Introduction: Heart transplant (HT) survival has barely improved in the last decades, which is unsatisfactory for many HT recipients. The development of anti-human leukocyte antigen (anti-HLA) antibodies in HT patients is associated with a cardiac allograft dysfunction. The mechanisms leading to this damage are unclear. The Multimodality Evaluation Of Antibody-Mediated Injury In Heart Transplantation (LEONE-HT) study aimed to thoroughly describe the damage inflicted on the myocardium by anti-HLA antibodies. Methods and analysis: The LEONE-HT study is a cohort study with a cross-sectional approach in which HT patients with positive anti-HLA antibodies are compared with coetaneous HT patients with negative anti-HLA antibodies. All patients will undergo a state-of-the-art multimodal assessment, including imaging techniques, coronary anatomy and physiology evaluations and histological and immunological analyses. The individual and combined primary outcomes of structural graft injuries and longitudinal secondary outcomes are to be compared between the exposed and non-exposed groups with univariate and multivariable descriptive analyses. Ethics and dissemination: The LEONE-HT study is carried out in accordance with the principles set out in the Declaration of Helsinki and the International Conference on Harmonization guidelines for good clinical practice and following national laws and regulations. The study design, objectives and participant centers have been communicated to clinicaltrials.gov (NCT05184426). The LEONE-HT study counts on the support of patient associations to disseminate the objectives and results of the research. This study was funded by the Spanish Ministry of Science and Innovation and the Spanish Society of Cardiology.  In receThe management of patients with anti-HLA antibodies is not well-defined due to the lack of solid evidence, with no reliable data about “which antibodies are of clinical relevance, which patients to treat, and which treatments are most effective or safe” . Thus, aThis is why a detailed description of antibody-mediated chronic injuries is a necessary first step to lead us to develop evidence-based diagnostic and care protocols. An antibody-mediated injury is produced through the activation of the complement system, which leads to leukocyte migration, increased phagocytosis and a T cell-mediated immune response. The rapamycin pathway promotes cytoskeletal remodeling and immune cell proliferation and migration. This immune response finally leads to allograft vasculopathy and a late allograft failure. The time from the antibody detection to myocardial damage varies between patients from a few months to several years .The vascular wall is an interface between the graft and the immune system of the recipient. The evidence suggests that antibody-mediated injuries are predominantly related to vascular damage . In kidnConversely, the diagnosis of antibody-mediated injuries in HT relies solely on optic microscopy (OM) and immunohistochemistry (IHC) techniques  because In this study, we aimed to describe the tissue damage inflicted on the myocardium by anti-HLA antibodies in heart transplant patients through a multimodal evaluation, including advanced imaging techniques, tissue and immunological analyses and coronary anatomical and physiological assessments. Endothelial cell vacuolization, subendothelial widening and duplicated basal membranes .An impaired myocardial perfusion evaluated with cardiac magnetic resonance (CMR)-based quantitative perfusion sequences and with invasive parameters (a pressure guidewire) of microvascular damage (coronary flow reserve and an index of microvascular resistance) .Increased serum and plasmatic markers of fibrosis: fibroblast growth factor 23 (FGF-23), C-terminal propeptide of procollagen type (PICP) and collagen type 1 C-terminal telopeptide (CITP) ,15.Increased T2 recovery times in CMR T2 mapping sequences due to intracellular edemas and increased native T1 recovery times and calculated extracellular volume as a sign of myocardial fibrosis .Subclinical parameters of a myocardial dysfunction  .Signs of coronary allograft vasculopathy in patients with long-term exposure, including a reduced fractional flow reserve (guidewire pressure) and increased intimal thickness (intravascular ultrasound (IVUS)) .Diastolic and systolic dysfunctions in patients with an established chronic rejection.Graft injuries related to a chronic exposure to anti-HLA antibodies debuts with microvascular damage and an impaired myocardial perfusion, leading to inflammation, fibrosis and, ultimately, an impaired myocardial function. This microvascular and myocardial damage may be identified early with TEM and with different diagnostic techniques commonly employed in daily clinical practice. The severity of the expected findings will progress depending on the time from the HT and will be more severe in those patients with positive anti-HLA antibodies. Specifically, we expect to find : EndothePrimary objective: To determine whether, in HT patients, a structural graft injury is modified or accelerated in patients exposed to anti-HLA antibodies compared with non-exposed contemporary HT patients through a thorough evaluation with TEM, OM and IHC techniques.