Choosing or altering the planned statistical analysis approach after examination of trial data (often referred to as ‘p-hacking’) can bias the results of randomised trials. However, the extent of this issue in practice is currently unclear. We conducted a review of published randomised trials to evaluate how often a pre-specified analysis approach is publicly available, and how often the planned analysis is changed.A review of randomised trials published between January and April 2018 in six leading general medical journals. For each trial, we established whether a pre-specified analysis approach was publicly available in a protocol or statistical analysis plan and compared this to the trial publication.n = 31, 35%) and analysis population , followed by the use of covariates  and the approach for handling missing data . Many protocols or statistical analysis plans were dated after the trial had begun, so earlier discrepancies may have been missed.Overall, 89 of 101 eligible trials (88%) had a publicly available pre-specified analysis approach. Only 22/89 trials (25%) had no unexplained discrepancies between the pre-specified and conducted analysis. Fifty-four trials (61%) had one or more unexplained discrepancies, and in 13 trials (15%), it was impossible to ascertain whether any unexplained discrepancies occurred due to incomplete reporting of the statistical methods. Unexplained discrepancies were most common for the analysis model (Unexplained discrepancies in the statistical methods of randomised trials are common. Increased transparency is required for proper evaluation of results. The results of a clinical trial depend upon the statistical methods used for analysis. For example, changing the analysis population or method of handling missing data can change the size of the estimated treatment effect or its standard error. In some instances, these differences can be large and may affect the interpretation of the trial –6. If inGuidelines such as ICH-E9  , The Lancet, New England Journal of Medicine (NEJM), and PLOS Medicine. We searched for articles in PubMed with a publication type of ‘randomized controlled trial’ or categorised with the MeSH term ‘random allocation’, or including the keyword ‘random*’ in the title or abstract, restricted to the aforementioned included journals and publication period. The full search strategy is shown in Appendix 1 in Additional file In this review, we examined randomised controlled trials published between January and April 2018 in six general high-impact medical journals: Articles were eligible for inclusion if they reported results from a phase 2–4 randomised trial in humans. Exclusion criteria were pilot or feasibility study, phase 1 trial, non-randomised study, secondary analysis of previously published trial, cost-effectiveness as the primary outcome, more than one trial reported in the article, results of an interim analysis, or if the protocol or SAP was not in English.One author screened the title and abstract of each paper for eligibility. The full texts of these articles were then assessed independently by two reviewers to confirm eligibility. For all eligible studies, one author searched the main text, supplementary material, and references to identify whether a protocol and/or SAP was available.Data was extracted onto a pre-piloted standardised data extraction form by two reviewers independently  if one outcome was listed as the primary, we used this; (b) if no outcomes or multiple outcomes were listed as being primary, we used the outcome that the sample size calculation was based on; and (c) if no sample size calculation was performed or sample size was calculated for multiple primary outcomes, we used the first clinical outcome listed in the objectives/outcomes section. We identified the primary analysis as follows: (a) if a single analysis strategy was used, or multiple strategies were used with one being identified as primary, we used this; (b) if multiple strategies were used without one being identified as primary, we used the first one presented in the results section.For each article, we extracted general trial characteristics, whether protocols or SAPs were available, including the given dates of these documents and, if available, the blinding status of trial statisticians. For published protocols or SAPs, we used the date of publication. For protocols or SAPs available as supplementary material with the results article or elsewhere, we used the given date on the documents. For articles with a protocol or SAP, we compared the method of analysis in the trial publication against the method specified in the earliest available protocol or SAP which included some information on the analysis of the primary outcome . We assessed the following four analysis elements: (i) analysis population , (ii) the statistical analysis model, (iii) use of baseline covariates in the analysis, and (iv) the method for handling missing data. We chose these elements as they are specified in the SPIRIT guidelines and have been used in previous reviews , 26.We evaluated two types of discrepancies for each analysis element. The first, termed a ‘change’, occurred when the analysis element in the trial publication was different to that specified in the original analysis plan. The following examples would constitute changes: (a) if an intention-to-treat analysis population was originally specified, but a per-protocol analysis was used; (b) if the functional form of the statistical analysis model was changed, such as from a mixed-effects regression model to generalised estimating equations (GEE); (c) if the original analysis plan specified the analysis would not adjust for baseline covariates but the trial publication adjusted for one or more patient characteristic; or (d) if a complete case analysis was originally specified, but multiple imputation was used.The second discrepancy, termed an ‘addition’, occurred when the original analysis plan gave the investigators flexibility to subjectively choose the final analysis method after seeing trial data. This could occur if the original analysis plan (i) contained insufficient information about the proposed analysis or (ii) allowed the investigators to subjectively choose between multiple different potential analyses. The following examples would constitute additions: if the original analysis plan stated that (a) both a per-protocol and intention-to-treat analysis population would be used, without specifying which was the primary analysis ; (b) either parametric or non-parametric methods would be used depending on distributional assumptions, but did not define an objective criteria for assessing distributional assumptions (as the investigators could then present whichever method gave the most favourable result); (c) the analysis would adjust for important baseline covariates, but did not define how these covariates would be chosen ; or (d) multiple imputation would be used, but did not define what the method of imputation would be, or what variables would be included in the imputation model .We classified each discrepancy as being ‘explained’ or ‘unexplained’. Discrepancies were classified as explained if they had been specified in a subsequent version of the protocol or SAP  or if the trial publication explained that an alteration to the pre-specified analysis approach had been made. Otherwise, discrepancies were classified as unexplained.The main outcome measures were (i) the number of trials with a publicly available pre-specified analysis approach for the primary outcome , (ii) the number of trials with no unexplained discrepancies from the publicly available pre-specified analysis approach, and (iii) the total number of analysis elements for each trial with an unexplained discrepancy.Secondary outcomes were, for each analysis element described earlier, (i) the number of trials with at least one unexplained discrepancy (either change or addition), (ii) the number of trials with at least one unexplained change, and (iii) the number of trials with at least one unexplained addition.Outcomes were summarised descriptively using frequencies and percentages. We performed two pre-specified subgroup analyses, where we summarised outcomes separately according to trial funding status  and type of intervention . Two post hoc subgroup analyses were performed. The first summarised outcomes according to whether a pre-specified analysis approach was made available prior to the publication of the results article . The second summarised outcomes between trials with a standalone SAP available versus those without (regardless of the availability of a protocol).All statistical analyses were performed using Stata version 15 .Our search identified 197 articles, of which 101 were eligible see Fig. . GeneralProtocols were available for 90 trials (89%) . SAPs were available for 46 trials (46%) . Of the 90 trials with an available protocol, the earliest version available was dated before recruitment began for 45 (50%) trials, 19 (21%) were dated during recruitment, 8 (9%) were dated after recruitment ended, and 18 (20%) did not have a date. Of the 46 trials with an available SAP, the earliest version of the SAP was dated before recruitment began for 9 (20%) trials, 13 (28%) were dated during recruitment, 13 (28%) were dated after recruitment ended, and 11 (24%) did not have a date.Overall, only 11 trials (11%) stated in the trial publication, protocol, or SAP that the statistician was blinded until the SAP was signed off, and 10 (10%) stated the statistician was blinded until the database was locked.Overall, 89 of 101 trials (88%) had a publicly available pre-specified analysis approach for the primary outcome. Eleven trials did not have an available protocol or SAP, and one trial had a protocol with no information on the analysis and no SAP. The document containing the original analysis plan  was dated before the start of recruitment for 41 of 89 (46%) trials, during recruitment in 19 (21%) trials , and after the end of recruitment in 8 (9%) trials . In 21 trials (24%), no date was available.For 46 of 89 trials (52%), the document containing the original analysis plan was made publically available prior to the trial publication , whereas the document for the remaining 43 trials (48%) was only made publically available at the point the trial was published .n = 5, explained discrepancies only n = 17). A further 54 trials (61%) had one or more unexplained discrepancies .Of the 89 trials with an available pre-specified analysis approach, only 22 (25%) did not have any unexplained discrepancies  or two  unexplained discrepancies. Only 11 (12%) had three and 2 (2%) had four unexplained discrepancies. Unexplained discrepancies were most common for the statistical analysis model  and analysis population , followed by the use of covariates  and handling of missing data . Table Most trials had one n = 5, 28% orOverall, 29 trials (33%) had at least one explained discrepancy. Most discrepancies were explained in a later version of the protocol or SAP; only 2 trials explained a discrepancy in the trial publication. Of the 29 trials with an explained discrepancy, only 6 (21%) stated that the statistician was blinded until the SAP was signed off, and 4 (14%) until the database was locked.A total of 43/61 (66%) trials that had no for-profit funding had at least one unexplained discrepancy, compared to 11/28 (45%) trials that had some for-profit funding. One or more unexplained