Introduction: The purpose of this epidemiologic study was to analyze the care provided by our institution to middle-aged and geriatric hip fracture patients throughout the pandemic to examine for any differences compared to pre-pandemic care and across the pandemic stages.Methods: Consecutive patients >55 years old treated for hip fractures at our institution between October 2014 and January 2022 were analyzed for demographics, coronavirus disease 2019 (COVID-19) and vaccination status at admission, injury characteristics, hospital quality measures, and outcomes. Patients were divided into three separate cohorts: Pre-COVID-19 (PRECOV), COVID-19 Pre-Vaccine (PREVAX), and COVID-19 Post-Vaccine (POSTVAX). A sub-analysis removed COVID-19-positive patients across the study period. Comparative analyses were conducted.Results: A total of 2,633 hip fracture patients were included. For the overall cohort, there was no difference in the rate of inpatient deaths between the PRECOV, PREVAX, and POSTVAX cohorts (p=0.278). PRECOV had a significantly lower 30-day mortality rate compared to PREVAX or POSTVAX (p=0.012). Differences in complication rates for surgical site infection, urinary tract infection, and anemia  were seen between cohorts. PRECOV had the longest length of hospital stay (p<0.01). PREVAX patients required more ICU level of care (p<0.01). When removing COVID-19-positive patients, all three cohorts had similar inpatient (p=0.872) and 30-day mortality rates (p=0.130).Conclusion: The care of patients treated for hip fractures did not change throughout the pandemic at our institution. The elevated mortality rate due to the effects of COVID-19 seen in the pre-vaccine cohort decreased over time as the understanding of COVID-19 improved and the vaccine was introduced. We recommend continuation of the same hip fracture care protocols as used pre-pandemic. The coronavirus disease 2019 (COVID-19) pandemic has greatly affected the world since it first began at the end of 2019. From the early days of late winter/early spring of 2020 to May 10, 2023, there have been 765,903,278 confirmed cases of COVID-19 globally with 6,927,378 deaths [The onset of the COVID-19 pandemic had a large impact on healthcare systems and caused hospitals to be stretched for resources. High volumes of infected individuals caused many hospitals to restructure their departments and staffing requirements to increase the availability of beds, staff, and treatments available for COVID-19-infected patients -5. TheseThe presence of the COVID-19 pandemic coupled with the well-documented risk for morbidity and mortality associated with hip fractures highlights a significant challenge facing providers today -10. MultThe development of the vaccine by the end of 2020 marked a significant timepoint in the pandemic’s evolution, allowing providers to combat the acute effects of COVID-19 infection more effectively. Multiple studies have demonstrated the vaccine’s efficacy in mitigating the occurrence of admission, inpatient complications, and deaths secondary to COVID-19 infection ,15.The purpose of this epidemiologic study was to analyze the care provided by one multi-center institution for patients treated for hip fractures during the pandemic with their subsequent outcomes, examining potential differences with pre-pandemic care and across the different stages of the pandemic.New York University (NYU) Langone Health Office of Science and Research Institutional Review Board  approved the use of a geriatric trauma database to review all patients, aged 55 or older, who sustained a hip fracture via a low-energy mechanism of injury between October 2014 and January 2022 . The study was conducted at NYU Langone Health and Jamaica Hospital Medical Center, New York, United States. All patients were treated at one academic medical center that included three Level 1 trauma centers, one university-based tertiary care referral hospital, and one orthopaedic specialty hospital.All centers within our institution followed a similar four-day hip fracture treatment protocol -18. For The hip fracture protocol utilized within this study included medicine co-management of all patients and concrete, well-delineated tasks for all members of the care team including nurses, doctors, social workers, and patient care managers, as well as both physical and occupational therapists. The goals of this protocol include immediate medical preparation for surgery followed by rapid surgical intervention with early identification of discharge barriers and coordination of physical therapy and occupational therapy for rehabilitation purposes. Likewise, at their time of arrival at the Emergency Department (ED), patients were rapidly screened by the initial treatment team and evaluated for medical clearance. If medically appropriate, patients were then transferred to a tertiary care orthopedic hospital with hospitalist co-management.Demographics were collected for each patient that included age, sex, body mass index (BMI), sex, hip fracture classification per AO/OTA guidelines, baseline ambulatory/functional status, and comorbidities as represented by the Charlson Comorbidity Index (CCI) . Injury Hospital quality measures collected for each patient included length of stay (LOS) and need for intensive care unit (ICU) level of care. Complications recorded during each patient’s index hospitalization included sepsis/septic shock, pneumonia, deep vein thrombus/pulmonary embolism (DVT/PE), myocardial infarction (MI), acute renal failure/acute kidney injury (ARF/AKI), stroke, surgical site infection (SSI), decubitus ulcer, urinary tract infection (UTI), acute respiratory failure (ARF), anemia, and cardiac arrest. Mortality measures collected included inpatient mortality and mortality within 30 days of discharge .Demographic data were characterized through descriptive summary statistics. Continuous variables were compared with the Mann-Whitney U test. Categorical variables were compared with chi-square test. Comparison of both categorical and continuous variables was done using independent sample t-tests and ANOVA. Patients were divided into three separate cohorts: Pre-COVID-19 (PRECOV), COVID-19 Pre-Vaccine (PREVAX), and COVID-19 Post-Vaccine (POSTVAX). The POSTVAX time period was defined as after December 1, 2020, to capture the period once the first vaccine was publicly available for the United States. Statistics were calculated with IBM SPSS Statistics for Windows, Version 25.0 . Significance was defined with an alpha of 0.05.Between October 2014 and January 2022, 2633 patients aged 55 years and older who sustained a hip fracture via a low-energy mechanism were seen in one healthcare system and enrolled in an IRB-approved geriatric trauma database. The PRECOV cohort included 1727 patients seen between October 2014 and February 2020. The PREVAX cohort included 400 hip fracture patients who presented after the onset of the pandemic  but before the first administration of the COVID-19 vaccine . The POSTVAX cohort included 506 hip fracture patients that presented after the first vaccine administration , 53 patients (5.8%) were COVID-19-positive, and 177 (19.5%) patients were vaccinated with at least one dose at the time of admission. Patients’ ambulatory status at admission included: 1777 (67%) were community ambulators, 751 (29%) were household ambulators, and 105 (4%) were non-ambulatory. The most common AO/OTA fracture classifications included: 1397 (53%) 31A, 1138 (43%) 31B. The most common interventions included: short intramedullary nail  and hemiarthroplasty .Differences between the PRECOV, PREVAX, and POSTVAX cohorts included a larger proportion of community ambulators in the PRECOV cohort  and a larger proportion of household ambulators in the PREVAX cohort . Significant differences were also seen in regard to the fracture classifications and treatments chosen throughout the pandemic . The PRECOV cohort had a significantly lower 30-day mortality rate compared to the PREVAX or POSTVAX cohorts . Patients who were COVID-19-positive in the PREVAX cohort experienced a higher rate of inpatient  and 30-day mortality  as compared to COVID-19-positive patients in the POSTVAX cohort; however, this difference was not significant.Among inpatient complications, there were significant differences in rates of surgical site infection , UTI , and anemia  between the three cohorts. The PRECOV cohort had the longest length of hospital stay . The PREVAX cohort had a significantly higher percentage of patients need ICU-level care   and 30-day mortality rates  , UTI , and anemia  between the three cohorts. PRECOV cohort had the longest LOS . The PREVAX cohort had a significantly higher percentage of patients that need ICU-level care  . However, since the initial surge of cases in the spring of 2020, the medical community’s understanding and management of COVID-19 have improved to combat the virus and subsequent variants . WidesprThe 30-day mortality rate was found to be significantly elevated in the PREVAX cohort. This may be explained by the shorter LOS seen as patients may have been discharged at an earlier stage in their recovery. Patients may have missed inpatient and outpatient physical and occupational therapy sessions that would have been instrumental to their recovery. With the travel restrictions in place as well as what we have seen anecdotally in the early stages of the pandemic, patients were less willing to travel to appointments for fear of infection. Additionally, patients in the PREVAX cohort experienced a change in discharge location similar to those reported in the literature as many facilities restricted which patients they admitted . While pIn order to compare immediate hip fracture care among the three cohorts accurately, we reexamined inpatient mortality without the influence of COVID-19. We found that there was no difference in inpatient mortality between the three cohorts when COVID-19-positive patients were removed. This highlights our institution's continued ability to provide effective hip fracture care to patients, despite the ongoing backdrop of the COVID-19 pandemic. In addition, there was no significant difference in 30-day mortality for any of the three cohorts, demonstrating that even during the COVID-19 pandemic, our patients did not experience a significantly higher mortality rate as compared to pre-pandemic. It is reassuring to see that the high level of care provided for hip fracture patients did not change, even with the disruptions caused by COVID-19.Patients who presented in the PREVAX cohort were more likely to require an elevated level of care. Our institution experienced an elevated rate (24%) of patients requiring ICU admission during the COVID-19 pandemic prior to the vaccine. This aligns with literature from the early days of the pandemic, demonstrating similar rates of ICU admission for ventilatory support and around-the-clock care in the backdrop of high rates of COVID-19 positivity . While tThis study has several limitations. The data we examined only encompassed one health system’s experience. While our health system is located in a diverse metropolitan area, our patient population may be different from those in other urban or rural areas. Especially during the pandemic, major metropolitan areas were often hotspots for COVID-19, meaning our experience may not be reflective of all places. Additionally, this is a retrospective study. As the pandemic continues to fluctuate and change, our findings may not fully represent the impact of new changes in COVID-19 variants.The care of patients treated for hip fractures did not change throughout the pandemic at our institution. The elevated mortality rate due to the effects of COVID-19 seen in the pre-vaccine cohort decreased over time as the understanding of COVID-19 improved and the vaccine was introduced. We recommend the continuation of the same hip fracture care protocols as used in the pre-pandemic period. Additionally, we urge providers in all specialties to recommend vaccination as all geriatric patients at risk for fracture would benefit from COVID-19 vaccination. While many studies have discussed the impact of the COVID-19 pandemic on fracture care, these have all focused on the early months of the pandemic. Therefore, we sought to expand the discussion regarding hip fracture care in a longitudinal scope closer to the present day.


