Acute earthquake period (EQ1): admissions between April 25th (including) until June 5th 2015Post-acute earthquake period (EQ2): admissions from May 16th until August 17th 2015Post-earthquake (post-EQ): admissions from June 6We classified age into four groups , and used the 21 ICD-10 disease categories for our analyses. We calculated length of stay (LOS) as the number of days between hospital admission and hospital discharge. We defined the following four study periods:14. It was also the time period required for other public services, such as schools, to reopen20. But most earthquake-related hospital admissions occur in the first days following the disaster2, and there were repeated aftershocks, the strongest on May 12th. Hence, we divided the critical six-week period in two smaller time intervals to increase precision and capture these particularities.We determined a six-week critical period divided into two three-week sub-periods for multiple reasons. First, six weeks after the earthquake, national health actors considered the critical period to be over, and health services started shifting back to a regular systemFirst, we made a descriptive overview of the variables and tested for bivariate associations between sex, age groups, earthquake periods, and ICD-10 category, using chi-square tests.21, which was the case in our sample: the variance-to-mean ratio ranged from 9.77 in pre-EQ to 15.86 in EQ1. We compared differences in Length of Stay in the four study periods using a Negative Binomial regression, and adjusted for age group, sex, and ICD-10 category. We also calculated LOS differences across the four periods for individual ICD-10 categories consisting of more than 10% of total admissions, adjusting for age group and sex. Outliers, i.e. admissions with a LOS longer than six months, were removed from the analysis (n = 4).Length of hospital stay generally follows a right-skewed distribution curve. Negative binomial regression is suitable to analyze LOS, with the advantage over Poisson regression in case of overdispersion of countsTo evaluate how the probability of admission for a given ICD-10 category changed with earthquake period, in relation to all other ICD-10 categories, we carried out binary logistic regressions, adjusting for age group and gender. We assumed the outcome as a Bernoulli variable, where being admitted for a given ICD-10 category was a success and for any other ICD-10, a failure. We only included ICD-10 categories that followed the one in ten rule, in this case with a minimum of 70 observations, and each time we compared one category with all the others grouped. We tested for interaction terms between control variables.th and July 6th, respectively). Splines are more flexible around the knots and this allows to fully capture non-linear trends23. We assessed the model’s goodness-of-fit by visual inspection of its residuals, where a good fit means 95% of the deviance residuals are between −2 and 2 and no big outliers are present.We calculated the median number of daily admissions for weekdays and Saturdays in the pre-EQ period, since Saturday is the only non-working day in Nepal. We considered these values as the baseline number of daily admissions. We computed the difference between this baseline and the actual number of daily admissions during the three following periods, and used Generalized Additive Models (GAM) to model these differences and to capture non-linear behaviour. We used a variable reflecting Saturdays and linear B-splines, with interior knots at transition dates between EQ1 and EQ2, and between EQ2 and post-EQ (May 16We used R software (version 3.5.0) to perform all analyses and considered α = 0.05.2/074/075).Personal identifiers were used solely in the data verification process, and removed immediately after. We submitted this research protocol to the Tribhuvan University – Institute of Medicine’s ethics committee. Since this study used secondary data from routinely generated information, the Tribhuvan University – Institute of Medicine’s ethics committee deemed that informed consent was not necessary, and they provided clearance to undertake this study (Ref. 381(6-11-E)th and August 17th 2015. Supplementary Figs. We included 9,596 admissions occurring between March 15Table Women accounted for 56% of all admissions, while children and adolescents under 15 years of age represented 17% of all admissions.LOS distribution was right-skewed, ranging from 0 to 175 days, with an average of 7.7 days (sd 9.59), a median of 5 days, and an interquartile range of 3 to 9 days. Table In comparison to pre-EQ, LOS for admissions due to injury and due to contact with health services were 57.3%  and 21.0%  longer during EQ1, respectively. In contrast, LOS for admissions related to respiratory diseases decreased by 21.6% in EQ1.In the bivariate analyses, earthquake period was significantly associated with age groups and ICD-10 categories . The share of small children (0–4years) was relatively low in EQ1, while the share of older people (50 years and older) relatively increased in EQ2 and post-EQ.Table We checked for interaction effects of earthquake period with age group and with sex, but these models were not significantly better, so we discarded them.In the pre-EQ period, the median number of daily admissions was 68 on weekdays and 45 on Saturdays, which we considered the baseline for further analyses. As shown in Fig. The model residuals do not show a particular remaining pattern although a big outlier is present , length of stay (LOS) for admissions was significantly longer than in other periods. This was mostly due to injury admissions, in which LOS highly increased. There were higher odds of admission due to injuries and contact with health services, compared with the pre-EQ period, and relative to other admissions. There was also a significant relative decrease of admissions due to infectious diseases, neoplasms, pregnancy-related conditions, ear and mastoid diseases, digestive diseases, respiratory diseases, skin diseases, and genitourinary diseases. Finally, the overall number of