discrepancy occurred for 25 of 47 (53%) pharmaceutical intervention trials, 10 of 12 surgical trials (83%), 9 of 9 (100%) psychological/behavioural/educational trials, 9 of 19 (47%) other trials, and 1 of 2 trials (50%) with multiple intervention types.Fewer trials with a SAP available had unexplained discrepancies than trials without an available SAP, though this figure was still high (SAP available 22/46 [48%] with ≥ 1 unexplained discrepancy vs. no SAP 32/43 [74%]). Trials with a SAP still had a relatively high number of additions to the analysis method, indicating that methods were not being adequately pre-specified within these SAPs .A total of 34/46 (74%) trials in which the original analysis plan was made available prior to the trial publication had at least one unexplained discrepancy, compared to 20/43 (47%) trials where the analysis approach was only made available at the time of trial publication.See Additional file In our review of 101 trials published in high-impact general medical journals, we found that most had a pre-specified analysis approach for the primary outcome available in either a protocol or SAP. This is essential to allow transparent assessment of whether inappropriate changes were made to the statistical methods. However, most pre-specified statistical analysis approaches were available in a document that was dated after the trial had begun, or had no date available. It is therefore possible that the analysis approach in these documents may have already been changed from the pre-trial version. Moreover, there was poor reporting around the blinding status of statisticians rendering it impossible to verify that the pre-specified analysis approach had not been written after seeing unblinded trial data. In this case, even if no alterations were made to the pre-specified approach, results still may be subject to bias.Only 25% of trials did not have any unexplained discrepancies between the trial publication and the pre-specified analysis approach, and only 6% had no discrepancies at all. Most trials had at least one unexplained discrepancy (61%), with 32% of trials having two or more. In 15% of trials, it was impossible to assess whether there were unexplained discrepancies due to poor reporting of the statistical methods used. Of note, 33% of trials had one or more explained discrepancies; however, less than a quarter of these trials reported that the statistician was blinded to treatment allocation until the analysis plan was finalised or the database was locked. These alterations may therefore have been made based on unblinded trial data, despite being explained. It was also surprising that only two trials explained a discrepancy in the trial publication, despite requirements by the CONSORT [Our results are broadly consistent with previous reviews. Spence et al.  evaluateThe key issues we identified in this study were (i) low availability of pre-trial protocols and analysis plans, (ii) poor pre-specification of statistical methods within protocols and analysis plans, (iii) frequent unexplained discrepancies in the final trial publication, (iv) poor reporting of the blinding status of statisticians in relation to modifications of analysis methods or access to trial data, and (v) poor descriptions of the actual analysis methods used in the final publication. Increased adherence to guidelines such as SPIRIT, CONSORT, and the guidelines for Statistical Analysis Plans , 27, 28 Our study had some limitations. We only included articles from six high-impact medical journals; it is likely that trials published in other journals may have lower availability of protocols and SAPs and higher rates of unexplained discrepancies. Protocols and SAPs were identified via searching the main text, supplementary material, and references of articles; it is possible that we might have missed a few articles, but we expect this number to be very small, as in our experience when protocols/SAPs are available they are referenced to within the paper. Comparisons were based on the first available protocol or SAP; however, many were dated after the trial had begun, so there may have been discrepancies before this that we missed. We also assumed the dates available on protocols and SAPs included as part of the supplementary material alongside trial publications were accurate. However, we had no way to verify this, and it is possible that some of these dates are incorrect, which could imply a higher proportion of analysis approaches were written later on in the life cycle of the trial than what we have reported here.In conclusion, unexplained discrepancies in the statistical methods of randomised trials are common. Increased transparency around the statistical methods used in randomised trials is required for proper evaluation of trial results.Additional file 1. Protocol and data extraction form – contains protocol and data extraction form used for this study.Additional file 2. Supplementary material – contains additional methods and results.


Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome (ARDS) but it is unknown whether prone positioning improves outcomes in mechanically ventilated patients with moderate to severe ARDS due to COVID-19.A cohort study at a New York City hospital at the peak of the early pandemic in the United States, under crisis conditions. The aim was to determine the benefit of prone positioning in mechanically ventilated patients with ARDS due to COVID-19. The primary outcome was in-hospital death. Secondary outcomes included changes in physiologic parameters. Fine-Gray competing risks models with stabilized inverse probability treatment weighting (sIPTW) were used to determine the effect of prone positioning on outcomes. In addition, linear mixed effects models (LMM) were used to assess changes in physiology with prone positioning.P< 0.005). Using LMM to evaluate the impact of positioning maneuvers on physiological parameters, the oxygenation-saturation index was significantly improved during days 1–3 (P< 0.01) whereas oxygenation-saturation index (OSI), oxygenation-index (OI) and arterial oxygen partial pressure to fractional inspired oxygen (PaO2:FiO2) were significantly improved during days 4–7 .Out of 335 participants who were intubated and mechanically ventilated, 62 underwent prone positioning, 199 met prone positioning criteria and served as controls and 74 were excluded. The intervention and control groups were similar at baseline. In multivariate-adjusted competing risks models with sIPTW, prone positioning was significantly associated with reduced mortality (SHR 0.61, 95% CI 0.46–0.80, Prone positioning in patients with moderate to severe ARDS due to COVID-19 is associated with reduced mortality and improved physiologic parameters. One in-hospital death could be averted for every eight patients treated. Replicating results and scaling the intervention are important, but prone positioning may represented an additional therapeutic option in patients with ARDS due to COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has had a profound impact on global public health. The ongoing COVID-19 pandemic has presented numerous clinical management challenges further compounded by overwhelmed health systems. The initial critical care experience in Hubei province, and more broadly in China, inadequately informed preparations for what has been seen in Europe and North America., 4Several interventions for ARDS have been evaluated over the last two decades. In particular, prone positioning is one of few therapeutic interventions for patients with severe ARDS that has demonstrated improved oxygenation and a survival benefit. Awake prThe South Bronx is a socioeconomically disadvantaged borough in New York City (NYC) that had the highest per capita COVID-19 case count in the United States at 2941 per 100,000 residents with very high hospitalization and death rates., 12 The We sought to determine if patients on mechanical ventilation with moderate to severe ARDS who underwent standardized prone positioning had lower mortality and improved within-person physiologic changes. As we rapidly evaluate drugs and interventions for COVID-19, it is crucial to understand if serial prone positioning could be a complementary therapeutic intervention for the most critically ill.A cohort design with participants from the peak of hospitalizations for COVID-19 in exposed (prone positioning) and non-exposed (non-prone-positioning) groups. During the COVID-19 pandemic, much of the hospital was converted into make-shift intensive care units and virtually all inpatients had confirmed COVID-19. During this time, a multidisciplinary prone team including personnel from the United States Air Force Medical and Nursing Corps, the United States Army, civilian contractors, and hospital occupational and physical therapy was assembled to offer positioning maneuvers which were otherwise rarely done due to crisis operations. Details of the prone positioning process, including peri-maneuver checklists, team size and roles, supplies and team schedule are included in a priori): arterial oxygen partial pressure to fractional inspired oxygen (PaO2:FiO2) <150 mm Hg, positive end-expiratory pressure (PEEP) >10 cm of water and FiO2 > 0.6. The ultimate decision for initiating and discontinuing positional movements was made by the primary team overseeing and coordinating care for each patient. Prone positioning was not mandatory, but was routinely available, 24 hours a day, seven days a week. The study received institutional review board approval (IRB # 20 – 007).Participants were identified from a single level 1 trauma hospital in the South Bronx, New York City, and were included across all hospital services . All sequential adult patients (>17 years of age) were included if they were intubated, had not undergone prone positioning by others, met criteria for prone positioning, and had confirmed SARS-CoV-2 infection by real-time real-time reverse transcription-polymerase chain nasal swab  from March 25 through May 2, 2020. The prone team offered positional services for mechanically ventilated patients who met the following criteria , acute physiology and chronic health evaluation (APACHE-II) score and vasopressor use were the primary confounders by indication. In the mortality analysis, the APACHE-II score was evaluated at the time of intubation., oxygenation saturation index (OSI), PaOtubation. BMI and Characteristics of the cohort were summarized using descriptive statistics as appropriate. Fine-Gray models were used to assess the association between prone positioning and death, accounting for hospital discharge as a competing risk. ParticipLinear mixed models (LMMs) were used to assess the association of prone vs supine positioning with physiologic parameter levels among the exposed. Outcomes were natural log transformed to meet normality assumptions. The LMMs included nested random effects for participant and positioning episode, and allowed for autocorrelation of the residuals. In addition to estimating overall positional effects, we also estimated these effects in days 1–3 and 4–7 of each episode. Pearson correlation coefficients were also used to characterize degree of agreement for OI, OSI, PaO2:FiO2 and SpO2:FiO2, to support clinical utility in practice. Analyses were performed in Stata .During the study period, 335 individuals were intubated and placed on mechanical ventilation. Sixty-two underwent prone positioning while 