○An assessment of the predictive performance of different diagnostic tools  to detect tissue damage using TEM, OM and IHC techniques as the combined reference standard. ○An estimation of the risk of adverse outcomes as a function of time from anti-HLA antibody positivity.Secondary objectives:To evaluate these hypotheses, we propose the following study objectives: Our study base is a large dynamic cohort of HT patients assembled in the region of Madrid, Spain. All patients have received an HT and continue their follow-up in one of the three participating centers: University Hospital 12 de Octubre; University Hospital Gregorio Marañón; and University Hospital Puerta de Hierro. The local protocols for the follow-up and treatment of the HT patients are equivalent for the three participating centers. For every HT patient in whom anti-HLA antibodies are detected at or since the start of the study (exposed), we will select a comparator (non-exposed) with negative anti-HLA antibodies, a normal functioning graft and whose HT date is close to that of the exposed patient .A confirmatory test for every patient with positive anti-HLA antibodies will be performed at least three months after the first determination and before the multimodal assessment. A regular anti-HLA antibody determination is performed as part of the local follow-up protocols for HT patients to confirm the patient allocation before evaluating the outcomes for the secondary endpoint. All patients will be appropriately informed about the risks of the scheduled test. For the selection of non-exposed patients, we will prioritize those patients who had a routine assessment planned within the local follow-up protocol .With the inclusion of a contemporary case of an HT comparator patient (with negative anti-HLA antibodies), we will reduce the possibility of a bias induced by shorter or longer follow-up periods that may under- or over-estimate the effect of anti-HLA antibodies in myocardial injuries. The inclusion and exclusion criteria are defined in For our primary objective, we estimated the required sample size based on previous TEM findings for kidney transplants . To findFor the secondary objectives, we obtained the estimation based on the previous data of intimal thickness assessments with IVUS: a change of intimal proliferation > 0.5 mm from the baseline to one year after HT was deemed a surrogate marker of allograft vasculopathy . To findWe expect that, in 10% of the exposed patients, we won’t be able to detect any tissue damage with the current diagnostic tools. Assuming a 10% dropout rate, we plan to recruit 45 patients per group between the 3 participant hospitals.The recruitment of non-exposed patients may be hampered by difficulties in finding a coetaneous patient for every exposed patient. A contingency plan has been established with an agreement with three additional HT centers in Spain that would start recruiting patients in the case of difficulties in achieving the estimated sample size. We designed a cross-sectional approach to address the primary objective of this project. All exposed and non-exposed participants will undergo a multimodal evaluation that consist of an endomyocardial biopsy and analyses with OM, TEM and IHC as well as transthoracic echocardiography and CMR, right-heart catheterization, coronarography, IVUS, a pressure guidewire assessment, an immunological study and the determination of the serum markers of fibrosis . ad hoc protocol. The Spanish National Center for Cardiovascular Research acts as the imaging core lab, where every included patient is transferred for the imaging acquisition and further analysis. A multidisciplinary team consisting of pathologists, interventional cardiologists, cardiac imaging experts, immunologists and biologists of the host institution  will analyzed all performed tests ; linear C4d staining (%) (IHC); and endothelial edema and vacuolization (semi-quantitative score), subendothelial widening (pm) and basal membrane duplication (TEM). ○2) (OM).Microvascular damage assessment: Index of microcirculatory resistance and coronary flow reserve (pressure guidewire), a CMR-based quantitative perfusion assessment (mL/min/100 g of tissue) and the capillary density (capillaries/mm○CMR T2 mapping (ms) to detect intracellular edemas .