Ursodeoxycholic acid (UDCA) may reduce susceptibility to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection by downregulating angiotensin-converting enzyme 2 (ACE2), based on recent experimental investigation. This study aimed to determine the potential protective effect of UDCA against SARS-CoV-2 infection in patients with chronic liver disease.Patients with chronic liver disease receiving UDCA (taking UDCA ≥1 month) at Beijing Ditan Hospital between January 2022 and December 2022 were consecutively enrolled. These patients were matched in a 1:1 ratio to those with liver disease not receiving UDCA during the same period by using a propensity score matching analysis with nearest neighbor matching algorithm. We conducted a phone survey of coronavirus disease 2019 (COVID-19) infection during the early phase of the pandemic liberation (from 15 December 2022 to 15 January 2023). The risk of COVID-19 was compared in two matched cohorts of 225 UDCA users and 225 non-UDCA users based on patient self-report.vs. control 94.2%, p = 0.002), more mild cases , and shorter median time from infection to recovery . Logistic regression analysis showed that UDCA was a significant protective factor against COVID-19 infection . Furthermore, diabetes mellitus  and moderate/severe infection  were more likely to prolong the time from infection to recovery.In the adjusted analysis, the control group was superior to the UDCA group in COVID-19 vaccination rates and liver function indicators, including γ-glutamyl transpeptidase and alkaline phosphatase (p < 0.05). UDCA was associated with a lower incidence of SARS-CoV-2 infection (UDCA 85.3% UDCA therapy may be beneficial in reducing COVID-19 infection risk, alleviating symptoms, and shortening the recovery time in patients with chronic liver disease. However, it should be emphasized that the conclusions were based on patient self-report rather than classical COVID-19 detection by experimental investigations. Further large clinical and experimental studies are needed to validate these findings. Since the liberation of epidemic control on 7 December 2022, there has been a sudden increase in the number of coronavirus disease 2019 (COVID-19) infections in China. Currently, COVID-19 infections were mostly mild due to the attenuated pathogenicity of the virus . HoweverUrsodeoxycholic acid (UDCA) is used as a cholagogic drug, which may provide health benefits to many illnesses. UDCA has been confirmed to possess protective effects on hepatocytes, including reducing cholestasis, improving liver function, and alleviating hepatic fibrosis . A recenMultiple pieces of evidence revealed that preexisting chronic liver disease was associated with poor prognosis in patients with COVID-19 . Data frA single-center cohort study was conducted at Beijing Ditan Hospital. Patients with chronic liver disease  receiving UDCA (taking UDCA ≥1 month) from January 2022 to December 2022 were included in the UDCA group. Patients (hepatitis B and autoimmune hepatitis) not receiving UDCA during the same period were enrolled in the control group. Exclusion criteria included the following: 1) repeated COVID-19 infection; 2) complicated with multiple chronic liver diseases; 3) concomitant with other coexisting chronic viral infections, such as cytomegalovirus and acquired immunodeficiency syndrome; 4) accompanied by other severe infections; 5) with active or suspected malignancy or history of malignancy; 6) pregnancy and breastfeeding; 7) age ≤ 18 years.This study was approved by the institutional review board of Beijing Ditan Hospital  and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. As this study was conducted during the epidemic, the patients were unable to come to the hospital to sign the informed consent forms, but verbal informed consent was obtained from all patients during the telephone survey.Demographic variables included age, sex, body mass index, personal history, clinical complications, COVID-19 vaccination, and UDCA administration information. The most recent laboratory investigations prior to SARS-CoV-2 infection were collected for liver function indicators , serum lipid parameters triglyceride and total cholesterol levels, blood routine indices , glycosylated hemoglobin, and estimated glomerular filtration rate. These variables were collected from electronic medical records.We then conducted a phone survey to investigate SARS-CoV-2 infection in these patients during the early phase of epidemic liberation (from 15 December 2022 to 15 January 2023). The main data collected were COVID-19 vaccination, SARS-CoV-2 infection, symptom characteristics, treatment, prognosis, time from infection to recovery, and progression of liver disease during COVID-19 infection.Chronic liver disease was defined as the progressive deterioration of liver function for more than 6 months, including the synthesis of clotting factors, detoxification of metabolic waste products, proteins, and the secretion of bile. All patients in this study had a disease duration of more than 6 months.Overweight/obesity referred to a body mass index greater than 24.Drinking was defined as current alcohol use or abstinence from alcohol for less than 6 months.Recovery time referred to the time from infection to symptom disappearance. Day 1 referred to the time when patients reported COVID-19-related symptoms, such as fever and cough.http://www.nhc.gov.cn/). Based on the disease severity, COVID-19 was categorized into four types: mild, moderate, severe, and critical cases.The diagnosis and classification criteria of COVID-19 referred to the 10th edition of the Chinese protocol for the treatment of SARS-CoV-2 infection  from the lower and upper tails of the propensity score due to a lack of common support. Propensity score matching was performed using an SPSS-R plugin for R packages .Given the inherent selection bias of patients with different chronic liver diseases, we controlled the confounding by performing a propensity score matching analysis. The propensity score was calculated with an t-test or the Mann–Whitney U test. Categorical variables were presented as percentages (%), and their statistical analysis was performed by the chi-square test or Fisher’s exact test. Logistic regression analysis was used to determine the factors influencing SARS-CoV-2 infection in the matched cohorts (450 cases) and potential risk factors for recovery time in patients with symptomatic infection in the UDCA group (190 cases). The reference variables were as follows: age < 50 years, no diabetes mellitus, unvaccinated/partially vaccinated, UDCA dose ≤0.75 g/day, UDCA duration <1 year, and mild infection. The results were presented as odds ratio (OR) and 95% confidence interval (CI). Statistical analysis was performed with SPSS (version 26), and figures were generated using GraphPad Prism (version 9.4.1). A two-sided p of less than 0.05 was considered significant.Continuous variables were expressed as mean ± standard deviation (M ± SD) or median (interquartile range) in case of skewed distribution. The difference between groups was analyzed by Student’s 2. The proportions of smoking and drinking were relatively low. Approximately one-fifth to one-quarter of the people had at least one chronic complication, such as hypertension and cardiovascular disease. Furthermore, approximately 23% of the patients had varying degrees of cirrhosis.A total of 225 patients with the chronic liver disease treated with UDCA were matched to 225 patients with chronic liver disease not receiving UDCA. Baseline characteristics before and after propensity score matching of the population are listed in In terms of liver function indicators, there were significant differences in γ-glutamyl transpeptidase and alkaline phosphatase between the two groups (p < 0.05), indicating that the UDCA group had poorer hepatic conditions. No difference was observed in blood routine counts, serum lipid parameters, glycosylated hemoglobin, and estimated glomerular filtration rate. The COVID-19 vaccination rate in the UDCA group was lower than in the control group (p < 0.001). In terms of vaccination doses, the control group received a higher proportion of the three doses . However, no difference in the incidence was observed between age groups and vaccination groups (p > 0.05), as shown in We included 450 matched patients in the logistic regression analysis. Through multivariate analysis, UDCA administration had an OR of 0.32 , a protective factor against SARS-CoV-2 infection. A total of 404 patients developed COVID-19, with an overall infection rate of 89.8%. We further assessed the impact of UDCA use, age stratification, and vaccination status on the incidence of COVID-19. In the UDCA group, the rate of COVID-19 infection was remarkably lower than in the control group  . Although there was no significant difference in the proportion of moderate and severe cases between the two groups, their frequency was higher in the control group. Notably, there were no critical cases in either group.Most of the patients chose the SARS-CoV-2 antigen test. The UDCA group outperformed the control group in terms of signs and symptoms of SARS-CoV-2 infection. The maximum body temperature in the control group was higher than that in the UDCA group . There was no significant difference in the proportion of outpatient and inpatient visits between the two groups (p > 0.05). Specifically, nine patients were seen in the outpatient clinic mainly because of unremitting symptoms of COVID-19. In the control group, two patients were hospitalized due to advanced age and liver disease progression, and three inpatients had worsening infection symptoms. The principal reasons for hospitalization in the UDCA group were acute decompensation of cirrhosis (two cases) and severe COVID-19 infection (one case). In addition, UDCA had the advantage of not requiring medication  and a shorter time from infection to recovery . Fortunately, there were no deaths in either group  , excluding two asymptomatic infection cases. The median time from infection to recovery was 5 days  (Given that the angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin-II receptor blockers (ARB) could augment ACE2 expression, we analyzed whether ACEI/ARB treatment would have an impact on the incidence and recovery time of COVID-19 in patients with chronic liver disease complicated by hypertension (n = 127). These patients were divided into two groups according to whether they were on ACEI/ARB or not. However, there was no significant difference in incidence and recovery time between the two groups (p > 0.05)  patients with chronic liver disease were generally susceptible to SARS-CoV-2 infection, with a total of 404 (89.8%) developing COVID-19; 2) UDCA was a protective factor against SARS-CoV-2 infection, with a significantly reduced incidence in UDCA users; 3) for patients with chronic liver disease, UDCA administration may have some benefits in improving clinical outcomes, such as reducing symptoms and severity and shortening the time from infection to recovery; 4) diabetes mellitus and moderate/severe infection were associated with longer recovery time in UDCA users rather than UDCA dose and duration.https://m.163.com/dy/article/HRVHIEFN0534I43Y.html). Preventing SARS-CoV-2 infection and mitigating harm are the common goals of all humankind  reported by UDCA is mainly administered to patients with chronic liver disease, which could effectively treat hepatitis-related hepatic dysfunction and cholestatic liver disease . We therImportantly, we also observed that diabetes mellitus and moderate/severe infection evidently prolonged the time from infection to recovery in UDCA users. However, the dose and duration of UDCA were independent of the recovery time. The possible reasons for this were that some of the patients interrupted the UDCA administration during the infection, or the relatively low daily dose failed to provide adequate protection against COVID-19. Several studies have suggested that diabetes mellitus, as a pro-inflammatory disease, increases the risk of severe SARS-CoV-2 infection and prolonged recovery . FurtherSince the COVID-19 pandemic, there has been growing evidence that patients with COVID-19 frequently present with hepatic damage . A retroSome limitations should be taken into account. First, the relatively small number of patients, and the fact that the study was conducted in a single center, means that the efficacy and safety of UDCA against COVID-19 need to be further validated in large-scale multi-center studies. Second, there was case selection bias due to the inclusion of different chronic liver diseases, including chronic hepatitis B and autoimmune liver disease. To address that, we controlled the confounding by performing a propensity score adjustment analysis using the nearest neighbor matching algorithm. However, there may be potential differences in the susceptibility to SARS-CoV-2 among various chronic liver diseases. The residual and unmeasured confounding probably persist, and our findings did not prove causation. Moreover, because most of the infected individuals did not seek medical attention, this study lacked the evaluation of laboratory indicators and imaging examination following infection. This may be related to the following reasons: 1) the liberation of the epidemic and the sudden increase in infections led to the shortage of medical resources; 2) most of the infections were mild and recovered spontaneously due to the diminished pathogenicity of SARS-CoV-2. Accordingly, we were unable to analyze the possible protective mechanisms of UDCA on COVID-19 due to the lack of patients’ blood samples. We were also unable to assess the effect of UDCA on viral shedding time, as few individuals undergo SARS-CoV-2 ribonucleic acid or antigen re-test during universal infection. Finally, it should be emphasized that the present study was based on self-reports rather than classically established COVID-19 by experimental investigation, which may affect the accuracy of the conclusions.Patients with chronic liver disease were generally susceptible to SARS-CoV-2 infection. UDCA had a protective effect on COVID-19, including reducing the infection risk, mitigating symptoms, and shortening the time from infection to recovery. The daily dose and duration of UDCA were independent of recovery time, whereas diabetes mellitus and moderate/severe infection may prolong the course of COVID-19 in UDCA users. Therefore, UDCA might be beneficial in preventing SARS-CoV-2 infection in patients with chronic liver disease, especially cholestasis and hepatic dysfunction. However, these findings need further corroboration and validation in other cohort samples and experimental investigations.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethical Review Committee of the Beijing Ditan Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.YLi was the first author of this study. Study design: YLi. Data collection: NZ. Analysis of data: YLin and XC. Drafting of the manuscript: YLi. Critical reversion of the manuscript: XL. Material and technical support, and study supervision: XL. All authors have read and approved the manuscript.