199 who did not undergo positioning changes, but met criteria to do so, were selected as contemporary controls. Seventy-four individuals were excluded for failing to meeting prone positioning criteria or for having undergone prone positioning by providers outside of the standard protocol. A study flow diagram depicts the inclusion and exclusion of participants across groups in Compared to contemporary controls, the prone positioning group had fewer deaths and a longer time to death in those who expired, in spite of similar length of stay and ventilator-free days. Estimates of the association between prone positioning and mortality are summarized in aO2:FiO2, SpO2:FiO2 and PaO2. For days 4–7 of prone positioning, improvement was seen in the PaO2:FiO2, SpO2:FiO2 and PaO2. Only the OI failed to show improvement at any time and OSI did not show improvement for days 4–7. During crisis operations with enhanced infection control and use of transport ventilators for routine ventilation, it may be difficult to obtain PaO2 and mean airway pressure values and so proxy variables may be helpful. We therefore looked at Pearson correlation coefficients amongst ratios and indices. Overall, PaO2:FiO2 and SpO2:FiO2 are moderately correlated (p = −0.51), and OSI and OI, and OSI and SpO2:FiO2, are closely correlated . The correlations did not differ when split into days 1–3 and 4–7. Results are summarized in aO2:FiO2 . No clear evidence for interaction between positioning and time was found.In analyses using LMMs to estimate the association of positioning with physiological indices, 19 of 62 exposed participants contributed more than one episode. In these analyses, prone vs supine positioning was significantly associated with overall improvement in PaO2:FiO2 . In modeWe report results from a comprehensive cohort study assessing the potential benefits of prone positioning in COVID-19 patients with moderate to severe ARDS. We found a nearly 40% reduction in mortality with prone positioning, an effect that appears sustained on cumulative incidence curves. With respect to physiologic parameters, there were meaningful changes across all ratios and indices to suggest that prone positioning is associated with improvements in within-person physiology and that the benefit may persist beyond three days. Our findings across both analyses were robust to various adjustments, modifications, sensitivity analyses and nested comparative testing.Fundamentally, this study has three key findings. First, we demonstrated a mortality benefit with prone positioning with a number needed to treat of eight. The durability of the finding is important, and ensuring that it can be replicated in other settings will be essential to justify a recommendation, but we have no evidence to attribute the survival benefit in the intervention arm to bias. Second, it appears that there is a benefit to additional days of prone positioning beyond 3 days. The effect seen with 4–7 days of prone positioning may be heavily influenced by a smaller group that realized a differential benefit, but 34 of 89 positioning sequences resulted in at least four days of intervention, representing a relatively large proportion of individuals. Third, prone positioning resulted in significant changes in physiologic parameters which may support the underlying hypothesis that prone positioning improves ventilation-perfusion matching. AdditionOur results are consistent with recent multi-center data suggesting a mortality benefit of prone positioning in patients with ARDS whether intubated or not., 19, 20 Some limitations of this study should be noted. First, this is a single center retrospective cohort study in a resource constrained environment under crisis operations. As a result, although patients had critical care needs, they were frequently cared for in ad-hoc intensive care units by non-critical care personnel. The decision to initiate or discontinue the intervention under study was left to the treating primary team without defining endpoints. We attempted to address any residual confounding through IPTW and no differences in the results were noted. If the prone team was consulted and the patient had moderate to severe ARDS and met criteria for prone positioning, it was felt that they could benefit from the intervention in addition to lung protective ventilation. Although this was pragmatic for this setting, if prone positioning is implemented elsewhere, the prone teams could consider establishing an opt-out approach with tailored entry and exit criteria, normal cadence of evaluation for candidacy for prone positioning and a mechanism for real-time data capture and quality control assessments. Finally, the results may not be readily generalizable to all populations, in particular those with milder disease and those that don’t reflect the ethnic diversity seen in the Bronx. The institutional mortality proportion was high (>75%) and therefore the impact of the intervention may be attenuated in the setting of advanced interventions .There are also some notable strengths of this work. We were able to collect detailed physiologic data in a structured manner to systematically evaluate the impact of the intervention. Also, our population has been gravely understudied in the COVID-19 pandemic and we’ve been able to contribute significantly to both describing their clinical course as well as critical care interventions for socioeconomically marginalized minority populations. Regarding outcome, we were able to include all patients who would have been eligible for prone positioning as controls creating a sound counterfactual for a contemporaneous comparison of both exposed and unexposed. Finally, compared to existing literature for patients with COVID-19, this study provides results for a large intervention group.In summary, we present data supporting prone positioning as an intervention to prolong survival and improve physiologic parameters in patients on mechanical ventilation with moderate to severe ARDS due to COVID-19. The findings should be replicated across institutions, but prone positioning may be an important consideration for health systems, particularly in the setting of an evolving suite of complementary interventions in the care of such vulnerable patients.


Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distresssyndrome (ARDS) but it is unknown whether prone positioning improvesoutcomes in mechanically ventilated patients with moderate to severe ARDSdue to COVID-19.A cohort study at a New York City hospital at the peak of the early pandemicin the United States, under crisis conditions. The aim was to determine thebenefit of prone positioning in mechanically ventilated patients with ARDSdue to COVID-19. The primary outcome was in-hospital death. Secondaryoutcomes included changes in physiologic parameters. Fine-Gray competingrisks models with stabilized inverse probability treatment weighting (sIPTW)were used to determine the effect of prone positioning on outcomes. Inaddition, linear mixed effects models (LMM) were used to assess changes inphysiology with prone positioning.P < 0.005). Using LMM toevaluate the impact of positioning maneuvers on physiological parameters,the oxygenation-saturation index was significantly improved during days 1-3(P < 0.01) whereas oxygenation-saturation index(OSI), oxygenation-index (OI) and arterial oxygen partial pressure tofractional inspired oxygen (PaO2: FiO2)were significantly improved during days 4-7 .Out of 335 participants who were intubated and mechanically ventilated, 62underwent prone positioning, 199 met prone positioning criteria and servedas controls and 74 were excluded. The intervention and control groups weresimilar at baseline. In multivariate-adjusted competing risks models withsIPTW, prone positioning was significantly associated with reduced mortality(SHR 0.61, 95% CI 0.46-0.80, Prone positioning in patients with moderate to severe ARDS due to COVID-19 isassociated with reduced mortality and improved physiologic parameters. Onein-hospital death could be averted for every 8 patients treated. Replicatingresults and scaling the intervention are important, but prone positioningmay represent an additional therapeutic option in patients with ARDS due toCOVID-19. In the setting of critical COVID-19 illness, SARS-CoV-2infection often results in severe pneumonia and hypoxemia with many patientsdeveloping acute respiratory distress syndrome (ARDS).2 Hypoxemic respiratory failure with ARDS has poor outcomes overall andCOVID-19 associated ARDS is no exception.4Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause ofcoronavirus disease 2019 (COVID-19), has had a profound impact on global publichealth. The ongoing COVID-19 pandemic has presented numerous clinical managementchallenges further compounded by overwhelmed health systems. The initial criticalcare experience in Hubei province, and more broadly in China, inadequately informedpreparations for what has been seen in Europe and North America.5 Awake prone positioning outside of the intensive care unit (ICU) is safe andmay decrease respiratory rate and improve oxygenation with early applicationpotentially delaying need for intubation in patients with COVID-19.68 In the ICU setting, prone positioning of patients receiving non-invasiveventilation or high-flow nasal canula, with or without sedation, may also be beneficial.8 Physiologically, prone positioning may improve matching of ventilation andperfusion, but studies have not linked physiologic changes to clinical outcomes,especially in COVID-19.10Several interventions for ARDS have been evaluated over the last 2 decades. Inparticular, prone positioning is one of few therapeutic interventions for patientswith severe ARDS that has demonstrated improved oxygenation and a survival benefit.12 The pressing challenge that COVID-19 brought to NYC necessitated externalsupport through the United States Departments of Defense and Homeland Security,re-distribution and up-training of local hospital staff, support from clinicalvolunteers, and augmentation through healthcare worker staffing agencies. Given thehigh volume of critically-ill patients admitted to the hospital, a multidisciplinaryteam was assembled to provide prone positioning given the support for the practicein other populations with ARDS.The South Bronx is a socioeconomically disadvantaged borough in New York City (NYC)that had the highest per capita COVID-19 case count in the United States at 2941 per100,000 residents with very high hospitalization and death rates.We sought to determine if patients on mechanical ventilation with moderate to severeARDS who underwent standardized prone positioning had lower mortality and improvedwithin-person physiologic changes. As we rapidly evaluate drugs and interventionsfor COVID-19, it is crucial to understand if serial prone positioning could be acomplementary therapeutic intervention for the most critically ill.A cohort design with participants from the peak of hospitalizations for COVID-19in exposed (prone positioning) and non-exposed (non-prone-positioning) groups.During the COVID-19 pandemic, much of the hospital was converted into make-shiftintensive care units and virtually all inpatients had confirmed COVID-19. Duringthis time, a multidisciplinary prone team including personnel from the UnitedStates Air Force Medical and Nursing Corps, the United States Army, civiliancontractors, and hospital occupational and physical therapy was assembled