○CMR T1 mapping (ms) and CMR extracellular volume quantification (%) as well as TTE-based global longitudinal strain (%) to identify myocardial remodeling and fibrosis.○Fibroblast growth factor 23 (R.U./mL), procollagen type 1 carboxy-terminal propeptide (µg/L) and collagen type 1 C-terminal telopeptide (µg/L) as the serum markers of fibrosis and remodeling.○FFR (coronary physiology) and IVUS-based intimal thickness (mm) as early markers of coronary allograft vasculopathy. ○Rate of antibody-mediated rejection, anti-HLA antibody seroconversion, heart failure and death. For the secondary objectives: The study data are collected and managed using REDCap electronic data capture tools hosted at the Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12). REDCap (Research Electronic Data Capture) is a secure, web-based software platform designed to support data capture for research studies ,21. A clinical research coordinator (CRC) conducts the data management at the host institution. The CRC is responsible for reviewing the eCRFs and ensuring compliance with the registration protocol. Of the sample, 10% will be randomly selected for the data verification. The individual and combined primary outcomes of the structural graft injury and longitudinal secondary outcomes will be compared between the exposed and non-exposed groups with univariate and multivariable descriptive analyses. The predictive performance of CMR, TTE, coronary physiology parameters and IVUS will be assessed with univariate and multivariable modelling. A parsimonious approach will be implemented to propose a standard assessment protocol, including a predictive score based on the study results. All statistical analyses will be obtained using dedicated software .Despite an improvement in the short-term survival of HT patients, the current life expectancy remains unsatisfactory for most of them. The development of anti-HLA antibodies is associated with a worse survival. As the mechanisms leading to these poorer outcomes are unknown, delving into them can help to improve the quality of life and survival of HT patients. https://www.menudoscorazones.org, accessed on 20 September 2022), the Hospital 12 de Octubre Association of Transplanted Patients and the Corazón y vida Foundation . For the dissemination of this study, we count on the support of several patient associations. HT patients are especially aware of the importance of research in healthcare and we regularly collaborate with patient associations such as the Menudos Corazones Foundation . The local investigator of each participating center will inform the patient about all pertinent aspects of the study, including the favorable written report of the Ethics Committee for Clinical Research. The ethics committee will be also informed of any event that may have affected patient safety or the continuation of the study.The design of this study and the participating institutions were declared to the United States National Library of Medicine  (NCT05184426).The results obtained from this research project will continue to be communicated to the scientific community through the participation of the researchers in national and international conferences. Other publications regarding the main results are expected, as are several publications regarding the technical particularities of the diagnostic techniques employed.Developing anti-HLA antibodies is associated with a worse prognosis in heart transplant recipients. The mechanism by which these antibodies lead to a graft dysfunction has not been clearly defined. The LEONE-HT study aims to carry out a detailed description of the damage that these antibodies inflict on the transplanted heart to improve knowledge about this phenomenon and thus be able to contribute to the development of diagnostic protocols and the description of possible therapeutic targets.


The Cell Death and Survival section of the Frontiers in Cell and Developmental Biology was launched in year 2013 to serve as an open-access venue for outstanding research and commentary in the field. This section has witnessed a dramatic increase in manuscript submission and publication of high-quality original research manuscripts and reviews addressing important Research Topic in cell death and survival in the last year. The published papers in our section cover a broad swath of topics in the area cell death and survival, from fundamental molecular and cellular biology to clinical application of cell death and survival principles. To give the broad readership of this journal an overview of the activities in the section, a Research Topic called Editor’s Pick for section editors to highlight significant contributions to the field provides a brief introduction to some of the cutting edge research and influential reviews in our section. Due to a limit on the