admissions decreased significantly in EQ1 in comparison to the pre-EQ scenario, remaining significantly low in EQ2, and returning to pre-EQ trends in post-EQ.24, or because many severe cases were probably referred to TUTH since it is a reference hospital, and it was functional after the earthquake15. In addition, external factors may have increased LOS: a disrupted transport network, or destroyed housing, may have delayed patient discharge until an improvement of the situation. Nevertheless, LOS for respiratory diseases substantially decreased in EQ1. This is probably because such admissions were shortened to ensure all earthquake victims in need were admitted and appropriately treated. These findings are in line with a study in a rural hospital in Nepal describing the patient load in the three weeks after the earthquake, which found that earthquake-related conditions had significantly longer hospitalizations compared to other non-earthquake related admissions17.The fact that injury admissions occurring in EQ1 were particularly long may relate to earthquake injury characteristics linked with severity and frequent complicationset al., the need for hospital care due to earthquake injuries is concentrated in the days after the earthquake, while other elective and less urgent conditions are deferred2. Shortly after, there is a need for hospital care for trauma-related complications, which could explain why admissions due to factors influencing health status and contact with health services are high in EQ1 and EQ2. A previous study identified this diagnostic category in some of the earthquake victims who were admitted in TUTH15. The high probability of admissions in this category in post-EQ may be due to an accumulation of interventions that re-started after weeks of being interrupted, such as the donation of organs and tissues by healthy donors. Admissions due to congenital diseases, musculoskeletal diseases, and neurological diseases increased in post-EQ, which could also correspond to an accumulation of elective care interrupted in EQ1 and EQ2. An unexpected finding was the sustained decrease of respiratory conditions in post-EQ in comparison to pre-EQ, as the literature reports medium-term increases of respiratory diseases after earthquakes5. The fact that our full study period corresponds to warmer months in Nepal, and that the population is relatively young, could be an explanation for this observation. Severe respiratory diseases requiring in-hospital treatment are less often expected, also because people were not particularly exposed to water or humidity during this earthquake – as opposed to hurricanes or earthquakes followed by tsunamis, characterized by an increase of pneumonia cases25. As such, most respiratory consequences of the 2015 earthquake would be reflected in outpatient care. A study in Patan Hospital, in the vicinity of Kathmandu, showed that there were significantly more emergency visits due to cardiovascular, psychiatric, respiratory, and hematologic conditions in the four months after the earthquake, compared to the same period one year before19.There was a relative increase of admissions due to injuries and due to other factors influencing contact with health services in EQ1; while admissions due to other ICD-10 categories substantially decreased. As explained in a conceptual model suggested by von Schreeb 3. An ethnographic study in rural Nepal showed that, after the earthquake, several women preferred delivering at home rather than at a health facility, making it more difficult to refer to a hospital when needed26. The transfer from basic delivery facilities to higher levels of care was disrupted after the earthquake due to road destruction26, which could further explain a sustained decrease in pregnancy-related admissions at TUTH. There are reports of operational mobile reproductive health clinics set up by the United Nations Population Fund (UNFPA) and the Ministry of Health in the initial response phase to address specific maternal care needs26, but there is very little information describing long term changes in maternal health. Some admissions related to the outcome of delivery, however, are categorized under “factors influencing health status and contact with health services”, which merits a more detailed examination combined with a more in-depth analysis of the category Pregnancy, Childbirth, and the Puerperium. In addition, more comprehensive studies can shed light on the external context’s influence on maternal healthcare. Regardless of this, it is essential to ensure appropriate care for pregnant women in the months following a disaster.Our findings show that pregnancy-related admissions decreased immediately after the earthquake, and remained low in the long-term. A study following typhoon Haiyan also identified an immediate decrease of pregnancy-related admissions, suggesting that lack of access may have caused an increase in unsafe deliveries with lack of adequate care27. Yet, our final analysis showed that, despite an initial peak of admissions on the fifth day after the earthquake, the total number of admissions dropped in the weeks after the earthquake, and slowly increased back to baseline levels after six weeks. During this six-week period, our model estimates that there were 381 fewer admissions than if the pre-EQ trend had continued. TUTH was functioning immediately after the earthquake, with victims and visitors arriving within 20 minutes after the shake. The emergency department put in place its mass casualty triage system, and increased its space by using other buildings as specific triage color areas. It is possible the emergency department treated many of the victims, who in normal circumstances would have been admitted, in an outpatient basis. Despite the lack of documentation to support this, it is likely that only the most severe cases were admitted in the hospital, which themselves were very resource-consuming, suspending all other non-urgent admissions. This is supported by the fact that admissions occurring during EQ1 had higher LOS, but would benefit from more