tooffer positioning maneuvers which were otherwise rarely done due to crisisoperations. Details of the prone positioning process, including peri-maneuverchecklists, team size and roles, supplies and team schedule are included in2:FiO2) < 150 mm Hg, positiveend-expiratory pressure (PEEP) ≥10 cm of water and FiO2  ≥  0.6.Patients with a do not resuscitate order were not explicitly excluded from thestudy. In addition, continuous venovenous hemodialysis (CVVHD), nitric oxide orextracorporeal membrane oxygen (ECMO) were not available in the facility andintermittent hemodialysis or paralytics were rarely used. The ultimate decisionfor initiating and discontinuing positional movements was made by the primaryteam overseeing and coordinating care for each patient. Prone positioning wasnot mandatory, but was routinely available, 24 hours a day, 7 days a week. Thestudy received institutional review board approval (IRB # 20-007). The pronepositioning service was advertised through critical care ,hospital medicine, and physical medicine and rehabilitation leadership. Theseservices had knowledge of and direct access to every patient who wasmechanically ventilated in the hospital, even when they were not the primaryteam.Participants were identified from a single level 1 trauma hospital in the SouthBronx, New York City, and were included across all hospital services . All sequential adult patients (>17 years of age)were included if they were intubated, had not undergone prone positioning byothers, met criteria for prone positioning, and had confirmed SARS-CoV-2infection by real-time reverse transcription-polymerase chain nasal swab from March 25 throughMay 2, 2020. The prone team offered positional services for mechanicallyventilated patients who met the following criteria (established a priori):arterial oxygen partial pressure to fractional inspired oxygen , acute physiology and chronic health evaluation (APACHE-II) score andvasopressor use were the primary confounders by indication. In the mortalityanalysis, the APACHE-II score was evaluated at the time of intubation.13 BMI and age were categorized for ease of interpretation and clinicalutility. The study team obtained the study data through manual electronicmedical record chart abstraction .The primary exposure was positional maneuvers, defined as regular alternationbetween prone and supine positioning. The primary outcomes of interest werein-hospital mortality and, among exposed patients, differences in physiologicalparameters in prone vs supine position. In the mortality analysis, every patienthad at least 30 days elapse following initiation of, or meeting criteria for,prone positioning. Follow-up of unexposed controls began when the participantfirst met prone positioning criteria during the 2 weeks after intubation. In theanalysis of positioning effects on physiologic parameters among exposedpatients, repeated measures of the oxygenation index (OI), oxygenationsaturation index (OSI), PaO14 Participants remaining in the hospital at the end of follow-up werecensored. The proportional sub-distribution hazards assumption was assessedvisually through cumulative incidence curves. To minimize confounding byindication, we used standard regression adjustment as well as a doubly robustapproach adding stabilized inverse probability treatment weights (IPTWs) to thefully adjusted model.1517 A sensitivity analysis was done to identify changes in results byexcluding controls that died within 48 hours of intubation. In addition, numberneeded to treat was calculated by the inverse of the averaged absolute riskdifferences at 30 days, for all participants at their actual and counterfactualvalues of prone positioning, and in combination with their observed covariate values.18Characteristics of the cohort were summarized using descriptive statistics asappropriate. Fine-Gray models were used to assess the association between pronepositioning and death, accounting for hospital discharge as a competing risk.Linear mixed models (LMMs) were used to assess the association of prone vs supinepositioning with physiologic parameter levels among the exposed. Outcomes werenatural log transformed to meet normality assumptions. The LMMs included nestedrandom effects for participant and positioning episode, and allowed forautocorrelation of the residuals. In addition to estimating overall positionaleffects, we also estimated these effects in days 1-3 and 4-7 of each episode.Pearson correlation coefficients were also used to characterize degree ofagreement for OI, OSI, PaO2: FiO2 and SpO2: FiO2, to support clinical utility inpractice. Analyses were performed in Stata .During the study period, 335 individuals were intubated and placed on mechanicalventilation. Sixty-two underwent prone positioning while 199 who did not undergopositioning changes, but met criteria to do so, were selected as contemporarycontrols. Seventy-four individuals were excluded for failing to meet pronepositioning criteria or for having undergone prone positioning by providers outsideof the standard protocol. A study flow diagram depicts the inclusion and exclusionof participants across groups in Compared to contemporary controls, the prone positioning group had fewer deathsand a longer time to death in those who expired, in spite of similar length ofstay and ventilator-free days. Estimates of the association between pronepositioning and mortality are summarized in aO2: FiO2,SpO2: FiO2 and PaO2.For days 4-7 of prone positioning, improvement was seen in thePaO2: FiO2, SpO2:FiO2 and PaO2. Only the OI failed to showimprovement at any time and OSI did not show improvement for days 4-7. Duringcrisis operations with enhanced infection control and use of transportventilators for routine ventilation, it may be difficult to obtainPaO2 and mean airway pressure values and so proxyvariables may be helpful. We therefore looked at Pearson correlationcoefficients among ratios and indices. Overall, PaO2:FiO2 and SpO2: FiO2 aremoderately correlated (p = −0.51), and OSI and OI, and OSI andSpO2: FiO2, are closely correlated.The correlations did not differ when split into days 1-3 and 4-7. Results aresummarized in aO2:FiO2. In models allowing positioning effects to differ in days 1-3 and4-7, prone positioning was associated with improved OSI during days 1-3 (p <0.01) as well as improved OSI, OI and PaO2:FiO2 during days 4-7 . No clear evidence for interaction between positioning and timewas found.In analyses using LMMs to estimate the association of positioning withphysiological indices, 19 of 62 exposed participants contributed more than 1episode. In these analyses, prone vs supine positioning was significantlyassociated with overall improvement in P2:FiO2. In modWe report results from a comprehensive cohort study assessing the potential benefitsof prone positioning in COVID-19 patients with moderate to severe ARDS. We found anearly 40% reduction in mortality with prone positioning, an effect that appearssustained on cumulative incidence curves. With respect to physiologic parameters,there were meaningful changes across all ratios and indices to suggest that pronepositioning is associated with improvements in within-person physiology and that thebenefit may persist beyond 3 days. Our findings across both analyses were robust tovarious adjustments, modifications, sensitivity analyses and nested comparativetesting.10 Additionally, we demonstrated the utility of relatively accessible clinicalinformation in the ICU as reasonable surrogates to monitor changes inphysiology.Fundamentally, this study has 3 key findings. First, we demonstrated a mortalitybenefit with prone positioning with a number needed to treat of 8. The durability ofthe finding is important, including for a longer time period, and ensuring that itcan be replicated in other settings will be essential to justify a recommendation,but we have no evidence to attribute the survival benefit in the intervention arm tobias. Second, it appears that there is a benefit to additional days of pronepositioning beyond 3 days. The effect seen with 4-7 days of prone positioning may beheavily influenced by a smaller group that realized a differential benefit, but 34of 89 positioning sequences resulted in at least 4 days of intervention,representing a relatively large proportion of individuals. Third, prone positioningresulted in significant changes in physiologic parameters which may support theunderlying hypothesis that prone positioning improves ventilation-perfusion matching.620 There are recommendations for prolonged prone positioning of 12-16 hoursdaily for mechanically ventilated adult patients with COVID-19 and refractoryhypoxemic respiratory failure,21 but the optimal duration of the intervention, its impact on physiologicparameters and details regarding how to organize and structure an intervention teamduring a crisis have not been completely evaluated. We acknowledge that pronepositioning in mechanically ventilated patients is a resource-intensiveintervention, particularly in overwhelmed healthcare systems during pandemicconditions. Before adopting prone positioning techniques, staff education andcommitment is paramount. If justified by hospitalized patient volume, we recommendidentifying personnel and assigning them to a dedicated prone team and tailoringreadily available checklists to institutional needs and constraints  and therefore theimpact of the intervention may be attenuated in the setting of advancedinterventions  or the added attention andcare of a multidisciplinary team could in and of itself change patient’s outcometrajectories, even though they were not involved in care decisions and did notintervene beyond prone positioning. Finally, there may be channeling bias due todisease severity or survivor bias, resulting in lower or higher probability ofexposure, respectively. The net result of this could be a bias toward or away fromthe null. We attempted to address this by ensuring that the populations werecomparable at baseline and by systematically including all possible patients. Atintubation, there was little difference in the quick SOFA, Charlson Comorbidityindex and the APACHE-II scores suggesting that the groups were similar, at leastwith respect to severity of illness.There are also some notable strengths of this work. We were able to collect detailedphysiologic data in a structured manner to systematically evaluate the impact of theintervention. Also, our population has been gravely understudied in the COVID-19pandemic and we’ve been able to contribute significantly to both describing theirclinical course as well as critical care interventions for socioeconomicallymarginalized minority populations. Regarding outcome, we were able to include allpatients who would have been eligible for prone positioning as controls creating asound counterfactual for a contemporaneous comparison of both exposed and unexposed.Finally, compared to existing literature for patients with COVID-19, this studyprovides results for a large intervention group.In summary, we present data supporting prone positioning as an intervention toprolong survival and improve physiologic parameters in patients on mechanicalventilation with moderate to severe ARDS due to COVID-19. The findings should bereplicated across institutions, but prone positioning may be an importantconsideration for health systems, particularly in the setting of an evolving suiteof complementary interventions in the care of such vulnerable patients.