Enzymes are used to treat a wide variety of human diseases, including lysosomal storage disorders, clotting disorders, and cancers. While enzyme therapeutics catalyze highly specific reactions, they often suffer from a lack of cellular or tissue selectivity. Targeting an enzyme to specific disease-driving cells and tissues can mitigate off-target toxicities and provide novel therapeutic avenues to treat otherwise intractable diseases. Targeted enzymes have been used to treat cancer, in which the enzyme is either carefully selected or engineered to reduce on-target off-tumor toxicity, or to treat lysosomal storage disorders in cell types that are not addressed by standard enzyme replacement therapies. In this review, we discuss the different targeted enzyme modalities and comment on the future of these approaches. Targeted enzymes can drive cell type-selective reactions that are not yet possible with other therapeutic approaches. These modalities work by bringing two biomolecules or cells together, thereby driving novel interactions and reactions, such as directing immune cells to specific target cells or targeted protein degradation. Molecular glues and PROTACs promise to expand the druggable proteome by promoting degradation of proteins of interest whose disease-driving functions cannot be inhibited by small molecules or blocked by antibodies. These bifunctional molecules direct a ubiquitin ligase to a protein of interest, which induces its ubiquitination and initiates a cascade that leads to its eventual degradation by the proteasome.5,6 This approach applies the lessons of proximity-induced reactions from other bifunctional therapeutics: the enzymes have low activity while circulating the body, and the high local molarity enabled by the targeting moiety drives the reaction on only target cells.Current bifunctional therapeutics are fundamentally limited to the chemistries that our enzymes or cells can inherently perform in the same subcellular location as the enzymatic target. This reaction space can be expanded both by targeting human enzymes to non-endogenous locations or by directing non-human enzymes to disease-driving cells or macromolecules. These targeted enzymes offer a unique approach to further expand the druggable space beyond what is possible with other bifunctional therapeutics. In some recent examples, enzymes have been engineered and targeted to modify cell surfaces of only disease-driving cells.versus other diseases, so these are discussed separately.This review focuses on current targeted enzyme strategies, including enzyme replacement therapies (ERT) and those that rely on proximity-induced reactions. There are four general approaches for current targeted enzyme therapies : (i) cel2.Enzymes are powerful therapeutics that can catalyze highly specific reactions within complex biological mixtures, avoiding molecules that are structurally and chemically similar to the desired reactant. This specificity is sometimes further tuned by cellular conditions, such as enzymes that are pH sensitive or rely on different cofactors for activity. Given their inherent catalytic nature, enzyme therapeutics can be effective when as little as a single copy is delivered to the cells of interest.7 and pancreatic enzymes (such as trypsin) for cancer therapies in the early 20th century.8,9 Enzymes from diverse biological sources continued to be used to treat a wide variety of disease until 1962,8 when the Kefauver–Harris Amendments to the Federal Food, Drug, and Cosmetic Act were passed. These amendments required therapeutics to be effective in addition to passing the prior safety requirements, establishing the groundwork for the modern United States Food and Drug Administration (FDA) approval system.10 This understandably and correctly limited the number of approved enzymes. The introduction of modern recombinant DNA technology and heterologous protein expression in 1977 further simplified production of therapeutic enzymes.11 This work came to fruition in 1987 with the first FDA approval of a recombinant enzyme drug, Alteplase .12,13 Now many therapeutic enzymes consist of recombinant human enzymes that are used to treat lysosomal storage disorders (LSDs).8Enzymes are old therapeutic interventions, starting with digestive aids (such as amylase and pepsin) in the late 19th century13,14 Since the Kafuever–Harris Amendment required expensive clinical trials, most pharmaceutical companies focused on large disease populations, and thus rare diseases that could be treated by ERT were often ignored. This changed with the Orphan Drug Act in 1983, which provided incentives – including market exclusively, tax credits and grants, and fast-tracking of development and approval – to companies to develop therapies for diseases affecting fewer than 200 000 people,15 thereby facilitating the development of enzyme therapies for rare diseases.16 In 1990, Pegademase , became the first FDA approved ophan drug designated therapeutic enzyme. It is a modified form of bovine adenosine deaminase used to treat severe combined immunodeficiency caused by adenosine deaminase deficiency.12,13In 1966, the concept of ERT was first proposed, in which an exogenous enzyme is administered to replace a missing or mutated version.17 Some of the main factors that limit their expanded use are high levels of immunogenicity – discussed later – and inability to distinguish between substrates in diseased cells versus healthy tissue.18,19 The latter problem can be addressed with targeting.Since the 1990s, there has been continual use and FDA approval of novel enzyme therapeutics, but they still only make up less than 15% of the currently licensed FDA-approved biologics .20 Therefore, M6P is commonly used to target ERTs to the lysosome. To date, there are no known comparable methods to target enzymes specifically to other subcellular locations, such as the nucleus or cytosol. While not discussed more here, this is an unsolved problem limiting delivery of therapeutics more broadly, and there are ongoing efforts to address better delivery methods.21The earliest and most used targeting modality is mannose-6-phosphate (M6P), which binds the cation-independent mannose-6-phosphate receptor (CI-M6PR) to traffic proteins to the lysosome. This modification is used in cellular homeostasis to retain lysosomal digestive enzymes but can be hijacked for lysosomal delivery of recombinantly expressed enzymes.18,19 As discussed in the sections below, different techniques are used against cancer, in which off-targeting is often toxic, as compared to targeted ERT, in which off-targeting is often non-toxic but limits efficacy to desired cells.There are currently four FDA approved enzymes that are targeted to specific organs and cells. These therapeutics use antibody and antibody fragments, other proteins (such as cytokines), and peptides (such as deca-aspartate) as targeting agents . Preclin3.e.g. sialic acids and PD-L1),22,23 metastasis drivers (e.g. integrins),24 and receptors that initiate proliferation and anti-apoptosis signals (e.g. HER2).25 Therapeutically, these markers are used as targeting epitopes for antibody–drug conjugates (ADCs), checkpoint inhibitors, and targeted enzyme therapeutics.Cancer cell surfaces are often distinct from healthy cells. These changes can drive cancer progression through overexpression of immune inhibitory motifs  an antibody or other protein that specifically binds to cancer cells, and (ii) a cytotoxic protein that kills the cells upon its internalization. ligands .12There are currently three FDA approved enzyme-based immunotoxins: moxetumomab pasudotox  for relapsed or refractory hairy cell leukemia, denileukin diftitox  for cutaneous T-cell lymphoma, and tagraxofusp  for plasmacytoid dendritic cell neoplasm .12pseudomonas exotoxin A (PE) fused to an anti-CD22 disulfide-stabilized Fv antibody fragment, while both denileukin diftitox and tagraxofusp consist of diphtheria toxin (DT) fused to interleukin-2 and interleukin-3, respectively 44–47 have used LMB-100, which is a de-immunized variant of the toxin, engineered from SS1P though T-cell and B-cell epitope mapping, point mutagenesis, and domain deletions.43,48,49 One final benefit of enzyme-based immunotoxins is that they can be expressed as single fusion proteins, simplifying production as compared to a biologic chemically conjugated to a synthesized small molecule.Furthermore, the use of an enzyme allows for directed evolution and other mutagenesis strategies to optimize the construct, contrasting the structure–activity studies required for small molecules. An example of this is the engineering of PE based immunotoxins to treat mesothelioma. Early clinical trials (start dates 2000–2011)3.2via an antibody against a cancer-specific non-internalizing receptor. The enzyme is selected or engineered to not catalyze extracellular reactions within a person other than prodrug activation. In the second step, a non-toxic prodrug is administered, where it is specifically activated in the tumor microenvironment by the antibody-enzyme conjugate. The activated drug is taken up into nearby cells, killing them  in clinical trials was 28.5 days.53,57,58 In ADCs, the entire biomolecule is endocytosed, and a small amount of the activated drug escapes the lysosome. This is an inefficient process that is still being optimized. In ADEPT, only the activated drug needs to be internalized, a far easier cytosolic delivery challenge. However, extracellular activation of the prodrug increases the risk of off-tumor toxicity through toxic drug uptake into nearby non-cancer cells or even leakage of the toxic drug out of the tumor microenvironment. Therefore, prodrugs have been designed such that the corresponding activated drugs have short half-lives.53,59ADEPT is proposed to solve delivery challenges of ADCs.Pseudomonas sp. has been evaluated in clinical trials.27,53,58,60 CPG2 cleaves reduced and non-reduced folates, and a non-targeted version was approved in 2012  to cleave excess circulating methotrexate in renal dysfunction  from function .61 This prodrugs .27,56,5753,55,56 However, repeated dosing of CPG2 ADEPT has been tolerated with co-administration of the immunosuppressive agent cyclosporine A.64 In an orthogonal approach, de-immunization through B-cell epitope mapping and mutagenesis of the antibody-enzyme conjugate yielded an ADEPT system that elicited antidrug antibodies in only 23% of tested patients,65 as compared to the 97% of patients observed with the non-engineered construct.55,56 Despite these limitations, ADEPT offers a unique approach to targeted delivery of cytotoxic small molecules.Like with immunotoxins, all ADEPT systems are immunogenic. Antidrug antibodies have been observed in all CPG2 ADEPT clinical trials, limiting the drug to one dose.3.3The third class of cancer-targeted enzyme therapies has distinct design principles and mechanisms of action as compared to the previous two. In this approach, a non-toxic enzyme is targeted to tumors, where it is not internalized but rather directly modifies the cell surface. This allows for altering of cancer surface markers that are difficult to drug with other approaches.Since these enzymes act upon endogenous cell surface markers, there is a risk for deleterious on-target off-tumor activity. Relying on the principles of proximity induced reactions, low activity enzymes with weak substrate binding are optimal for this targeted approach. Such enzymes are minimally active when circulating the body, but when the enzymes are targeted to cancer cells, the high local molarity of substrates drive a pseudo-intramolecular reaction. Therefore, these enzymes need to be selected or engineered such that binding to cancer cells through the targeting agent drives the enzymatic reaction, rather than intrinsic binding of the enzyme to free substrate .Work from our group and others has focused on two main cancer targets, sialic acids and mucins, but these approaches are applicable to other substrates.3.3.166 These sialoglycans have distinct functions and signaling networks based on their structure and the linkage