number of papers allowed for Editor’s pick, many high-quality research manuscripts and insightful reviews published in our section deserve as much attention. We highlight the following publications to showcase the breadth and depth of these scholarly contributions to our section. Moreover, we thank all contributors to our section and know that the growth in Frontiers in Cell and Developmental Biology has been driven by these important contributions.Naim and Kaufmann). Szczesniak et al. used TurboID-mediated proximity labeling to probe the Bok interactome. They reported that Bok interacts with, and is proximal to proteins involved in various cellular functions such as mitochondrial fission, endoplasmic reticulum-plasma membrane junctions, and surprisingly, Mcl-1. The physical and functional interactions between Bok and Mcl-1 were further verified. Importantly, these authors showed that Bok interactome is distinct from those of Mcl-1 and Bak. This report has provided important functional insights into Bok in apoptotic and non-apoptotic intracellular processes and will instigate additional research into this important area.The function of the Bcl-2 family member Bok (Bcl-2 related ovarian killer) remains mysterious. Although Bok was initially classified as a pro-apoptotic protein due to its sequence homology to Bax and Bak, accumulating evidence suggests that Bok is involved in many other cellular functions  was known to be upregulated in colorectal cancer (CRC) but its function in tumorigenesis was unknown . Yang etLei et al.). Zhang et al. investigated the expression and prognostic value of a newly identified immune checkpoint receptor Human endogenous retrovirus-H Long terminal repeat-Associating protein 2 (HHLA2) in 22 types of solid tumors. HHLA2 was reported to be expressed in all 22 tumors at the mRNA level and 12 types of these tumors at the protein level. Furthermore, HHLA2 was found to have the highest transcript levels in kidney clear cell carcinoma (KIRC) and was positively correlated with better survival and high CD8 expression in this group of patients. This study reveals a potential immunotherapeutic target for KIRC treatment.Immune checkpoint inhibitors such as anti-PD-1 and PD-L1 antibodies have revolutionized cancer therapy by overcoming immune exhaustion , delays the premature senescence progression in mouse embryonic fibroblasts. The authors further investigated molecular mechanisms underlying DNMT3L-mediated chromatin surveillance through epigenetic regulation. This study is significant as a new epigenetic reinforcement strategy may be used for overcoming premature senescence in a variety of diseases.Cellular senescence, a stable exit from the cell cycle in response to different stresses and cellular replication, is tightly linked to physiological and pathological processes such as aging and cancer . PrematuXiong et al. studied the molecular network regulating neuronal cell death after HIE and identified a novel long non-coding RNA (lncRNA) Vi4 (TCONS00044054) as a key regulator of neuron survival and apoptosis after HIE. Furthermore, the investigators demonstrated that a regulatory network consisting of Vi4, miR-185-5p, and the target gene Igfbp3 plays an important role in neuronal death and survival after HIE, highlighting potential strategies for therapeutic intervention of HIE.Hypoxic ischemic encephalopathy (HIE) causes severe disability and death in ∼400,000 newborns worldwide each year . Xiong eScutellaria baicalensis have potent antitumor activities  may play roles in gastrointestinal (GI) tract function and be involved in GI cancers and inflammatory bowel diseases . Wang etXia et al. investigated the T cell compartment in Beclin 1/Atg6 conditional knockout mice and found a diminished naïve T cell population, increased effector T cells and MDSCs, and severe colitis in aged mice. Interestingly, the reduced population of naïve T cells was rescued by crossing the conditional Beclin 1/Atg6 KO mice onto a TCR transgenic background, which also led to a normalized population of effector T cells. These data provide support for context-dependent roles of Beclin1/Atg6 in T lymphocyte survival and differentiation.Autophagy plays an essential in regulating T lymphocyte survival and homeostasis . Xia et Hu et al. tested the antitumor activities of intratumorally injected IL-27 using recombinant adeno-associated virus (rAAV)-based delivery. The authors demonstrated that intratumoral injection of rAAV-IL-27 led to strong antitumor activities in several mouse tumor models. The investigators further showed that IL-27 induces infiltration of CD8+ T cells and CXCR3 expression and imparts synergistic antitumor activities with anti-PD-1 administration or adoptively transferred T cells. These interesting preclinical data suggest