Small intestinal hemangiomas are uncommon tumors that frequently present with gastrointestinal bleeding (GIB). Diagnosis, detection, and treatment can be challenging and may require surgical intervention. An 81-year-old female presented with melena. Video capsule endoscopy revealed active bleeding in the proximal jejunum and push enteroscopy identified a polypoid nodule with central umbilication. The patient underwent laparoscopic resection and jejunal submucosal hemangioma was detected. Submucosal hemangiomas are a rare cause of GIB. As the most common site of submucosal hemangiomas is the mid-jejunum, they are not easy to detect. Surgical intervention is usually required for a definitive diagnosis and definitive treatment. Intestinal hemangiomas are uncommon tumors, comprising only 0.05% of all intestinal neoplasms . These bThe diagnosis of intestinal hemangioma can be challenging due to their location, and often no preoperative diagnosis is identified. Endoscopic resection is feasible for small lesions. However, surgical resection is the preferred treatment modality for large lesions or small lesions not amenable to endoscopic treatment. A case of an 81-year-old female with a jejunal hemangioma that was identified by capsule endoscopy and push enteroscopy before undergoing surgical resection is presented herein.An 81-year-old female presented with a one-week history of melena. The development of melena was associated with fatigue, pallor, palpitations, and shortness of breath on exertion. Hematemesis, hematochezia, nonsteroidal anti-inflammatory drug (NSAID) use, use of anticoagulants or antiplatelet agents were denied. Presenting blood pressure was 130/70 mmHg and the heart rate was 106 beats/minute. Her physical examination was unremarkable except for pallor and lower limb edema. Admission hemoglobin was 4.7 g/dL, which was lower than her baseline of 8 g/dL. Transfusions were administered to maintain hemoglobin above 7 g/dL, and a proton pump inhibitor infusion was initiated. Esophagogastroduodenoscopy (EGD) was performed; benign-appearing and non-bleeding polyps were detected in the gastric fundus and body Figure .The patient’s hemoglobin continued to trend downward, and melena continued. Subsequently, colonoscopy did not detect significant abnormalities other than moderate diverticulosis, internal hemorrhoids, and old melenic liquid throughout the colon and ileum but no active bleeding Figure . Due to ongoing transfusion requirements, a video capsule endoscopy was performed, and active bleeding was identified shortly after the capsule passed the duodenum. Push enteroscopy was then performed, and a polypoid nodule with central umbilication and red spot was detected in the proximal jejunum Figure . The lesion was biopsied and post-biopsy bleeding developed Figure .Hemostasis was achieved by injection of epinephrine (1:10000 concentration) and application of three hemostatic clips. After endoscopic intervention, the patient’s hemoglobin remained stable, and further transfusions were not required.Due to concerns for malignancy and further bleeding a laparoscopic segmental jejunal resection was performed. Histological examination of the resected specimen confirmed a jejunal submucosal hemangioma with surrounding hemorrhage, an organizing hematoma, and no evidence of dysplasia or malignancy. The hemoglobin remained stable after surgery, and the patient was discharged home. On follow up, her hemoglobin normalized, her fatigue improved, and her melena resolved. Intestinal hemangiomas are rare tumors and they are therefore an uncommon cause of GI bleeding. Unlike arteriovenous malformations, hemangiomas are neoplastic lesions secondary to endothelial hyperplasia . They maThe presentation of intestinal hemangioma is variable, with the most common presenting symptom being GI bleeding and anemia -8. BleedThe diagnosis of intestinal hemangiomas is challenging as no imaging study can provide a definitive diagnosis. Due to their location in the small intestine, they are often not detected during upper endoscopy or colonoscopy. The advent of newer endoscopic techniques such as capsule endoscopy, antegrade and retrograde single and double-balloon enteroscopy, and push enteroscopy have allowed for enhanced detection of these lesions . While tPush enteroscopy, unlike capsule endoscopy, also plays a role in the management of intestinal hemangioma, offering an opportunity for biopsy, hemostatic intervention, and definitive treatment -13. As oThis case was presented as a poster at the American College of Gastroenterology annual meeting .Submucosal hemangiomas are a rare cause of GIB. They can present with melena and severe anemia. As the most common site is mid-jejunum, it is difficult to detect submucosal hemangiomas. In this case, the lesion was detected by video capsule endoscopy and was reached by push enteroscopy. Surgery is usually required for a definitive diagnosis and for definitive treatment.


Results showed that low N concentrations (5‐ and 20‐mM N) promoted mean final germination proportion of all eight species by 4.4% and 6.4%, but high concentrations (40 mM N) had no effect. The mean germination rate was decreased 2.1% and 5.1% by higher N concentration (20‐ and 40‐mM N) levels, but germination start time showed the opposite trend, delayed by 0.7, 0.9, and 1.8 d for the 10, 20, and 40 mM N treatments. Final germination proportion, mean germination rate, and germination start time were significantly different among species in response to N concentration treatments. The final germination proportion of Allium tenuissimum and Chenopodium glaucum were suppressed by increased N concentration, whereas it increased for Potentilla bifurca, Plantago asiatica, and Setaria viridis. Our findings provide novel insights into N deposition‐induced species loss based on seed germination factors in semi‐arid grassland communities.Seed germination plays an important role in mediating plant species composition of grassland communities under nitrogen (N) enrichment. Shifts of plant community structure with N‐enhanced deposition in terrestrial ecosystems have occurred globally. Despite numerous studies about the effects of enhanced N deposition on mature plant communities, few studies have focused on seed germination. Using a laboratory experiment, we report the effects of five N concentrations, including 0, 5, 10, 20, and 40 mM N (NH Low N concentrations promoted mean final germination proportion of all species. The responses of eight species to N addition were differences. Mean germination rate was lower at higher N concentration levels. The long‐term mean annual precipitation is 385.5 mm, with ~ 90% distributed May–October. The mean annual temperature is 2.1°C, with a range from −17.5°C in January to 18.9°C in July. According to FAO classification, the soil type is Haplic Calcisols, with an average of 16.95% clay, 20.30% silt, and 62.75% sand. Four perennial forbs (2.24NO3). Each N treatment level had 3 replicates of 50 seeds. NH4NO3 is a common source of N in grassland experiments. The emergence of the radicle was the criterion for germination , germination rate (GR), and germination start (GS).The FGP is the proportion of sown seeds that germinated  and n is the number of seeds used in an experiment  between seed sowing and the start of germination  was used to determine the relationship between pairs of FGP, GR, and GS. All statistical analyses were performed using the R statistical software environment .Two‐way analysis of variance (ANOVA) was used to determine the statistical significance of N treatment, species, and their interactions on FGP, GR, and GS. The Duncan's post hoc tests were used for multiple comparisons when a significant effect was detected. One‐way ANOVA was used for multi‐comparisons of the effect of N concentration on FGP, GR, and GS of each species, followed by Duncan's post hoc tests. A linear model (3A. frigida had the highest GP (95.3) and GR (28.1). C. glaucum had the earliest GT. P. bifurca had the lowest FGP (16.1) and GR (1.9), and the latest GT (5.3).Two‐way ANOVA showed that both N addition and species had a significant effect on final germination (FGP), germination rate (GR), and starting time (GT). Moreover, there was an interaction effect of N and species on FGP, GR, and GT Table . A. frig3.1p < .05, Figure p < .05; Figure A. tenuissimum, P. bifurca, P. asiatica, and C. glaucum. Compared to the control, FGP of A. tenuissimum was suppressed by 28.7% under the N4 treatment, and FGP of P. bifurca was enhanced by 17.3%, 19.3%, and 14.6% under the N1, N2, and N3 treatments, respectively. FGP of P. asiatica was enhanced by 26.0%, 16.7%, 26.0%, and 26.0% under the N1, N2, N3, and N4 treatments, respectively, and FGP of C. glaucum was suppressed by 22.0% under the N4 treatment , and annuals and biennials (AB) functional groups  and two AB (P. asiatica and S. viridis). Moreover, higher nitrogen significantly delayed the mean GS of all species, as well as for the PF and AB functional groups. Water uptake is a fundamental requirement for the initiation and completion of seed germination . Mengzhou Liu: Formal analysis ; Methodology . Xudong Huanag: Formal analysis ; Funding acquisition (lead). Wei Hu: Funding acquisition (supporting). Ning Qiao: Investigation (supporting). Hongquan Song: Investigation . Bing Zhang: Conceptualization ; Data curation ; Formal analysis ; Investigation . Rui Zhang: Funding acquisition (supporting). Zhongling Yang: Data curation ; Funding acquisition (supporting). Yinzhan Liu: Conceptualization ; Investigation . Yuan Miao: Data curation . Shijie Han: Formal analysis . Dong Wang: Conceptualization (lead); Data curation (supporting); Formal analysis (supporting); Funding acquisition (supporting); Investigation (supporting); Methodology (supporting); Project administration (supporting).