of the sialic acid to the rest of the glycan chain, but they generally act as immune inhibitory motifs through binding to sialic-acid-binding immunoglobulin-like lectins (Siglecs) on immune cells, paralleling classical immune checkpoints.67 A common phenotype of cancer is hypersialylation of different glycoconjugates, cloaking the tumor from immune cell killing ,69,70 and chimeric antigen receptor (CAR) T-cells.71Sialidases  are enzymes that remove sialic acids, or negatively charged monosaccharides commonly appended to the end of glycan structures, exposing underlying sugars. killing .23 ThereVibrio cholerae sialidase was conjugated to an anti-HER2 antibody , antibody , left.68in vivo use as compared to T-sia 1.5 This antibody-enzyme conjugate used a less active Salmonella typhimurium sialidase that lacks the previously mentioned lectin domains  to 2000-fold (T-sia 2)  , right.5in vivo cancer models, as was observed with T-sia 2 delaying HER2+ tumor growth in a mouse breast cancer model.5 Follow-up in vivo work used a further optimized antibody-enzyme conjugate from Palleon Pharmaceuticals (E-301), comprised of two Salmonella typhimurium sialidase domains fused to the anti-HER2 antibody, trastuzumab. In multiple mouse tumor models, E-301 activated the adaptive immune system, repolarized tumor-associated macrophages, increased immune cell infiltration, and synergized with classical check-point blockade strategies.73 Further optimization of the antibody–enzyme conjugate by Palleon Pharmaceuticals led to E-602, which is currently being investigated in phase 1/2 clinical trials against a variety of different cancers.74Targeted sialidases can also reduce tumor burden in et al. and Szijj et al. both demonstrated that conjugation of sialidases to BiTEs enhanced T-cell killing of the target cancer cells as compared to unconjugated BiTEs.69,70 Yang et al. further evaluated the efficiency of BiTE-sialidases in controlling tumor burden in two cancer xenograft mouse models in immune deficient mice injected with human peripheral blood mononuclear cells and one syngeneic mouse model of melanoma. In all three models, the BiTE-sialidase reduced tumor burden as compared to vehicle control and the BiTE alone.69 In an orthogonal approach, Durgin et al. engineered CAR T-cells to secrete sialidase when activated by tumor cells. In multiple mouse tumor models, sialidase secreting CAR T-cells extended mouse survival and reduced tumor burden as compared to non-sialidase secreting CAR T-cells.71Sialidases have also been targeted specifically to the tumor-immune cell synapse through BiTEs, or bispecific antibodies that bridge a cancer cell and a T-cell and thus enhance T-cell killing of the target cancer cell, and CAR T-cells . Yang et3.3.2O-glycosylated glycoproteins that are commonly upregulated in cancers and have been shown to drive tumor progression through multiple immune-inhibitory and biophysical mechanisms.75 Mucins are difficult to drug with classical therapeutic and other bifunctional approaches, because they are a protein class comprised of repeated peptide domains highly modified with variable, branching, non-genetically encoded, and biosynthetically complex glycans, leading to an extended “bottle brush” conformation that can alter signaling and membrane biophysics.76,77We and others have applied the lessons learned from targeted sialidases to specifically degrade mucins on cancer cells using targeted mucinases. Mucins are a class of densely 78,79 Paralleling the antibody-sialidase work, we fused an engineered mucinase to an anti-HER2 nanobody to specifically degrade mucins on cancer cells. To avoid toxic on-target off-tumor mucin cleavage, the highly active enzyme was engineered to have reduced activity and binding to cell surfaces, consistent with the design principles demonstrated above for targeted sialidases. The resulting conjugate degraded mucins specifically on HER2+ cells, potentiated natural killer cell killing of only target cells, and reduced metastasis and primary tumor burden in multiple HER2+ mouse tumor models  cell line. The tethered mucinase disrupted the mucin barrier at the interface between the immune and target cancer cells, which lead to increased target cell killing.80 Consistent with the effectiveness of the sialidase secreting CAR T-cells, this mucinase-tethered CAR-NK cell work demonstrates the potential of improving cellular therapies with targeted enzymes.Similar to the CAR T sialidase work, Park Sialic acids and mucins only represent a small fraction of targets that could be specifically altered by directing exogenous enzymes to cancer cell surfaces. These approaches therefore provide a blueprint for more broadly modifying the altered cell surface microenvironment in cancer.4.In contrast to many therapeutics that target cancer cells, targeted enzymes for other disorders are generally less toxic. The targeting is used to enhance delivery to organs that are either most affected by the disease or least treated with other therapeutics. Therefore, targeting can be less selective, and the enzyme is not required to be engineered to limit off-target activity. This approach is being used to transport enzymes across the BBB and to organs such as bones and muscles.4.181Lysosomal storage disorders are characterized by enzyme deficiencies that lead to a toxic buildup of undegraded biomolecules in the lysosome. The frontline treatment for many LSDs is ERT, in which the missing enzyme is administered to restore wild type cellular function. However, many LSDs have pronounced neurological symptoms and pathophysiologies, and enzymes injected intravenously cannot readily cross the BBB to reach the central nervous system .8181 One successful approach is hematopoietic stem cell therapy. When administered at a young age, hematopoietic stem cell transplantation is the gold standard for addressing the neurological components of some LSDs, including severe Hurler syndrome.82 Another successful approach has been to target ERTs to receptors on brain capillary endothelial cells, causing receptor-mediated endocytosis and transcytosis across the BBB .et al. functionalized poly-(lactide-co-glycolide) nanoparticles with brain targeting peptides, including transferrin. Galactosylceramidase was loaded into these nanoparticles and successfully delivered to the central nervous system in a mouse model of Krabbe disease.91Other approaches beyond antibody targeting have been used to hijack endogenous receptor-mediated transcytosis pathways. For example, Del Grosso Unlike in the cancer therapeutics space, off-target delivery is not a problem for ERT targeting the brain, because peripheral tissues are also enzyme deficient and there is a therapeutic benefit in restoring wild type cellular function in these cells as well , top. Ho4.2via engaging CI-M6PR. This strategy is not effective for delivery of non-lysosomal enzymes or if the target cells do not express CI-M6PR at high enough levels for sufficient uptake. Recent work on therapeutics for hypophosphatasia and Pompe disease demonstrate approaches to address both limitations.Enzymes used in ERT are often functionalized with M6P for their efficient delivery to lysosomes 92 Hypophosphatasia is primarily a disease of defective bone and tooth mineralization, which can manifest as fractures, tooth loss, skeletal pain, and rickets.92,93 Injection of the untargeted ERT was not effective, so the field turned to bone targeting approaches.94Hypophosphatasia is caused by a deficiency of tissue non-specific alkaline phosphatase, a cell surface protease with enhanced expression in bones, liver, and kidney. This enzyme cleaves extracellular inorganic pyrophosphate to generate inorganic phosphate, which is required for hydroxyapatite crystallization, the mineral component of bone and teeth.et al. demonstrated that attaching six aspartic acids (Asp)6 to a fluorophore caused the fluorophore to bind to hydroxyapatite in vitro and specifically targeted it to bones and teeth in rats.95 A similar targeting motif, (Asp)10, was conjugated to tissue non-specific alkaline phosphatase to specifically target it to bones in vivo. The fusion construct  was further engineered through removing the enzyme's C-terminal glycosylphosphatidylinositol-anchor and attaching the human IgGγ1 Fc domain.94 In a phase 2 clinical trial for patients under 5 years old comparing 39 treated patients with 48 historical controls, Asfotase Alfa improved survival to age 1 from 42% to 95% and to age 5 from 27% to 84%.96 In older children (6–12 years old) treatment lead to substantial skeletal muscle healing.93 Asftoase Alfa was approved by the FDA for treatment of infantile and juvenile onset hypophosphatasia in 2015  are not successfully treated by the standard ERT .100 In a randomized, double-blind phase 3 trial comparing Nexviazyme to the standard of care Lumizyme, Nexviazyme lead to greater improvements in respiratory and motor muscle function, indicating improved skeletal muscle delivery.101 Nexviazyme was approved by the FDA in 2021 for the treatment of late-onset Pompe disease.12 Similar approaches to increase consistent targeting of acid alpha-glycoside to CI-M6PR include fusion to insulin-like growth factor II, another CI-M6PR ligand,102 or expression of this enzyme in a proprietary cell line that leads to higher M6P content.103A second-generation acid alpha-glycoside ERT  was designed for enhanced CI-M6PR targeting and uptake through modification with synthetic M6P-bearing glycans.et al. fused acid alpha-glycoside to an antibody against CD63, an internalizing receptor enriched on skeletal muscles.104 The fusion protein trafficked to lysosomes in cellulo in a M6P-independent manner and removed more glycogen in muscle tissues than the standard ERT in a mouse model of Pompe disease. This approach of targeting an enzyme to a cell type-specific internalizing receptor is already being used in the cancer space (see immunotoxin section above). However, as nicely demonstrated by Biak et al., this modality of targeted enzyme therapeutics has great value in other tissues and cells. CD63 was selected for skeletal muscle delivery, because it was known to be expressed highly on skeletal muscles (necessary for uptake), minimally expressed in the liver (necessary to avoid rapid liver clearance), and traffics between the cell surface and lysosome .104 For similar approaches to target enzymes specifically to other tissues, it is important that the targeting agent and receptor are carefully selected.An orthogonal approach is to bypass CI-M6PR altogether. Baik 5.8 While there can be great benefit in targeting these to specific cells and organs of interest, there is even greater therapeutic potential if we look beyond human enzymes to those from other animals, bacteria, viruses, plants, and fungi. Even considering only bacteria from human microbiotas, there are enzymes that perform therapeutically-relevant reactions with previously unseen specificity, such as selectively converting blood group antigens, or degrading only densely O-glycosylated mucins.79,105 Therefore, enzyme-based therapies could expand not only the list of druggable targets but also the ways in which these therapeutics modify human biology.Most FDA approved enzyme-based therapeutics are recombinant human enzymes.106 Two of many such examples include production of inflammasome-inhibiting enzymes and secretion of highly selective IgA proteases.107,108 Novel therapeutics that alter immune responses could be beneficial in treating autoimmune disorders and cancer, improving efficacy of vaccinations, and reducing immunity to other therapies.One example of this is the direct modulation of host immunity. Microorganisms have evolved effector molecules, including enzymes, to alter host immune responses through interactions with the innate immune system, autophagy, complement proteins, cytokines and chemokines, adaptive immune system, and cellular death pathways.82,109 Techniques to deimmunize enzyme therapies fall into two categories: (i) reducing the immunogenicity of the enzyme itself or (ii) modulating the immune system during