that intratumoral delivery of IL-27 may be useful alone or in combination with other therapies for cancer treatment.Intratumoral administration of immunotherapies has the potential to maximize immune activating capability and minimize systemic toxicities of immunotherapeutic agents . Hu et aYuan et al. reviewed the pathways between ionizing radiation and ferroptosis and the critical roles of fatty acid metabolism in radiation-induced ferroptosis in tumor cells. Importantly, the authors provided thoughtful perspectives on the implications of the interplay between fatty acid metabolism, ferroptosis and ionizing radiation for future clinical development of novel cancer treatment modalities. Many clinical drugs have known cardiotoxicities due to their ability to induce various forms of cell death in cardiomyocytes. Ma et al. first reviewed the different forms of cell death including apoptosis, autophagy, necrosis, necroptosis, pryoptosis and ferroptosis in cardiomyocytes. These authors further analyzed the underlying mechanisms of cardiomyocyte death induced by three major classes of clinical therapeutics: anti-tumor, anti-diabetic, and anti-viral drugs. This review has offered both an excellent theoretical overview and practical guidance for physicians in therapeutic application of these clinical drugs. In a more targeted review by Zhai et al., the roles of one specific form of cell death, ferroptosis, in cardiomyopathy are discussed in depth. The potential for utilizing the ferroptosis pathway as a diagnostic and therapeutic target for patients suffering from cardiomyopathy was carefully analyzed. T lymphocytes are essential for the antitumor efficacies of immunotherapies. However, primary and acquired resistance to immunotherapies are common and may be at least partially caused by T cell dysfunction in the tumor microenvironment of cancer patients. Zhang et al. provided a comprehensive update on the molecular mechanisms of T cell dysfunction and exhaustion in the tumor microenvironment and potential strategies to overcome these defects. In summary, the above reviews have provided timely information and thoughtful insights in a wide array of topics with high scientific and clinical significance.In addition to the above nine original research papers, we would like to highlight several insightful reviews published in our section. One example focused on ionizing radiation as a major cancer treatment modality, highlighting its ability to induce ferroptosis and alter fatty acid metabolism in tumor cells.


Assessing the naturalistic puff topography and associated nicotine consumption during e-cigarette use is important as such information will not only unveil how these products are being consumed in real-world conditions, but also enable investigators and regulatory bodies to conduct quantitative, accurate, and realistic harmful exposure and nicotine abuse liability risk assessments based on actual e-cigarette use. Conventional approaches cannot accurately, conveniently, and noninvasively determine e-cigarette puff topography in a natural use environment. Thus, novel technology-enabled systems that do not primarily rely on self-report mechanisms or intrusive measurements to monitor e-cigarette product use behaviors are highly desired.This study aimed to explore and demonstrate the feasibility of a novel puff recording electronic nicotine delivery system (PR-ENDS) device for measuring naturalistic puff topography and estimating nicotine consumption during the ad libitum use of products among smokers and vapers.An ancillary data analysis based on a completed parent study was conducted. The parent study was a 1-way randomized controlled open-label puff topography and nicotine pharmacokinetic assessment carried out in 24 healthy adults (12 smokers and 12 vapers). Participants were assigned a randomized product use sequence of a PR-ENDS device within 5 site visits for both controlled and ad libitum product use sessions. Blood samples were obtained for plasma nicotine analysis, and questionnaires were administered at various time points. During the ad libitum use session, puff topography was measured using a Clinical Research Support System (CReSS) device as a benchmark, as well as the PR-ENDS device with a built-in puff recording feature.ad lib (plasma concentration-time curve for 1-hour ad libitum use) substantiated the potential of using this device as a pragmatic, noninvasive, and convenient means for estimating nicotine intake in the human body without blood collection.There were no significant differences in representative puff topography parameters  between the PR-ENDS and CReSS devices at the populational level across different device powers, e-liquid nicotine strengths, and flavors. The nicotine consumption estimated by the PR-ENDS