Efficacious HIV-1 vaccination requires elicitation of long-lived antibody responses. However, our understanding of how different vaccine types elicit durable antibody responses is lacking. To assess the impact of vaccine type on antibody responses, we measured IgG isotypes against four consensus HIV antigens from 2 weeks to 10 years post HIV-1 vaccination and used mixed effects models to estimate half-life of responses in four human clinical trials. Compared to protein-boosted regimens, half-lives of gp120-specific antibodies were longer but peak magnitudes were lower in Modified Vaccinia Ankara (MVA)-boosted regimens. Furthermore, gp120-specific B cell transcriptomics from MVA-boosted and protein-boosted vaccines revealed a distinct signature at a peak (2 weeks after last vaccination) including CD19, CD40, and FCRL2-5 activation along with increased B cell receptor signaling. Additional analysis revealed contributions of RIG-I-like receptor pathway and genes such as SMAD5 and IL-32 to antibody durability. Thus, this study provides novel insights into vaccine induced antibody durability and B-cell receptor signaling. In the case of experimental HIV vaccines, efficacy is correlated with Envelope (Env)-specific antibody responses4. In particular, Env IgG3 titers that mediate antibody-dependent cellular phagocytosis (ADCP) and antibody dependent cellullar cytotoxicity (ADCC) are correlated with decreased risk of infection6. However, these responses decay more than 10-fold 6-months post-vaccination, paralleling the decline in vaccine efficacy7. Estimates of HIV-1 specific IgG half-lives range from 25-213 days, depending on the vaccine regimen and antigen (excluding gp41)9. Interestingly, the Vaccine-Induced HIV Seropositivity/Reactivity (VISP/VISR) in non-infected HIV vaccine recipients is commonly observed for vaccines with a Gag or Env component10, indicating that antibody responses can remain detectable for several weeks to a year post-vaccination. However, positive results on diagnostic tests are qualitative in nature and thus not quantitative in estimating antibody longevity. Hence, in this study, we measured antibody responses in the VISP cohort by a quantitative assay, the Binding Antibody Multiplex Assay (BAMA), across vaccine regimens. Furthermore, we model antigen-specific antibody decay kinetics and immunoglobulin subclasses necessary to inform rational design of HIV-1 vaccines with improved durability.While vaccine-elicited antibody durability has been achieved for licensed vaccines such as yellow fever, measles, smallpox, and Hepatitis B, elicitation of long-lived, functional antibody responses with candidate HIV-1 vaccine regimens remains elusive12. Env-specific antibodies after gp120 protein vaccination correlate with the number of circulating short-lived memory B-cells13; however, in participants who control HIV without treatment, memory B cell responses did not correlate with plasma antibody titers14. Since memory B cells differentiate into plasma cells upon boost and studying the plasma cell compartment in human participants is difficult, memory B cells provide the best window into signaling underlying the generation of antibody responses.Vaccine induced antibodies are secreted by plasmablasts within days of exposure, followed by memory B cells and plasma cells. These B cell dynamics are noticeable in analyses of half-lives using antibody-titers24. Specifically, analysis of early innate PBMC signatures associated with antibody levels after one dose of recombinant HIV gp140 with TLR-4 agonist adjuvant revealed a negative correlation of mitotic cell division modules with antibody production and upregulation of plasma cell surface markers a week after vaccination23. Further, a recent study identified a B cell (CD14−CD20+) gene signature that predicts protection from infection after vaccination with Ad26-prime and gp140-boost in Rhesus Macaques and in RV-14424. Collectively, these results suggest that gene expression profiling, particularly from sorted B cells may unveil important insights into mechanisms of B cell differentiation across vaccine types.Additional insights into drivers of vaccine induced antibody responses from genome-wide transcriptional profiling have revealed gene signatures of B cell differentiation and early innate signaling. Early studies of gene expression showed type I interferon, inflamasome, and complement signaling in peripheral blood mononuclear cells (PBMCs) upon vaccinationIn the present study, we assessed the longevity of HIV-1 Env-specific IgG and IgG3 responses across 10 HIV vaccine regimens and quantified the effects of each vaccine type on durability using both linear and nonlinear mixed effects modeling (see Table 10. IgG or IgG3 in serum were assessed by a Binding Antibody Multiplex Assay (BAMA) for binding to a panel of consensus HIV Env proteins including gp120, gp140, gp41, and the V1V2 region of gp120 at peak (2-4 weeks post-last vaccination) at multiple post-vaccination time points. Vaccine immunogens varied by trial with DNA-MVA (HVTN 094 and 205) utilizing a gp150 immunogen, and Canarypox-protein (HVTN 097) and DNA-protein (HVTN 105) utilizing a gp120 immunogen.Five phase I or phase IIa HVTN trials 077 (DNA/Ad35 or Ad5), 094 (DNA/ HIV62B MVA), 097 (ALVAC/AIDSVAX B/E), 105 (DNA/AIDSVAX B/E), 205 (DNA/HIV62B MVA) were investigated in this study. Broadly, HVTN 094 and HVTN 205 were MVA-boosted trials with or without DNA priming, utilizing DNA expressing VLPs displaying gp160 in the prime and matched MVA-expressing VLPs displaying gp150 in the boost, HVTN 077 was Adenovirus (Ad)-boosted, and HVTN 097 and HVTN 105 were protein-boosted with subtype B MN gp120 and subtype A/E A244 gp120. HVTN 097 was primed with canarypox containing clade E 92TH023 gp120 while 105 was primed with DNA containing clade C ZM96 gp140. HVTN 094-T3 and 105-T2 were each primed twice with DNA followed by two MVA or protein boosts, respectively  window of the model.Robust responses against gp140 were elicited in both MVA  and protein-boosted trials . For gp140 and gp120 IgG as well as gp140 IgG3 antibody responses, MVA-boosted trials  displayed longer half lives than protein-boosted trials   baseline Net MFI at peak and 6 month timepoints. Post vaccination data points that were less than (pre-vaccination) baseline were not included. Only viral-boosted trials HVTN 077 (Ad5), HVTN 094 (MVA), and HVTN 205 (MVA) had sufficient post-vaccination data to be modeled. Newer trials  have not yet sampled for a sufficient period post-vaccination to observe long-lived responses, and other trials did not have sufficient participants with maintained antibody responses. DNA/MVA vaccination in HVTN 205 resulted in an asymptote between 103 Fig. . Uniquel103 Fig. , indicatnge Fig.  for MVA nge Fig. , panel DWe next investigated molecular signatures of durable responses in 26) was used with and without addition of FCRL genes to evaluate the role of FCRL genes in the regulation of BCR signaling. The FCRL genes were incorporated in BCR signaling pathways by mining primary literature28. Boolean Omics Network Invariant-Time Analysis (BONITA), which performs discrete-state modeling identified BCR pathway with FCRL genes to be significant at p 29 between MVA and protein boosted groups. PTPN6 and PTPN11 inhibit BCR signaling and were identified to have low impact on regulation of BCR pathway by BONITA irrespective of FCRL genes. The regulation identified by BONITA suggests that FCRL2 and FCRL3, which have very high impact scores, are required for activation of PTPN11 and PTPN6 while LYN can be activated by either Fig. Discrete dynamic modeling was employed to better understand changes in BCR signaling and how they relate to changes in FCRL genes. A network of BCR signaling , in agreement with previous vaccine studies33. IL32, shown to act as an immunosuppressant during HIV infection34, was accordingly identified as negatively correlated in both MVA- and protein-boosted trials  genes in B cells40. Taken together, this analysis shows that the durability of antibody responses is induced by a coherent transcriptional signature supported by previous studies of B cell gene expression.An association of B cell gene expression at peak antibody magnitude with antibody half life suggests potential molecular mechanisms responsible for long-lived antibody responses. This association could be dependent on the vaccine type used in each trial, and thus MVA- and protein-boosted trials were analyzed separately (see methods). Twenty-four genes were significantly associated with half-life in MVA  and in protein-boosted trials (HVTN 105) . These genes included tetraspanins (SNX13 and TSPAN33) which are involved in several signal transduction events required for B cell activationials Fig . Additio18. Genes highly expressed after MVA-boosted vaccination  were enriched in genes downregulated after LAIV compared to pre-vaccination. Further, genes highly expressed in durable vs transient subjects in protein-boosted 105-T2 (DDPP) were also enriched for genes downregulated after LAIV. Further, a signature of many immunoglobulin genes, TNFSF13B, and FCRL5 was observed seven days after vaccination with TIV or diphtheria toxoid22 as well as in protein-boosted HIV vaccines in our study. Thus, protein-boosted vaccination induces genes regulating BCR signaling.To compare gene signatures identified in this study with previous vaccine signatures we investigated influenza and diphtheria toxoid vaccine response. Comparison between live attenuated (LAIV) or protein (TIV) influenza vaccination revealed that LAIV produced lower magnitudes of HA-specific antibodies and gene expression at 1 week than TIV24 derived two gene signatures of HIV vaccine efficacy, one of 53 genes and the other of 200 genes from B cells following Ad26-gp140 vaccination in non-human primates. These signatures were validated in PBMCs from RV-144. Both signatures were enriched in genes with higher expression upon MVA boost than protein boost , which results in responses of the size of those in protein-boosted trials but with extended half lives in multiple years and decades.This study describes differences in half life of antibody response between MVA-boosted and protein-boosted HIV vaccinations. MVA-boosted vaccinations  lead to longer half life with lower-magnitude of antibody responses than protein-boosted HIV vaccines . Nevertheless, protein-boosted vaccines elicited a greater time-averaged mean antibody response to gp120. Promisingly, MVA-boosted vaccines led to half