dosing.The expanded use of non-human enzymes would come at a cost: immunogenicity. Almost every biologic on the market comes with the risk of eliciting anti-drug antibodies and immune reactions. However this risk is greater with foreign enzymes, especially those that remain in circulation or on cell surfaces instead of being quickly internalized.48,49,110 Another popular option is to shield the entire enzyme surface with modifications such as polyethylene glycol (PEG), reductive methylation, or polysialylation.110 This is exemplified by the FDA approval of both unPEGylated and PEGylated asparaginase from both Escherichia coli and Erwina chrystanthemi for the treatment of acute lymphoblastic leukemia.12 However, some patients also develop anti-PEG antibodies, so care must be taken with repeated administration of PEGylated foreign biologics.111,112One way to reduce the inherent immunogenicity of an enzyme is to modify the most immunogenic structures and features, such as was done the immunotoxin LMB-100. These features are identified through multiple techniques, including phage display, incubation with patient derived T- and B-cells, and even computational prediction algorithms.53,113,114 Another approach is to tolerize the immune system against the single novel agent rather than broadly suppressing it. For example, Selecta Biosciences developed rapamycin-encapsulated nanoparticles (ImmTOR) that when co-administered with immunogenic biologic therapies, induce tolerogenic dendritic cells and antigen-specific regulatory T-cells.115 In animal models, ImmTOR effectively mitigated antidrug antibody formation against different therapeutic biologics without affecting immune responses to unrelated antigens.115 ImmTOR is currently being tested in a phase 3 clinical trial for treatment of refractory Gout with uricase.116The second option is to not alter the enzyme itself and instead modulate the host immune system during treatment. For example, ADEPT and ERT clinical trials have used cyclosporine to broadly suppress the immune system during dosing.6.This review has discussed the therapeutic potential of targeting enzymes to specific cells and organ systems, but similarly to many other modalities, the future of proximity-induced enzymes is targeting a single biomolecule. The current approaches only target at the cellular level, and the molecular substrates are dictated by inherent enzymatic specificity. For example, DT and PE immunotoxins specifically modify eukaryotic elongation factor 2 because of their inherent specificity for the diphthamide post-translational modification and not because of targeting or engineering. With this system, we cannot easily redirect this activity to different post-translational modifications or protein targets, seriously limiting the modularity of targeted enzyme therapies.117 CRISPR/Cas9 is inherently a proximity-induced enzymatic system, but it is also being used to target other enzymes to specific DNA sequences, including cytidine deaminase for base editing,118,119 reverse transcriptase for gene alteration,120 and DNA polymerase for mutagenesis.121To more closely mirror the single target specificity of PROTACs and molecular glues, we need to build modular targeted enzyme systems, in which the substrate is exclusively dictated by the targeting agent and the chemical reaction exclusively dictated by the enzyme. Nature has already evolved such systems, which we are beginning to understand and manipulate for therapeutic purposes. The most powerful example is CRISPR/Cas9, in which we can easily redirect the DNA cutting capabilities of Cas9 to any sequence in our genome based on the identity of the guide RNA.122 engineering enzymes to have enhanced specificity for an amino acid sequence found in only one target protein,123,124 or fusing broadly-active enzymes to highly-specific antibodies with fast off-rates, which have enhanced cleavage of the target substrate relative to the untargeted enzymes.125While not yet translated to clinical use, several academic studies have used multiple strategies to engineer enzymes that target individual molecules rather than, for example, the cells that they occupy. Such approaches include splitting the enzyme into two non-functional units that only combine when bound to the target of interest,Despite these limitations, proximity-induced enzymes combine the power of current enzyme therapies with the single-cell specificity of current bifunctional approaches. This has so far led to four FDA approvals, which provide novel mechanisms to drug cancers and treat previously untreatable skeletal manifestations of enzyme deficiencies. As discussed in this review, these design principles and approaches can be applied to other disease areas, and hopefully shepherd in a new era of precision biologics.G. S. T. performed the literature search and wrote the manuscript. C. R. B. provided conceptualization and wrote the manuscript.C. R. B and G. S. T. are co-inventors on a patent related to the topic of this review, which has been filed by Stanford University (docket no. STAN-1929PRV). C. R. B. is a co-founder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Biosciences, Redwood Biosciences , OliLux Bio, InterVenn Bio, Neuravid, Firefly Bio, Valora Therapeutics, and GanNA Bio.


Arabidopsis transcription factor binding sites (TFBSs) prediction models, which now cannot meet the above demand of identifying the core motif.An emerging approach using promoter tiling deletion via genome editing is beginning to become popular in plants. Identifying the precise positions of core motifs within plant gene promoter is of great demand but they are still largely unknown. We previously developed TSPTFBS of 265 in-silico tiling deletion, and in-silico mutagenesis to identify the potential core motifs of any given genomic region.Here, we additionally introduced 104 maize and 20 rice TFBS datasets and utilized DenseNet for model construction on a large-scale dataset of a total of 389 plant TFs. More importantly, we combined three biological interpretability methods including DeepLIFT, For the results, DenseNet not only has achieved greater predictability than baseline methods such as LS-GKM and MEME for above 389 TFs from Arabidopsis, maize and rice, but also has greater performance on trans-species prediction of a total of 15 TFs from other six plant species. A motif analysis based on TF-MoDISco and global importance analysis (GIA) further provide the biological implication of the core motif identified by three interpretability methods. Finally, we developed a pipeline of TSPTFBS 2.0, which integrates 389 DenseNet-based models of TF binding and the above three interpretability methods.http://www.hzau-hulab.com/TSPTFBS/), which can support important references for editing targets of any given plant promoters and it has great potentials to provide reliable editing target of genetic screen experiments in plants.TSPTFBS 2.0 was implemented as a user-friendly web-server ( IPA1 gene can improve rice yield (Transcription factors (TFs) can activate or suppress transcription of genes by binding to the specific DNA sequences that are known as transcription factor binding sites (TFBSs), thereby playing an important role in gene expression. With the popularization of genome editing technology such as CRISPR-Cas9, it is now possible to genetically manipulate a TFBS to study how a TFBS regulates gene expression and then indirectly affects quantitative traits in plants . Recentlce yield . Thus, tce yield .in vivo and in vitro, have been developed for TFBS discovery. The in vitro methods mainly contain protein binding-microarrays (PBM) (in vivo methods primarily include chromatin immunoprecipitation sequencing (ChIP-seq) (With the rapid development of next-generation sequencing (NGS) techniques, a number of experimental methods, both ys (PBM) , systemays (PBM) , and DNAys (PBM) . The in hIP-seq) , chromathIP-seq) . In the hIP-seq)  and RoadhIP-seq) . They haBased on existing abundant experimental data, a series of data-driven computational methods have been proposed for predicting TFBSs varying from simple pattern matching methods to more complex models. As TFBSs are degenerate sequence motifs , early rIn addition to these PWMs-based methods, many machine learning-based methods also have been developed to predict TFBSs. For a famous example, LS-GKM  leverageArabidopsis TFs was produced by DAP-seq in 2016  We additionally introduced 104 maize and 20 rice TFBS datasets and utilized DenseNet for model construction on a large-scale dataset of a total of 389 plant TFs. We hope transfer predictability will be improved based on more powerful models trained on more TFBS datasets. (ii) Based on the trained models, we will combine three interpretability methods including DeepLIFT , in-silideletion , and in-agenesis  to identagenesis .https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137972) and 20 Oryza sativa TFs (https://biobigdata.nju.edu.cn/ChIPHub/), DAP-seq datasets for 265 Arabidopsis TFs .Here, we first downloaded large-scale datasets including ChIP-seq datasets for 104 Zea mays TFs  , which is composed of two convolution layers and all of these DenseLayers connect (with matching feature-map sizes) directly with each other. The TransitionLayer is mainly used to connect two DenseBlocks, including a convolution layer (kernel size = 1) and an average pooling layer (stride = 2). Finally, we flatten the output of the last DenseBlock and connect it to a fully connected layer to generate an output value. The maximum number of epochs to train was set to 80 and the learning rate was set to 0.001. We employed TensorFlow 2.0 to train models, and one can find the code at https://github.com/liulifenyf/TSPTFBS-2.0.We then adopted a powerful architecture of DenseNet to build TFBS prediction models for each dataset. As the best paper award in CVPR-2017, DenseNet draws on the idea of ResNet to closely connect each layer with all the previous layers . It beneWe compared the presented DenseNet models with other existing baseline methods, namely LS-GKM, DeepCNN, MEME-M1-MAX. For each method, we optimized the parameters and report the best results to perform a fair comparison. For LS-GKM, we first downloaded lsgkm from GitHub - Dongwon-Lee/lsgkm and then employed the function of ‘gkmtrain’ and the function of ‘gkmpredict’ with the default parameters. For DeepCNN, we designed the model including a convolution layer, a rectified linear unit (ReLU) layer, a max pooling layer and three fully-connected layers, and used sigmoid function for the output layer . The maxvia Blastp, and then utilized the prediction model of the most homologous TF  to perform trans-species prediction.For a given TF from other species, we computed the protein sequence similarity between this TF and all the TFs of TSPTFBS and TSPTFBS 2.0 respectively In this paper, the performance of all models were evaluated by ROC-AUC on the testing set to allow for fair comparisons with other existing baseline prediction methods. The performance measure is defined as follows:ROC-AUC: The metric indicates the area under the receiver operating characteristic curve. It contrasts False Positive Rate (FPR) against True Positive Rate (TPR). A ROC-AUC value around 0.5 suggests a random classifier. The calculation method of TPR and FPR is defined as follows:For the evaluation indexes of trans-species prediction, we used PPV , NPV  and recall as follows:TP (true positive) represents correctly predicted ‘positive’, TN (true negative) represents correctly predicted ‘negative’, FP  represents ‘negative’ incorrectly predicted as ‘positive’ and FN  represents ‘positive’ incorrectly predicted as ‘negative’.https://github.com/liulifenyf/TSPTFBS-2.0/blob/main/interpretability.py.We employed a powerful interpretability tool called ‘DeepLIFT’, a new approach to assign importance score to the inputs for a given output. Specifically, DeepLIFT first compared the prediction values of the given sequences and their reference sequences (The reference sequence refers to dinucleotide shuffle based on the input sequence) and then assigned