device suggested that this device can be employed as a convenient monitoring tool for estimating nicotine use without measuring e-liquid weight loss between puffs. The linear relationship between nicotine consumption estimated by the PR-ENDS device and the pharmacokinetic parameter AUCThe novel PR-ENDS device was feasible for assessing naturalistic puff topography and estimating nicotine consumption and intake in the human body during ad libitum use. Several key factors can influence users’ puff topography, including device power levels, e-liquid nicotine strengths, and flavors. The study results pave the way for further research in the real-time measurement of naturalistic puff topography and puffing behaviors in the real world. Electronic nicotine delivery systems (ENDSs) or electronic cigarettes (e-cigarettes) have become a global public health concern, with a significant increase in prevalence in the past decade -3. The uSo far, various techniques and strategies have been applied to measure the puff topography of e-cigarette use. The most commonly recognized method is the self-reporting survey -28, in wCompared to the self-reporting survey, video assessment, and puff-sensing devices, e-cigarettes with the a built-in puff recording feature are better positioned for naturalistic puff topography assessment, because (1) users can intuitively use the product with no interference or intervention from investigations and monitoring tools; (2) information about e-cigarette use, such as e-liquid and device power, can be simultaneously obtained in situ and integrated with puff topography to approximate user’s nicotine consumption; (3) users can view their own puffing data and track their own nicotine consumption in real time by connecting puff recording e-cigarettes with personal mobile devices, such as cell phones, via securely paired Bluetooth. This is especially valuable for those who want to gain the awareness to help quit or cut back nicotine use. By combining device-recorded puff data with laboratory nicotine emission results, the inhaled nicotine amount can theoretically be estimated, which otherwise must be done by collecting and analyzing multiple blood samples in a pharmacokinetic assessment. The application of e-cigarettes with a built-in puff recording feature provides a convenient and pragmatic avenue to bypass the nontrivial operations of blood specimen collection and thus makes it viable to directly assess the real-world nicotine use of e-cigarettes at large user population levels. In this report, a novel ENDS device with a built-in mechanism to record puff data , Shenzhen JWEI Electronics Co, Ltd) was investigated. We aimed to explore its feasibility in measuring puff topography during ad libitum use among smokers and vapers. The device’s capability in estimating nicotine consumption was further assessed based on comparison of the results calculated by the PR-ENDS device and blood sample analysis in nicotine pharmacokinetic assessment.This is an ancillary study based on a completed parent study. The parent study was a 1-way randomized controlled open-label puff topography and nicotine pharmacokinetic assessment in 24 healthy adult volunteers who either regularly smoke combustible cigarettes  or regularly vape e-cigarettes . The participants were recruited in the Los Angeles, California metropolitan area in fall 2021. The study protocol and informed consent form (ICF) were reviewed and approved by the Institutional Review Board (IRB) (Advarra IRB). Potential participants were examined for study eligibility during the initial screening step. The individuals were included if they (1) were healthy males or females within the ages of 21 to 65 years; (2) were either current smokers (≥10 per day) of factory-made combustible cigarettes (eCO >10 ppm at screening) for at least 3 continuous months and occasional users of e-cigarettes, but none in the 14 days before the screening visit, or current daily users of e-cigarette devices with an e-liquid nicotine concentration >0 mg/mL (eCO ≤10 ppm at screening) for at least 3 continuous months and occasional users of combustible cigarettes, but none in the 14 days before the screening visit; and (3) had urine cotinine >200 ng/mL at screening.Following the screening step, participants visited the study site 6 times, including 1 visit with their own usual brand combustible cigarettes or e-cigarettes and 5 visits with PR-ENDS devices prefilled with assigned freebase nicotine e-liquids in a randomized product use sequence. Participants were provided with a supply of their assigned PR-ENDS product to use at home before their next visit for familiarization. Each individual visit was separated by a minimum washout period of 24 hours. During visits, each participant had 2 use sessions. In the