lives longer than those for immunoglobulin molecules53. When gp41 is included in the boost (i.e. gp150 boost vs gp120 boost), gp41 responses were as robust as gp140 responses, with increased response longevity vs gp120 responses. High epitope similarity to gut bacteria may contribute to rapid, large gp41 responses and persistence of gp41-specific antibodies in early HIV infection and HIV vaccination55. Inconclusivity of this study with regard to V1V2 along with putative efficacy of V2-specific antibodies calls for a higher powered study of V2-specific antibody responses57, including ongoing efficacy trials.Antibody responses observed here and in primary trials vary considerably by antigen-specificity. We found responses to gp140 are most frequent and robust followed by gp41 (when included in boost) and gp120 with few responses to V1V260. Taken together, this evidence demonstrates that vector and priming strategies, independent of immunogen, play a critical role in determining the magnitude of the gp120-specific antibody response.In the current study, effects of immunogen on vaccine-elicited antibody durability was not explicitly tested because MVA and protein boosted regimens used gp150 and gp120 respectively. Moreover, protein-boosted regimens (HVTN 097 and HVTN 105) included in this study were boosted with AIDSVAX B/E (gp120 immunogen) whereas MVA-boosted regimens (HVTN 094 and HVTN 205) analyzed in this study were boosted with MVA/HIV62 (gp150 immunogen) matched to the DNA prime. Despite the differences in gp120 sequences previous studies suggest a stronger response regardless of the immunogen used. Moreover, viral-prime, protein-boost vaccine trials have demonstrated robust gp120-specific antibody response following both gp120 and gp140 boost61. Despite the caveats of the HVTN 910 (VISP/VISR) cohort , we have added further evidence that long-lived vaccine-induced antibody responses are achievable with current HIV vaccine regimens. Moreover, this evidence makes clear that future experimental vaccines studies should investigate persistence of antibody responses and underlying B cell transcriptional programs.The nonlinear model demonstrated that two different viral vectors (vaccinia and adenovirus) can lead to long-lasting antibody responses. Nonetheless, 62, their lifespan is likely imprinted at the time of differentiation12. Long term survival of antibodies has been shown to correlate to memory B cell numbers following meningitis vaccine63. Moreover, gp120-specific antibodies correspond to memory B cells following HIV vaccine13. Therefore, study of gene expression in memory B cells is an attempt to understand responses that might have led to lasting memory B cell and plasma cell responses. Though our study can not differentiate into responses driven by specific B cell types, it demonstrates the state of memory B cells that emerge from germinal centers at the same time as longer-lasting MVA-induced plasma cells. Given the difficulty of sampling lymph nodes in human participants and pre-selection of participants, memory B cell signaling provides the most expedient window into germinal center dynamics.Our study profiled gene expression in memory B cells, which develop upon multiple rounds of vaccinations and whose predecessors, at the minimum, would have contributed to the development of long-lived plasma cells. Although plasma cells develop distinct transcriptional programs from memory B cells67. Markedly high FCRL2, FCRL3, and FCRL5 were identified in the context of Hepatitis C vaccination68. T-bet and FCRL5+ memory B cells correlate with longevity of antigen-specific antibody responses69, however there are conflicting data on longevity and FCRL5+ memory B cells71. We found a weak negative correlation between Tbet and half life in MVA trials  and no other correlations. Finally, discrete state modeling shows that FCRL2 and FCRL3 can independently activate LYN which leads to downstream activation of the BCR signaling pathway27. Thus, the current study provides mechanistic insights into regulation of BCR signaling by FCRL.The current gene expression analysis indicated a clear and strong signature of difference between protein and MVA boosted trials including differences in FCRL genes. FCRL molecules have an excitatory role in marginal zone (B2) B cell signalling74. Unfortunately, dendritic cell-based therapeutic HIV vaccination studies demonstrated decreased immunogenicity with TLR4 activation75. Another adjuvant, MF59, used in HVTN 702 (NCT02968849) with gp120-boost following canarypox prime, increases strength, breadth, and durability of response to Influenza vaccine by activating the early innate response76. Thus, the data shown here calls for careful understanding of how TLR4 agonists affect antibody responses on their own and in the context of MF59.The transcriptomic signature of enhanced durability of MVA-boosted regimens  consisted of RUNX1, RUNX2, and TLR4 as well as higher levels of Toll-like Receptor and IL-1R signaling. Thus, use of TLR4 agonists could increase antibody durability. Indeed there are safe TLR4 adjuvants that have already been tested in HIV vaccination24 in MVA-boosted trials along with increased gp120 half-lives is encouraging for the development of MVA as a vector for HIV vaccination. Moreover, enrichment of this signature in subjects with durable responses in AIDSVAX B/E protein-boosted 105-T2 suggests that protective HIV vaccine signatures may also be associated with durable responses. The Ehrenberg et al. gene signatures was identified by measuring gene expression at varied time points in human PBMCs (RV144) and in B cells from non-human primate Ad26-gp140 efficacy studies24. However, there are many differences between our study and Ehrenberg et al24, including antigen specificity , timepoints assessed (2 weeks and 6 months vs multiple), and prime (DNA vs Ad26 or canarypox). Nonetheless, MVA-boosted regimens  produced a B cell gene signature associated with protection, but at lower levels of antibody production than the protein-boosted regimens in Ehrenberg et al. Thus, our data present a rationale for continued investigation of MVA+ protein regimens, including testing immunogen to improve magnitude and durability of antibody responses.Enrichment of HIV vaccine signatures associated with protection47, HVTN 09448, HVTN 09749, HVTN 10550, HVTN 20553 were previously acquired and data were used in this analysis with approval from the HVTN77. Analysis of Levey-Jennings charts indicate the comparability of historical controls across trials and time for positive controls including HIVIG and CH58 mAb. All methods were carried out in accordance with relevant guidelines and regulations, and informed consent was obtained from all subjects.Antibody levels measured by binding antibody multiplex assay (BAMA) were either acquired from the primary analysis or obtained de novo for the current study. We tested serum samples from HVTN trial 910 retrospectively with IRB approval . Longitudinal samples from HVTN 105 at month 12 were obtained with informed consent . Primary data for trials HVTN 07780, Con6 gp120, and gp70_B.CaseA_V1V2 at primary and longitudinal timepoints as described previously82. Briefly, samples were incubated with antigen-coated beads in BAMA assay diluent , 1% milk blotto (w/v), 0.05% Tween-20 (v/v) in PBS), washed, then incubated with either biotinylated anti-human IgG (Southern Biotech) or biotinylated anti-human IgG3  followed by washing and incubation with Streptavidin-PE (BD Biosciences). Binding peak magnitude (Median Fluorescence Intensity) was measured in duplicate using a Bio-Plex 200 (Bio-Rad) and averaged to obtain Mean Fluorescence Intensity (MFI). Positive controls included titrated HIV positive IgG (HIVIG), CH58 mAb  and Purified IgG3 human myeloma protein (Sigma) coupled beads. All experiments were conducted following Good Clinical Laboratory Practice (GCLP) guidelines with tracking of antigen performance through historical Levey-Jennings control charts.Serum samples were tested for IgG and IgG3 binding Ab responses to gp41, Con S gp140lme4 and statistical properties  computed using emmeans which estimates a reference grid based on the factors in the model then averages over other factors and propagates error so that confidence intervals and p-values can be computed for the factor of concern84. Residuals were well-distributed across time . Covariates such as BMI and sex were not included in the model since systematic bias was not observed in these variables , and insufficient sample size existed to consider covariates in our mixed effects models. Time-averaged means were obtained by dividing the area under the response curve by 540 days as log(magnitude) and Antibody decay was assessed by both linear and nonlinear mixed effects modeling of log-transformed or raw Net MFI values from BAMA respectively. Models used random effects to account for inter-individual variability but did not control for demographic variables. Linear mixed effects models included participants with at least two non-zero  was used during cell labeling to prevent gp120 from binding T cells. On average 350 gp120+ cells were obtained per sample.Participants were selected for B cell bulk RNA-sequencing by matching durable 89. BONITA was additionally used to assess the B Cell Receptor Signaling Pathway from which was taken from Wikipathways26 but originally created in NetSlim25. This pathway was manually converted into the format necessary for use with BONITA29. FCRL genes interact through immunoreceptor tyrosine-based activating motif (ITAM) and immunoreceptor tyrosine-based inhibitory motifs (ITIM) with LYN and PTPN molecules, respectively28. To recapitulate this behavior, activating edges were added from FCRL2-5 to LYN and activating edges were added from FCRL1-5 to PTPN6 and PTPN11. Note that PTPN6 and PTPN11 have inhibitory connections to downstream genes; activating them inhibits the rest of the pathway. The resultant networks (with and without FCRL genes) were then subjected to BONITA pathway analysis. Edge sets (.txt) and network files (.graphml) of these BCR networks are now available at https://github.com/Thakar-Lab/BONITA.Genes implicated in durable response were evaluated by measuring association between gene expression at the peak and half-life estimates from linear model by Spearman correlation. The genes with coefficient of variation Supplementary Data Table S1.Supplementary Data Table S2.Supplementary Data Table S3.Supplementary Data Table S4.Supplementary Information.