a DeepLIFT contribution score to each base of a given sequence according to decompose the difference by a backpropagation-like algorithm . One canin-silico tiling-deletion  of all possible mutations to a sequence  in 1000 negative samples to calculate their global importance and drew a heatmap of their differences of global importance with a sequence logo that has heights scaled according to the L2-norm at each position.Unlike the previously proposed model interpretability approaches for deep learning, such as attribution methods, global importance analysis (GIA) quantifies the population-level effect size that identified potential core motifs have on model predictions . GIA provia a R package of ‘motifmatchr’ with the following parameters: p.cutoff=1e-05  and employed a new model architecture of DenseNet , but in Arabidopsis and Oryza sativa it was significantly inferior to DenseNet (median AUC 0.912 in Arabidopsis and 0.961 in Oryza sativa). As a summary, this demonstrates that DenseNet has achieved greater predictability than other baseline methods on a total of 389 plant TFBS datasets from three plant model species.We previously have verified that the prediction performance of TSPTFBS is superior to other methods on 265 DenseNet   and then compared each TF prediction performance with TSPTFBS 1.0 and TSPTFBS 2.0 respectively.We next ask whether TSPTFBS 2.0 which integrated more datasets will perform better than TSPTFBS 1.0 on trans-species TFBS predictions. To this end, we conducted a comparative analysis of trans-species TFBS predictions between them under the idea of transfer learning . FirstlyFor the result, 100% (9 out of 9) of tomato TFs in TSPTFBS 2.0 achieved greater positive predictive values (PPVs) and negative predictive values (NPVs) than TSPTFBS 1.0, and 88.89% (8 out of 9) of tomato TFs achieved satisfied PPVs and NPVs between 0.8 and 1.0  for discovering potential core motifs within any plant promoters. More precisely, for a given TF model of interest, we allow the user to upload the promoter sequence (upstream 500 bp of plant gene TSS of interest) in our website, and the web-server will return the following results: (i) the predicted binding intensity of the uploaded promoter sequence of the given TF; (ii) numeric results of three interpretability methods—base contribution score from DeepLIFT, the difference of predicted value between WT (Wild Type) and the sequence after a 10 bp deletion from in-silico tiling deletion and the difference of predicted value between WT and the sequence after a point mutation from in-silico mutagenesis; (iii) a logo graph for intuitively displaying the above interpretability results.Although our method can theoretically detect core motifs of any genomic region, we focus on plant promoter region in view of its high importance. Finally, we developed a pipeline of TSPTFBS 2.0, which integrates 389 DenseNet-based models of TF binding and the above three interpretability methods. This pipeline was implemented as a user-friendly web-server  cis-regulatory deletion can increase both grains per panicle and tiller number (IPA1 (from -500 to TSS) and showed the scanning results in IPA1 has the above sequence motifs. And then we searched our 389 TF models to find the corresponding TFs with the same family of each of the above nine TFs. For bHLH TF family, there are a total of 14 TF models (10 bHLH Zm TFs and 4 bHLH At TFs). We plotted the predicted binding intensity of the promoter sequence of IPA1 from each of the 14 bHLH TF models in Zm to perform interpretability analysis. For TCP TF family, there are two TCP TF models of TCP 10 in Zm and TCP 23 in Zm, and we chose the model of TCP 10 in Zm to perform interpretability analysis.We next take an example of rice r number . An-1 bir number . To inveIPA1 into the two TF models of TCP10 in Zm and bHLH47 in Zm, and drew the corresponding logo graphs of their interpretabilities in in-silico tiling deletion and in-silico mutagenesis do not strongly support it. As the experimental result (IPA1-Pro9 (this editing event has a deletion of the above region) only significantly decreased the IPA1 level in shoot but not in young panicle, which leads a significant increasement of tiller number but no significant change of panicle weight. From IPA1-pro10 (it has a deletion of this peak) leads to increasements both in tiller number and in panicle weight. As for other scanning motifs by FIMO, we found no Zm TF models but only found At TF models. And we drew them in IPA1 implies that our webserver is helpful for identifying core motifs within plant gene promoter and for supporting references of gene editing.We input the promoter sequence of l result , we founArabidopsis, maize and rice and have proven that trans-species prediction on 15 TFs from other six plant species is feasible. We also developed three interpretability methods to identify base-resolution core motif. Finally, we took rice IPA1 gene , which has great potentials to provide reliable editing target of genetic screen experiments in plants.In conclusion, TSPTFBS 2.0 used DenseNet to improve the predictability of TFBS prediction, and further verified that the trans-species TFBS prediction capability of TSPTFBS 2.0 was significantly improved, which is the first contribution of the current work. The main contribution is that we combined three interpretability methods to identify the potential core motif within a TFBS, which will be a powerful tool for assisting plant scientists on providing candidate targets of genome editing. To be convenient for applications, TSPTFBS 2.0 integrates 389 DenseNet-based models of TF binding and three interpretability methods, and it was implemented as a user-friendly web-server (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/HC and LL: Data curation, formal analysis, and writing original draft preparation. YZ, KD and YG: Formal analysis. XH: Conceptualization, funding acquisition, and review and editing. All authors contributed to the article and approved the submitted version.


Small-cell lung cancer (SCLC) is an aggressive disease with distinct biological and clinical features. The clinical course of SCLC is generally characterised by initial sensitivity to DNA-damaging therapies, followed by early relapse and broad cross resistance to second line agents. Whilst there has been an enormous expansion of effective targeted and immune-based therapeutic options for non-small cell lung cancer (NSCLC) in the last decade, little improvement has been achieved in SCLC treatment and survival due, at least in part, to underappreciated inter- and intra-tumoral heterogeneity. Here we review the current treatment paradigm of SCLC including recent advances made in utilizing immunotherapy and the challenges of identifying a predictive biomarker for immunotherapy response. We examine emerging new targeted therapies, combination immunotherapy and future directions of SCLC treatment research. Small cell lung cancer (SCLC) is a poorly differentiated neuroendocrine tumour which represents approximately 15% of all lung cancer diagnoses . The staSCLC is a highly proliferative epithelial neuroendocrine tumour . A diagnTP53 and RB1, sometimes by complex genomic rearrangements. Targetable mutations in known oncogenes, including BRAF, PTEN, PIK3CA and SLIT2 were only found in rare cases which may provide a possible therapeutic opportunity of these individual patients. In contrast, a high frequency of mutations affecting known epigenetic regulators including histone-modifying genes and inactivating mutations in Notch family members were observed. These targets are a focus of intense ongoing investigation. There has been an increasing understanding of the molecular and genomic heterogeneity in SCLC as part of efforts to identify therapeutic vulnerabilities to improve outcomes. Key genomic profiling studies of SCLC, including comprehensive whole exome and whole genomic genome analyses were published in 2012 and 2015 ,8. SCLC ASCL1, NEUROD1 and POU2F3  and the International Association for the Study of Lung Cancer (IASLC) recommended the using of TNM staging system for SCLC as well as non-small cell lung cancer (NSCLC) . In clinFor patients with LS-SCLC, the current standard treatment is concurrent thoracic radiotherapy and chemotherapy with cisplatin and etoposide. This is based on a meta-analysis of 13 trials that showed a 14% reduction in the mortality in patients treated with radiotherapy in addition to chemotherapy . CisplatA small fraction of patients limited stage disease (TNM stage I-IIA) may be candidates for up-front surgical resection. A retrospective analysis from the National Cancer Database in the United States showed, when matched based on stage, there was an improvement in median overall survival (OS) to 38 months compared to 22 months for these node-negative patients who underwent initial surgery . TreatmeAbout 85% of patients with SCLC have extensive stage disease at the time of diagnosis . Until rp = 0.007) was celebrated as the most clinically meaningful survival prolongation in SCLC for decades. The addition of immune checkpoint inhibitors to this treatment has been the only improvement in SCLC for many years that has improved survival, as discussed below, and summarised in p = 0.032) in platinum-etoposide only patients. However, the addition of tremelimumab to durvalumab plus EP did not significantly improve outcome versus EP. This was the first report of a phase III trial evaluating dual ICB in combination with chemotherapy in ES-SCLC. The increased toxicity observed with the addition of a CTLA4 inhibitor resulting in lower total exposure to durvalumab could have contributed to the absence of significant benefit compared with platinum-etoposide. The CASPIAN trial published in 2019 was a randomised (1:1:1), open-label, three-arm trial assessing PDL1 blockade with durvalumab, with or without the CTLA4 inhibitor tremelimumab, in combination with platinum-etoposide (EP) in treatment-naïve ES-SCLC ,22. Consp = 0.0023). Although the OS hazard ratio favoured pembrolizumab plus EP, the prespecified significance threshold of p = 0.0128 was narrowly missed . The lack of a statistically significant OS benefit was unexpected based on the results of CASPIAN and IMpower133. The shorter than expected OS in KEYNOTE-604 may have been due to a high proportion of patients with brain metastases, larger tumour dimensions, and ≥3 metastases at baseline. Given this negative result, pembrolizumab is not approved for use in first line ES-SCLC. KEYNOTE-604 was published in 2020 and reported on pembrolizumab in combination with platinum-etoposide also as first line treatment for ES-SCLC . The addCheckMate 451, published in 2021, examined the potential benefit of maintenance nivolumab and ipilimumab for four cycles followed by nivolumab alone, or nivolumab alone after chemotherapy or placebo in patients who had responded the first line platinum-based chemotherapy . There wp = 0.0017, HR 0.72). In contrast to KEYNOTE-604, there were just five patients in each group with brain metastases which may go some way to explaining the improved OS in both the intervention and placebo arms. As identified by the authors, there was also a high proportion of patients who received subsequent systemic therapies beyond the clinical trial compared to the trials discussed above. Despite these limitations, as well as the restriction of this study to only include patients from China, it is clearly supportive of the addition of ICB to EP for ES-SCLC. Published in 2022, CAPSTONE-1 examined the novel PD-L1 inhibitor adebrelimab with EP chemotherapy . This stp < 0.001). Of note, 19.8% of patients in the trial were never smokers, a proportion higher than previous multinational trials with larger fractions of non-Asian patients. An improvement in OS was seen in this unique patient subgroup, consistent with that in the overall population. The most recent phase III study of ICB in addition to chemotherapy is ASTRUM-005 published in September 2022 which included 585 patients treated with EP +/− serplulimab, a fully humanised IgG4 monoclonal antibody