research exploring other contextual factors.It is known that earthquakes cause a sharp increase of medical needs due to injuries, many of them requiring surgical and in-hospital treatmentet al.’s conceptual model proposes2.Finally, there is no clear compensating bump for the total number of admissions in the beginning of the post-EQ period, but an accumulation of admissions of specific diagnostic categories may have happened, as indicated in the logistic regression, for congenital, musculoskeletal, and neurological diseases. This suggests an attempt to resume delayed work, as von Schreeb Our study had some limitations, including that it used a large dataset from routine hospital activities, not created for research purposes, with a limited number of variables. There may have been a categorization bias in the diagnosis classification, since it often influences financial compensation. However, by using broad ICD-10 categories, we reduced this bias. Data from the earthquake periods may be less complete because of the hectic situation at the time, and it is possible that not all information was systematically registered. Since we completed and verified most of our findings with an additional earthquake victim dataset, which we verified with original patient files, we believe this is a minor issue. Our study compared the post-disaster situation with a relatively short baseline period, and did not take seasonality into account. Seasonality could partially have been overcome by using annual trends from previous years, but this information was not available. Nevertheless, in the post-EQ period, the number of admissions returned to baseline, suggesting a fairly low seasonal variation of daily hospital admissions at TUTH in our study period. When we computed the baseline number of daily admissions, we differentiated for the only non-working day in Nepal. However, we could not capture variations related to festive holidays, strikes, or other events that may affect admission patterns.Earthquakes heavily disrupt the functioning of tertiary hospitals. Our study provides useful information for tertiary hospitals in seismic and low-resource settings. Because of the increase of injured patients with long admissions, hospitals should be prepared to quickly mobilize resources to treat high numbers of injuries in the event of an earthquake. But the overall number of admissions decreased in the weeks following the earthquake, mostly due to a strong decrease of admissions due to other causes, which were also shorter than usual immediately after the earthquake. To prevent negative health outcomes from conditions other than injuries, efforts are needed to ensure accessibility to the hospital and a quick return to normality. The relative decrease of pregnancy-related conditions is concerning because deliveries are expected to remain stable despite the earthquake, suggesting deliveries are taking place elsewhere or without the presence of a skilled attendant. While more research is needed to understand earthquake impact on maternal healthcare at the hospital level and beyond, a priority is to ensure community capacity to manage urgent maternal health needs in remote areas after earthquakes.Supplementary files.


To maximize the benefit of parent-directed, positive sensory exposures in the NICU, a structured sensory-based program titled the Supporting and Enhancing NICU Sensory Experiences (SENSE) program was developed that includes specific doses and targeted timing of evidence-based sensory exposures.The Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework was used to systematically evaluate the SENSE program as an implementation strategy. One-hundred preterm infants ≤32 weeks gestation were studied (61 receiving the SENSE program and 39 standard-of-care). Parent education time and infant sensory exposures were tracked, and parents completed a questionnaire that probed their perceptions about the SENSE program.p < 0.0001). Ninety-five percent of infants tolerated the SENSE program as defined, with 5% of infants requiring intermittent adaptations or the interventions being stopped for a period that typically lasted 1–2 weeks. Earlier parent education was related to more parent participation in SENSE program interventions (p = 0.04). Eighty-five percent of participants receiving the SENSE program had most of the sensory interventions completed by parents, as opposed to the medical or sensory support team. Seventy-two percent of infants had at least 100% of the auditory and tactile doses conducted over the length of stay. Parents reported acceptability.One-hundered thirty-one families were recruited, and 100 (76%) enrolled. The SENSE program was initiated at an average postmenstrual age of 29.8 (±2.4) weeks; 4.9 (±5.6) days after birth. The average number of education sessions with families was 4.8 (±3.7) amounting to 72.3 (±37.4) total minutes over hospitalization. The total time of logged tactile and auditory exposures among SENSE recipients over the length of hospitalization was a median (IQ range) of 9325  minutes over an average of 10.1 (±7.6) weeks of hospitalization. There were differences in the proportion of tactile and auditory exposure targets received by the infant among those receiving the SENSE program compared to standard-of-care  informed future strategies to aid maintenance during dissemination. The evidence to support positive sensory exposures  to improve outcomes for high-risk infants in the NICU and their families is well-understood –4. HowevDue to the complex environment and differences in culture across NICUs, there is significant variability in the application of sensory-based interventions , reducinSupporting and Enhancing NICU Sensory Experiences (SENSE) program is to engage parents in consistently providing positive, developmentally-appropriate, evidence-based sensory interventions to their high-risk infants in the NICU every day of hospitalization. The SENSE program includes specific doses and targeted timing  of sensory exposures such as skin-to-skin care, infant massage, auditory exposure, holding, and