UBE2O, an E2/E3 hybrid ubiquitin-protein ligase, has been implicated in the regulation of adipogenesis, erythroid differentiation, and tumor proliferation. However, its role in cancer radioresistance remains completely unknown. Here, we uncover that UBE2O interacts and targets Mxi1 for ubiquitination and degradation at the K46 residue. Furthermore, we show that genetical or pharmacological blockade of UBE2O impairs tumor progression and radioresistance in lung cancer in vitro and in vivo, and these effects can be restored by Mxi1 inhibition. Moreover, we demonstrate that UBE2O is overexpressed and negatively correlated with Mxi1 protein levels in lung cancer tissues. Collectively, our work reveals that UBE2O facilitates tumorigenesis and radioresistance by promoting Mxi1 ubiquitination and degradation, suggesting that UBE2O is an attractive radiosensitization target for the treatment of lung cancer. Lung cancer is the deadliest malignancy worldwide, of which non-small cell lung cancer (NSCLC) represents ~85% , 2. LungThe ubiquitin-proteasome system (UPS) mediates more than 80% of protein degradation in eukaryotes, which involves ubiquitin, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2s), ubiquitin ligase (E3s), and proteasome . Accumul−/− knockout mice are prone to form squamous cell carcinoma and malignant lymphoma [MAX interactor 1 (Mxi1) is located in the cancer hotspot region 10q24-q25 of human chromosomes . Mxi1 calymphoma , indicatlymphoma . This isIn this study, we first reveal that UBE2O is physically associated with Mxi1 in vitro and in vivo. We then show that UBE2O targets Mxi1 for ubiquitination and degradation at the K46 residue. Functionally, genetical or pharmacological inhibition of UBE2O significantly impairs lung cancer progression and radioresistance in vitro and in vivo. Clinically, UBE2O is remarkedly upregulated and negatively correlated with Mxi1 downregulation in lung cancer tissues.2. All plasmids were subcloned into entry vector and then transferred to destination vector with indicated Myc, HA, SFB, or GST tag for the expression using Gateway Technology (Invitrogen). K46R mutation was generated by the KOD Hot Start DNA Polymerase (Novagen) and validated by DNA sequencing.The human bronchial epithelioid cell line HBE, HEK293T, SMCC-7721, HeLa, and all human lung cancer cell lines including A549, H1299, H292, HCC827, and H1975 cells were purchased from American Type Culture Collection and cultured in RPMI1640 or DMEM with 10% FBS in incubator at 37 °C with 5% COAnti-UBE2O antibody (GTX119315) for immunohistochemical (IHC) analysis was purchased from GeneTex. Anti-UBE2O antibody (A301-873A) for immunoblotting was purchased from Bethyl Laboratories. Anti-Mxi1 antibody (HPA035319), anti-Flag antibody (F1804), and Cycloheximide (01810) were purchased from Sigma-Aldrich. Anti-Myc (sc-40) and anti-Mxi1 (sc-1042) antibodies were obtained from Santa Cruz Biotechnology. Anti-GAPDH (60004-1-Ig) antibody was obtained from Proteintech. Anti-HA (#3724) and anti-GST (#2624) antibodies were obtained from Cell Signaling Technology. Anti-ATM (A19650) and anti-phospho-ATM-Ser1981 (AP0008) antibodies were obtained from ABclonal. The proteasome inhibitor MG132 (474790) was purchased from Millipore. Arsenic trioxide (ATO) was a clinically available drug which was obtained from Beijing ShuangLu Pharmaceutical Co., Ltd., China .Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection. After 48 h of transfection, cells were collected and analyzed by western blotting. The siRNAs ON-TARGETplus SMARTpool for Mxi1 were purchased from Dharmacon. The siRNAs targeting UBE2O and β-Trcp were as follows:si-UBE2O#1: 5-GGUUGUAGAGUUGAAAGUUTT-3;si-UBE2O#2: 5-CCACCCAGUGUGAAACCAATT-3;si-β-Trcp: 5-AAGUGGAAUUUGUGGAACAUC-3 , 21.This assay was carried out as described previously , 23. In Cells were lysed in NETN buffer , and then cell lysates were separated by SDS-PAGE. For the exogenous co-immunoprecipitation, cell extracts were incubated with Myc beads (Santa Cruz Biotechnology), S beads (Novagen), or HA-beads (Millipore) overnight. For the endogenous binding, cell lysates were incubated with protein A/G agarose (Santa Cruz Biotechnology) plus IgG or anti-UBE2O/Mxi1 antibody overnight. After being centrifuged and washed with NETN buffer five times, the precipitates were analyzed by immunoblotting.The indicated plasmids and siRNAs were transfected into cells and proteasome inhibitor MG132 was added for 4 h before harvested. Cell lysates were incubated with S protein beads for co-immunoprecipitation, and ubiquitinated Mxi1 was detected by immunoblotting with anti-HA antibody.SFB-Mxi1, Myc-UBE2O, and HA-ubiquitin plasmids were co-transfected in HEK293T cells for 24 h. Cells were then harvested and lysed with NETN buffer. SFB-Mxi1 protein was enriched by S beads co-immunoprecipitation. The ubiquitination-modified lysine (K) sites were identified by mass spectrometry (MS) analysis.This method was carried out as previously described –25. In bsh-UBE2O#1: 5-CGATGATTCCTATGGCTTCTA-3;sh-UBE2O#2: 5-CGGGTCTCTTCTTCGATGATT-3 .The Cell-Light EdU Apollo567 in vitro kit was purchased from RIBOBIO, and the EdU assay was conducted as per the instruction manual. Briefly, cells transfected with indicated siRNAs or treated with ATO were suspended and seeded in a 96-well plate. After 2 h incubation with 50 μM EdU, cells were washed and fixed with 4 % formaldehyde for 30 min. EdU-Apollo was added for 30 min and cells were treated with 0.5% Triton × 100/PBS for 10 min. Cells were counterstained with DAPI and then detected by fluorescent microscope.This assay was performed as described previously . Cells tCells were plated in six-well plates for 24 h and incubated with CFSE (carboxy fluorescein succinimidyl ester) for 10 min. The CFSE labeling was stopped by the treatment with 40% cold bovine serum. Flow cytometry analysis was used to measure cell proliferation.Cells were plated in six-well plates and exposed to irradiation with indicated doses. After being cultured for 14 days, cell clones were fixed with 4% formaldehyde and treated with crystal violet solution for 30 min. Colonies with more than 50 cells were evaluated.The reagent kit (4250-050-K) for neutral comet assay was purchased from Trevigen, and this assay was carried out as per manufacturer’s instructions. Briefly, 4 h after irradiation, cells were collected and then combined with molten LMA agarose and immediately pipetted onto a slide. Subsequently, the slides were immersed and placed in 1× Neutral Electrophoresis Buffer at 21 V for 45 min and then immersed in DNA precipitation solution for 30 min. Samples were stained with SYBR for 10 min and viewed by a fluorescent microscope. The olive tail moment of at least 100 cells was calculated.Cells were transfected with indicated siRNAs or treated with ATO and then irradiated. After 4 h of irradiation, cells were fixed with paraformaldehyde and then blocked with 5% bovine serum albumin. After being incubated with Rad51 antibody and secondary antibody, samples were washed with PBS buffer for three times. Rad51 foci was detected under a laser confocal microscope. Cells with more than 10 foci were considered positive cells.6 sh-Control, sh-UBE2O#1 or sh-UBE2O#2 H1299 cells. For irradiation treatment, mice were irradiated with 10 Gy when the tumor volume reached 100  mm3. For ATO treatment, ATO  were administered to mice every day when the tumor volume reached 130 mm3. Tumor sizes and weights were monitored every 2 or 3 days.Experiments in xenograft mouse models were approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. This assay was carried out as previously described . BrieflyThe lung adenocarcinoma tissue microarray, which contained 82 paired carcinoma tissues and adjacent tissues, was purchased from Shanghai Outdo Biotech . IHC analysis was performed as previously described , 27, 28.t test. Correlations of UBE2O and Mxi1 expression in tumor tissues was analyzed using the Pearson chi-square test. Overall survival analysis was evaluated by the Kaplan–Meier method. P value of <0.05 was considered significant.Each experiment was performed at least three times independently, and the data were shown as mean ± SD unless stated otherwise. The statistical significance between two independent groups was analyzed by unpaired two-tailed Student’s To better understand how Mxi1 is controlled at the posttranslational level, we previously conducted a tandem affinity purification/MS analysis to identify Mxi1-binding proteins . Of themGiven that UBE2O is an E2/E3 hybrid ubiquitin-protein ligase and has been reported to mediate ubiquitination of several proteins, we therefore speculated that Mxi1 might be a ubiquitination substrate of UBE2O. As shown in Fig. To identify the potential ubiquitination site of Mxi1 by UBE2O, we performed a MS analysis and found the lysine 46 (K46) site in Mxi1 had a di-glycine modification Fig. , indicatOur previous study has clearly shown that Mxi1 is downregulated and inhibits lung cancer cell proliferation . As mentWe have previously found that overexpression of Mxi1 enhanced the radiosensitivity in lung cancer cells . Since UThe biological functions showed that UBE2O plays oncogenic roles in lung cancer and is expected to be an attractive target for anticancer and radiosensitization therapeutics. It has been reported that ATO can inhibit UBE2O activity by crosslinking adjacent cysteines in its catalytic domain . TherefoGiven that UBE2O depletion enhanced the radiosensitivity of lung cancer, we therefore wondered whether pharmacological inhibition of UBE2O with ATO had a similar effect. To verify our hypothesis, we performed the neutral comet and Rad51 foci experiments and found that ATO treatment significantly increased the length and DNA content in the comet tails as well as impaired the formation of Rad51 foci Fig. . In agreSince Mxi1 is a ubiquitination substrate of UBE2O, we determined whether Mxi1 is required for the functions of UBE2O in lung cancer. H1299 cells were transfected with siRNAs targeting UBE2O, Mxi1 or both Fig. . As seenP < 0.05). Importantly, we analyzed the association between UBE2O and Mxi1 expression in the paired lung adenocarcinoma tissues and found that there was a negative correlation between UBE2O and Mxi1 protein levels (P < 0.001)  Fig. . Taken tThe downregulation of Mxi1 expression has been found in human lung cancer , but theA growing body of evidence has shown that Mxi1 acts as an antagonist of proto-oncogene c-Myc through competing for Max and repressing the transcriptional activity of c-Myc . We prevUBE2O has been reported to play essential roles in cell proliferation and apoptosis, cellular clock function, and metabolic homeostasis , 31–33. In summary, our work identifies UBE2O as a novel E3 ubiquitin ligase targeting Mxi1 for ubiquitination and destruction, leading to tumorigenesis and radioresistance in vitro and in vivo, as proposed in Fig. Supplemental MaterialSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4