first controlled use session, participants started by taking 10 puffs, 30 seconds apart, followed by a period of 120 minutes to allow the nicotine plasma concentration to ramp down to baseline. In the second ad libitum use session, participants were allowed to take as many puffs as desired during a period of 60 minutes. Throughout both the controlled use session and ad libitum use session, blood samples were obtained for plasma nicotine analysis, and subjective effect assessment questionnaires were administered at various time points. A schematic flowchart to describe the parent study is shown in The investigated PR-ENDS device is an open refillable ENDS product with a removable 0.8-ohm coil and a 2-mL e-liquid capacity, and with 3 power settings . One unique design feature of the PR-ENDS device is that it can measure puff data  through a built-in mechanism (button-activated puff recording) and via securely connected Bluetooth. The puff duration is measured and recorded based on the time of pressing and holding the power button. The specific scope of the investigated PR-ENDS device includes 2 power settings (high and low), 2 nicotine strengths (12 mg/mL and 3 mg/mL), and 2 flavors (tobacco and menthol). Detailed information about the investigated product groups (groups A-D) is shown in ad lib) was calculated based on the time course of the measured nicotine concentration from 0 min to 60 min.During the 1-hour ad libitum use session, e-liquid weight loss for assessment of nicotine consumption was calculated based on the weight difference before and after use sessions. Blood samples were taken from the study subjects at the following time points: 0 min, 30 min, and 60 min of 1-hour ad libitum use. Nicotine plasma concentrations at those time points were obtained based on nicotine analysis from blood samples. The area under the plasma concentration-time curve for 1-hour ad libitum use . A 1-way or repeated measures ANOVA and Dunnett multiple comparison tests were conducted to assess stratified differences in variables among product use groups (groups A-E). The correlation coefficient (R2) was further calculated to assess the linear relationship between compared variables.Descriptive statistics, including means and SDs, were calculated for each variable. The box plots of targeted variables have been presented in figures. Two-sample This ancillary study is an independent research topic that uses data from the completed parent study conducted in 2021. The parent study protocol and ICF were reviewed and approved by the IRB (Advarra IRB). This ancillary study was also reviewed by the IRB (Advarra IRB) and was granted exemption.The IRB was constituted and operated in accordance with the principles and requirements described in the US Code of Federal Regulations (21 CFR Part 56). The IRB is compliant with the International Council for Harmonisation (ICH) guidelines.The IRB granted exemption for informed consent in the secondary analysis in this ancillary study. In the parent study, all subjects voluntarily accepted to participate in this study and signed the ICF after having fully comprehended the contents of this form and before any study-specific procedures were performed. The identity of the subjects was kept confidential. Each eligible subject was assigned a subject number, which was used on the case report form instead of the subject’s name. Only the deidentified data were available for this ancillary study.In the parent study, if the human subjects completed all 7 study visits, which included 6 testing visits, they were paid a total of US $1800 for participation at the completion of visit 7. The study participation payments were reported to the US Internal Revenue Service (IRS) as income. There was no payment for this ancillary study.The PR-ENDS device has the capability of recording the following 3 representative puff topography parameters: (1) number of puffs, (2) total puff duration (accumulated puffs within the 1-hour ad libitum use), and (3) average puff duration, for each individual user during product use. The puff durations measured by the PR-ENDS device have been proven consistent (with an accuracy within 0.1 second) by testing with preset puff duration parameters on puffing machines. Here, the presented assessment of puff topography provided further evidence for the puff recording feature in the PR-ENDS device.As shown in P=.25, vapers: P=.21) given the large variation in actual puff behavioral data.In the smoker group, the puff data recorded by the PR-ENDS device showed that higher device power  and nicotine strength  yielded lower values for the number of puffs, total puff duration, and average puff duration. Yet, a dissimilar trend was observed for the vaper group in that the power and nicotine strength only had limited