against the PD-1 receptor . The immTaking these pivotal immunotherapy trials together, there is clearly benefit from the addition of checkpoint inhibitors to platinum-based chemotherapy. As has been the case in other tumour types, the long tails of the Kaplan–Meier curves in these trials suggest that there is a small subset of patients, whom we are still ill-equipped to identify, that may have deep and durable responses to ICB. Unfortunately, there has not been any reliable or robust prognostic biomarkers identified. PD-L1 expression was not predictive of efficacy in these trials, and likewise tumour mutational burden (TMB) is of limited value ,25,27.Despite rapid and impressive initial response to first line platinum-etoposide with or without immunotherapy, virtually all patients eventually relapse. For recurrent disease, chemotherapy outcomes are poor and immunotherapy trials’ results have been disappointing, as summarised in p = 0.552) but improved symptom control. This was by comparison with the more toxic regimen of CAV  [Topotecan is the standard second line treatment for ES-SCLC based on a phase III trial published in 1999 which showed equivalent efficiency  ,34. Patients who experience progression ≥ 90 days from the completion of first-line chemotherapy are generally considered as platinum sensitive. A comparison of rechallenge with platinum-based chemotherapy compared to second line topotecan was published in 2020 involving 164 platinum sensitive recurrent SCLC patients in France . PFS wasIn patients not fit enough to consider any intravenous chemotherapy, oral topotecan has shown an ORR of 7% and increased survival from 13.9 weeks to 25.9 weeks with improved symptom control compared to best supportive care in a small, randomised study of 141 patients in 2006 . Other sLike all emerging therapies, ICB was first tested in patients recurrent ES-SCLC who had failed standard treatment. The modest efficacy observed led to the rapid launching of trials in the first line setting a few years later, as discussed in However, the phase III confirmatory studies did not show any statistically significant survival benefit when compared to chemotherapy in the second line setting with nivolumab (CheckMate 331) . The indLurbinectedin is an alkylating drug and an analogue of tetrahydroisoquinoline trabectedin which induces degradation of transcribing RNA Pol II causing DNA damage with demonstrated antitumour activity in animal models . In a siCompared to classic chemotherapy, antibody–drug conjugates (ADCs) are designed to achieve a wider therapeutic window with minimal off-target side effects. All ADCs have three main components: an antibody targeted against a tumour-associated antigen, a linker, and a cytotoxic payload. The ideal antibody target is a cell surface protein that is overexpressed by tumour cells but not on normal cells thus allowing more selective killing. The linker binds the antibody to the cytotoxic agents and is usually either of cleavable  or non-cleavable varieties. Several cytotoxic payloads have been used ranging from microtubule inhibitors (auristatin) to DNA cleavage agents  and topoisomerase inhibitors (camptothecin). Delta-like ligand 3 (DLL3) is a NOTCH ligand involved in regulating neuroendocrine differentiation which is expressed in up to 75% of SCLC, whilst exhibiting minimal to absent surface expression in normal tissues . Notch pRovalpituzumab tesirine  is the first DLL3-targeted antibody tethered to a cytotoxic payload (pyrrolobenzodiazepine) by means of a protease-cleavable linker. The first-in-human phase I trial published in 2017 elicited significant enthusiasm because of the efficacy results in ES-SCLC- an ORR rate of 18% and an ORR of 38% in DLL3-high (>50% expression) ES-SCLC . In addiFour independent studies were published in the Journal of Thoracic Oncology in 2021, namely MERU (Rova-T versus placebo as a maintenance treatment in patients with SCLC after platinum-based chemotherapy regardless of DLL3 expression) , TAHOE  is a transmembrane calcium signal transducer that is overexpressed in many epithelia cancers including triple-negative breast cancer (TNBC), NSCLC, and SCLC .Sacituzumab govitecan is a TROP-2 directed antibody bound to SN-38, the active metabolite of irinotecan and a topoisomerase I inhibitor, through a hydrolysable linker . Based oBRCA mutations or homologous recombination deficiency, aberrant expression of several genes implicated in DNA damage repair is a common observation. Nearly 100% of cases of SCLC have homozygous loss or inactivation of RB1, encoding the primary regulator of the G1-S cell cycle checkpoint and TP53, critical for multiple DNA damage response pathways [While SCLC is not characterised by pathways . The notPoly (ADP-ribose) polymerase (PARP) is an essential nuclear enzyme for the repair of single-stranded DNA (ssDNA) breaks through the base excision repair (BER) pathways . PARP inp = 0.009) and OS  were observed in patients with SLFN11-positive tumours treated with the TMZ and veliparib combination, as further discussed below. A combination of TMZ and olaparib has been studied in a phase II study of 50 patients with recurrent, previously treated SCLC [2 daily for seven days per 21-day cycle. There is evidence that lower continuous dose TMZ can potentiate the cytotoxic activity of PARP inhibitors by forming PARP-DNA-trapping complexes to induce cytotoxicity, proposing an alternative strategy of combing PARP inhibitors with lower dose, continuous TMZ. IMP4297-106 is a phase Ib/II open label study to assess the efficacy of senaparib with TMZ at 30mg flat dose daily, day 1–21 out of a 28-day cycle in patients with advanced solid tumours with a planned expansion cohort in ES-SCLC after first line platinum-based chemotherapy [PARP inhibitors are being extensively explored in various combinations for treating SCLC . Combinated SCLC . This stotherapy .Combination of PARP inhibitors with immune checkpoint inhibitors have had mixed results. Treatment of SCLC with olaparib led to an upregulation of PD-L1 expression in in vitro models and subsequent increased sensitivity to combination treatment . In a phSLFN11 (schlafen family member 11) has been identified as a novel predictive biomarker of response to talazoparib . In the p = 0.007); however, no statistically significant survival benefit. For relapsed patients, there was an ORR of 19% compared to 71% for the untreated patients. A 2017 meta-analysis of 1322 patients who received any anti-angiogenesis agents in randomised controlled trials similarly concluded that there was no improvement to PFS, OS, or ORR in combination with chemotherapy [A 2021 meta-analysis examined the role of bevacizumab with chemotherapy in first-line ES-SCLC . This inotherapy . p = 0.02), there was no statistically significant improvement in median OS . Anti-angiogenesis agents, namely bevacizumab, sorafenib and cediranib, have so far not had a meaningful impact as monotherapy for recurrent SCLC. Cediranib was examined in a phase II study of 25 patients who had progressed after platinum-based chemotherapy . This stSorafenib was trialled on 89 patients with recurrent disease in a phase II SWOG trial . This trAnlotinib monotherapy, an antioangiogenic multi-kinase inhibitor, has been retrospectively studied in refractory SCLC patients who failed 2 prior lines of treatment . Whilst C-kit expression has been reported in up to 88% of SCLC samples and high levels of expression have correlated with improved OS in retrospective data . DespiteThe transformation of NSCLC to SCLC has been identified as a cause of EGFR-targeting TKI resistance. This mainly occurs in the light smoker and histologically combined subtypes . DespiteBRAF, PTEN, PIK3CA, and SLIT2 are rare and their therapeutic value in SCLC is unknown. PIK3CA mutations were identified in 5 out of 28 SCLC samples [Other targetable mutations in known oncogenes, including  samples . PIK3CA  samples .Bispecific T-cell engagers have been shown to be effective in haematological malignancies  but the Targeting the same DLL3 protein, AMG 119 is a chimeric antigen receptor (CAR) T cell therapy currently in a phase I trial  with expSCLC has been a challenge for the era of personalised therapy due, at least in part, to underappreciated inter- and intra-tumour heterogeneity. In contrast to NSCLC, where biomarker selection for targeted and immune therapies has dramatically altered treatment approaches, clinical trials for SCLC have largely focused on unselected populations and have had predictably disappointing results in most cases. SCLC has one of the highest mutation loads with about 200 non-synonymous mutations per tumour. Conventional thought is that it should therefore be sensitive to immunotherapy, which has not been seen in trials thus far. The potential cause of this poor efficacy comprises a range of factors adverse to immunotherapy, such as low number of tumour infiltrating lymphocytes, low expressions of PD-L1 and MHC class, presence of immunosuppressive cell populations and cytokines, as well as avascular tumour areas from rapid tumour growth for immune evasion . While Ip = 0.0025) in the PACIFIC trial [Maintenance durvalumab as consolidation therapy in patients with locally advanced, unresectable NSCLC without disease progression after two or more cycles of platinum-based definitive chemoradiation therapy has shown significant OS benefit  in the DESTINY-Breast03 trial which compared to T-DM1 [ADCs are an appealing treatment tool given their broad applicability across different tumour types and the possibility to combine with other agents. The most successful example is ado-trastuzumab emtansine (T-DM1). This HER2-directed ADC incorporates the HER2 targeted actions of trastuzumab with the microtubule inhibitor DM1 . T-DM1 wto T-DM1 . More reto T-DM1 . These tto T-DM1  and inclThe failure of Rova-T development strategy in SCLC, on the other hand, also highlighted the dangers of moving directly from promising small phase I studies to large registration phase III studies without confirming the safety and efficacy data in phase II studies or larger expansion cohorts in seamless phase I/II trial. Although this approach has worked for agents targeting oncogenic driver mutation, we should not abandon the principles of good drug development with other agents. The risk is overly optimistic results based on biased phase I trials, with carefully selected relatively good prognosis patients not representative of ‘real world’ experience. Additionally, the over-estimated real benefit of the drug is based on the lack of a control arm.Combination trials of PARP inhibitors with additional agents have shown promise, with response rates as high as 40% in the olaparib and TMZ trials. Whilst limited by low numbers, the biological foundation for these combinations is being established. This combination is especially attractive in the relapsed setting, given the high prevalence of intracranial disease in SCLC and the demonstrated CNS-control in the available data so far . OngoingTP53 and RB1 offers great opportunities for the detection of SCLC ctDNA. For example, in a study of serial ctDNA samples from 27 patients, disease-associated mutations were detected in 85% of patients [One obstacle in SCLC research is a paucity of tissue for comprehensive molecular characterisation, biomarker analysis and a deficiency of clinically relevant model systems both for the study of basic biology of SCLC and the preclinical drug development to inform clinical trials. For example, in the successful IMpower-133 trial of atezolizumab, data on the PD-L1 status of most patients could not be obtained due of insufficient quality of tumour material . Liquid patients . AlthougIn summary, SCLC is a recalcitrant disease and difficult to treat, especially at the time of relapse. Combination therapies with strong scientific rationale in robust clinical trials should continue to be explored. Further research to understand the markers and clinicopathological characteristics that govern response or resistance to targeted and immune therapies represent a fundamental unmet need.