rocking. The SENSE program was developed to optimize parent engagement, while maximizing daily positive sensory exposures to improve infant development and parent-infant interaction. A systematic and scientific process of development of the SENSE program was undertaken using a synthesis of research related to sensory interventions (known to improve outcome), parent and health care professional focus groups and interviews, and expert input  were achieved across hospitlization, and differences in sensory exposures among those who did and did not receive the SENSE program were evident. Eighty-five percent of parents who received the SENSE program conducted the majority of the SENSE interventions, and when combined with the sensory support team, 91% of infants received all of the sensory experiences defined in the SENSE program across hospitalization.The SENSE program was feasible to implement with preterm infants across hospitalization using a research team to faciliatate programming. Use of the SENSE program led to more positive sensory exposures across hospitalization. Ninety-five percent of infants in a Level IV NICU could tolerate the SENSE program exposures as defined, and only 5% required occasional periods of modification or stopping interventions for medical instability. Of interest was that the earlier the SENSE program education and interventions occurred, the more parent engagement occurred. To our knowledge, we are the first study to report that earlier engagement leads to better parent participation in the NICU. Ninety-one percent of the auditory and tactile dose targets defined in the SENSE program occurred among those receiving the SENSE program. Seventy two percent of infants received more than the dose targets described in the SENSE program. While 3 h of auditory and 3 h of tactile exposures may sound like a large amount of additional sensory exposures to accomplish, families were able to find ways to incorporate them into daily life in the NICU to mitigate painful or procedural sensory experiences.Parents reported high acceptability. Previous work identified a subset of parents who voiced concerns about other personnel interacting with their infant, however, having health care professionals and/or the sensory support team nurture and provide extra attention to their infant in their absence was viewed as a positive factor among most parents who were surveyed. This parallels an increase in reports of NICU volunteers , 25, whoSENSE program education was an average of 73 min, which can be recuperated in standard billing by a neonatal therapist. The American Academy of Pediatrics recommends that Level III and IV NICUs have an occupational or physical therapist on staff , and theThere were limitations to this study. The sample size was pre-determined based on the 2 overarching cohorts, and power analysis was not conducted on implementation outcomes to ensure adequate sample size. Most of the outcomes are descriptive in nature. Enrollment in a study that is investigating different types and quantities of sensory exposures suggests that this is what parents should be doing and could also have impacted parent behavior. Log sheets used to track sensory exposures also are suggestive, even though the doses were not listed on the standard-of-care group logs, which could have led to more parent engagement due to power of suggestion. Despite these limitations, we were able to demonstrate significant differences in the proportion of auditory and tactile exposures completed with infants between the SENSE program group and standard-of-care group. This study was also limited by error introduced by logging and reporting of sensory exposures across multiple sources. Twenty-four hour videorecording was attempted to better capture the sensory environment, but was not feasible. Other limitations include subjective interpretation of survey questions and parent’s answers, lack of capture of other costs that were related to implementation , and lack of capture of other sensory exposures  when investigating fidelity. This manuscript includes a very large amount of data on sensory exposures across long periods of hospitalization, which increases the risk of error and/or omission. In addition, the large amount of data is managed by use of descriptive analysis, of which average or median values may not provide the same level of understanding as individual reports on the application of sensory exposures in the moment. There are also a multitude of implementation factors that can be investigated, and this study only reports on a few. However, we were able to use a systematic process to report implementation outcomes using the RE-AIM framework to speed the translation of research findings to the bedside.The SENSE program is an implementation strategy designed to ensure that appropriate positive sensory exposures are delivered to high-risk infants each day of NICU hospitalization in order to improve their health care experience and drive appropriate brain development. This study reports on implementation within the context of a research study, with research team personnel used to administer the SENSE program in a level IV NICU. Refinement of educational materials for parents as well as implementation materials for hospitals was undertaken, using knowledge gained from this study. Following this study, there was increased interest across the United States and abroad in the SENSE program, and care was taken to define a dissemination and implementation strategy to aid its uptake across different hospitals. Given that the study had a research team member administer the program, one important component of the implementation strategy that was detailed and included with SENSE program educational materials for distribution was the identification of a SENSE administrator at each site. This SENSE administrator oversees the education of parents, assesses tolerance of the infants, and provides access to program materials. The use of a facilitator, or SENSE administrator, is in line with frameworks that support the use of facilitation , 29. The