Early in the pandemic, the United States population experienced a sharp rise in the prevalence rates of opioid use, social isolation, and pain interference. Given the high rates of pain reported by patients on medication for opioid use disorder (MOUD), the pandemic presented a unique opportunity to disentangle the relationship between opioid use, pain, and social isolation in this high-risk population. We tested the hypothesis that pandemic-induced isolation would partially mediate change in pain interference levels experienced by patients on MOUD, even when controlling for baseline opioid use. Such work can inform the development of targeted interventions for a vulnerable, underserved population.Analyses used data from a cluster randomized trial (N = 188) of patients on MOUD across eight opioid treatment programs. As part of the parent trial, participants provided pre-pandemic data on pain interference, opioid use, and socio-demographic variables. Research staff re-contacted participants between May and June 2020 and 133 participants (71% response rate) consented to complete a supplemental survey that assessed pandemic-induced isolation. Participants then completed a follow-up interview during the pandemic that again assessed pain interference and opioid use. A path model assessed whether pre-pandemic pain interference had an indirect effect on pain interference during the pandemic via pandemic-induced isolation.Consistent with hypotheses, we found evidence that pandemic-induced isolation partially mediated change in pain interference levels among MOUD patients during the pandemic. Higher levels of pre-pandemic pain interference and opioid use were both significantly associated with higher levels of pandemic-induced isolation. In addition, pre-pandemic pain interference was significantly related to levels of pain interference during the pandemic, and these pain levels were partially explained by the level of pandemic-induced isolation reported.Patients on MOUD with higher use of opioids and higher rates of pain pre-pandemic were more likely to report feeling isolated during COVID-related social distancing and this, in turn, partially explained changes in levels of pain interference. These results highlight social isolation as a key risk factor for patients on MOUD and suggest that interventions promoting social connection could be associated with reduced pain interference, which in turn could improve patient quality of life. As these two public health crises collided, the COVID-19 crisis created a range of new risk factors for patients on medication for opioid use disorder (MOUD), including higher-than-average rates of COVID-19 infection, job insecurity, closure of opioid treatment programs that dispense medication, and social distancing or shelter-in-place orders limiting chance of bystander overdose rescue , 3, 4, 5One potential pathway through which social isolation may affect those with OUD is through its relationship with pain. Pain is a well-established predictor of OUD , 14, 15,Karos and colleagues  hypothesGiven the exceptionally high prevalence of pain interference among patients on MOUD (approximately 40% report chronic interfering pain) , it is i2.2.1clinicaltrials.gov Masked) that is focused on testing two implementation strategies for helping opioid treatment programs (OTPs) to implement contingency management, an evidence-based behavioral intervention in which patients earn prizes for meeting treatment goals . Participants provided informed consent prior to completing any study procedures. The study was approved by the involved university Institutional Review Board (IRB). Project [Masked] was a cluster-randomized type 3 implementation-effectiveness hybrid trial  to complete a supplementary survey on COVID-19-related impacts. After providing informed consent for this supplemental aspect of the main study, participants completed the Epidemic-Pandemic Impact Inventory ; a scale consisting of 92 binary (yes/no) items that inventory the impact of COVID across 10 life domains: employment, interpersonal conflict, social isolation, economic, emotional health, substance use, physical health, quarantining and physical distancing, infection exposure, and caretaking. The survey took about 15–20 minutes to complete, and patients received $20, which was added to the rechargeable gift card they had already received as part of Project [Masked]. Participants then completed routine Project [Masked] follow-up assessments. For the current study, each participant’s first follow-up assessment to follow the EPII assessment was used to calculate the effect of COVID-related impacts on subsequent pain and opioid use.2.32.3.1In the baseline survey, participants answered questions about their socio-demographics. Specific covariates included in the current analysis were selected based on well-documented associations with pain and opioid use: biological sex at birth , racial/ethnic identity, age in years, and annual income.2.3.2.At baseline and each follow-up assessment, the Timeline Followback  was used2.3.3At baseline and each follow-up assessment, participants self-reported pain interference on the Brief Pain Inventory . Focal i2.3.3In order to investigate how experiences of pandemic-induced isolation impacted participants, we counted the number of EPII endorsed isolation-related items. This included 10 items from the “social isolation” domain  and 3 items from the “quarantining and physical distancing” domain . Total possible scores on this isolation-related impact inventory ranged from 0 to 13. As the EPII signals endorsement of events, it is not advised to compute reliability statistics from this type of data .2.4t(186) = 2.790, p = .006). Further analyses examined whether condition (assignment to implementation strategy condition), timing of when the EPII was completed (before the 6 or 9-month follow-up), or contingency management dosage (number of sessions received) were associated with the focal items, but no significant associations were identified. Thus, data were pooled across conditions, and neither timing nor number of sessions were controlled for in analyses. For clarity, we refer to the baseline variables as “pre-pandemic” and the follow-up variables as “during the pandemic.”The analytical sample was determined as those who completed the EPII survey. A 71% response rate was obtained for the EPII, yielding an analytical sample of 133 respondents. Sensitivity analyses found that completers were representative of the full Project [Masked] sample, except that non-completers more often identified as male . When pre-pandemic pain interference was included as a predictor of pain interference during the pandemic, partial correlations demonstrated that the effect of age was no longer significant ; hence, age was not included as a covariate in the final analysis.Preliminary analyses determined that most of the sociodemographic variables  were not related to pain interference and were thus not included as covariates in analysis. The only sociodemographic variable that predicted pain interference was age, such that older participants reported greater experiences of pain interference pre-pandemic and during the pandemic  were predominantly Non-Hispanic White (83%), female (60%), and had completed high school (60%), with a median age of 34 years . On average, participants reported experiencing six pandemic-induced isolation-related experiences . In the baseline pre-pandemic assessment, participants reported a mean score of pain interference at 3.88 , indicating moderate interference from pain on daily activities. In the follow-up assessment during the pandemic, participants reported pain interference scores of 3.41 , indicating an overall small, but non-significant (p = .071) reduction in reported pain at follow-up across the sample. Participants from the analytical sample (p = 0.08). Importantly, results from the bootstrapped estimation of the indirect relation between baseline pain interference and pain interference during the pandemic via pandemic-induced isolation revealed a significant indirect effect , however this path was not statistically significant. When examining the path between pandemic-induced isolation and pain interference during the pandemic, the effect was positive, but not significant  and therefore we cannot make causal assertions about the observed effects. We can simply conclude that pandemic-induced social isolation partially mediated changes in pain interference during the pandemic. Third, we studied the effects of pandemic-induced social isolation and pain because these are known risk factors for overdose, but we did not track the actual rate of overdose in the sample. Finally, the analytical sample was drawn from eight opioid treatment programs (OTPs) in the New England region of the United States and should not be considered representative of OTPs throughout the United States.Despite these limitations, this study is among the first to show that pain interference prior to the pandemic predicted pandemic-induced isolation, and that this isolation in turn partially explained pain among patients on MOUD. Our path model further strengthens the argument for a bidirectional relationship between pain and social isolation. Understanding the relationship between social isolation and pain interference among patients with MOUD is important, given that pain is a well-established predictor of OUD, returning to opioid use, and worse treatment outcomes among patients receiving MOUD. Most importantly, this study highlights social isolation as a key risk factor for patients on MOUD and suggests that interventions promoting social connection could be associated with reduced pain interference, which in turn could predict greater quality of life. Future research would benefit from examining social isolation among a larger group of patients on MOUD to further explore as the complex, and likely bi-directional relationship between pain interference and social isolation.5.In summary, this study is among the first to demonstrate that social isolation during the early months of the COVID-19 pandemic partially explained pain interference among patients on MOUD, even when controlling for opioid use and pain interference at baseline. The current findings have at least two key clinical implications. First, given the high rates of pain interference and social isolation reported in this study, paired with the fact that MOUD patients are already less likely to seek medical care due to stigma and reports of substandard care  programs