effect on puff topography parameters. Such a difference might potentially indicate different puffing behaviors between smokers and vapers ,37. InteIt is essential to validate the observed puff topography parameters measured by the PR-ENDS device. Although the CReSS device is not suited perfectly for analyzing e-cigarette puff topography , it is sAs shown in P values of 2-sample t test comparisons (2) exist between data recorded by the CReSS and PR-ENDS devices , combinPreviously, it has been recognized that nicotine emission in e-cigarette use is strongly impacted by applied device power  and e-liIn order to prove the concept, nicotine consumption during the ad libitum use of a PR-ENDS device prefilled with designated e-liquids (groups A-E) was estimated. Specifically, nicotine consumption was calculated by integrating the PR-ENDS–measured puff topography data and laboratory nicotine emission results based on the following equation:(1)Nicotine consumption = (nicotine emission / testing puff duration) × total puff duration Specifically, nicotine consumption refers to the amount of nicotine consumed by PR-ENDS users during the ad libitum use session. The nicotine emission value  was obtaNicotine consumption was calculated to represent how much nicotine has been consumed during the ad libitum use session. The assumption applied in the calculation is that nicotine consumption is projected as linearly proportional to the puff duration measured by the PR-ENDS device, which means it is not needed to involve puff volume or puff flow rate (puff volume over unit puff duration) into the calculation. This assumption is deemed reasonable, as a previous study  has cleat tests and paired t tests of nicotine consumption between the PR-ENDS and e-liquid weight loss methods showed that no statistically significant differences were present.In order to validate the assumption that nicotine consumption can be estimated based on the puff data recorded by the PR-ENDS device and laboratory aerosol emission results, the nicotine consumption value was alternatively obtained by calculating e-liquid weight loss multiplied by prelabeled nicotine concentrations. A similar calculation of nicotine consumption using e-liquid weight loss has been rationalized in a previous study . As showP<.001) and vapers (P<.001), the nicotine consumption was significantly different among groups A-E. A further assessment with the Dunnett multiple comparisons test (control: group A) showed that device power  and nicotine strength  significantly impacted nicotine consumption . The results showed that for both smokers  relationship between nicotine consumption derived from the PR-ENDS device and AUCad lib was present. Of note, a similar linear relationship was identified between nicotine consumption derived from e-liquid weight loss and AUCad lib. These results, taken together, indicate that it is viable to use puff recording e-cigarettes, such as PR-ENDS devices, to directly assess puff topography, nicotine consumption, and intake in a natural use environment.For clinical validation of nicotine consumption derived from the PR-ENDS device, the plasma nicotine concentration  and AUClculated . By defiDespite the high relevance to nicotine abuse liability and inhalation health risk assessment, conducting noninvasive, accurate, and convenient measurements of e-cigarette puff topography is still challenging . Variousad lib provided further clinical validation of the noninvasive estimation of nicotine intake. Together, these results support the proposal that it is feasible to assess naturalistic puffing behavior and nicotine use with an e-cigarette having built-in puff recording features, such as a PR-ENDS device.The objective of this study was to evaluate the feasibility of a novel PR-ENDS device for measuring naturalistic puff topography and nicotine consumption during the ad libitum use of products. Based on the comparisons of puff numbers and puff durations measured between the PR-ENDS and CReSS (benchmark) devices, it has been clearly demonstrated that there is no significant populational difference in the recorded puff topography parameters between the 2 methods and across different device powers, e-liquid nicotine strengths, and flavors. The puff data recorded by the PR-ENDS device can be used to quantitatively estimate the nicotine consumption during natural use. This is achieved solely by integrating puff data with laboratory emission results in a mathematical model, with no need to physically collect e-liquid weight loss data. The linear relationship between nicotine consumption and the pharmacokinetic parameter AUCt tests of puff data from the CReSS and PR-ENDS devices indicated certain systematic discrepancies at the individual level  was approximately 2.04 seconds, which is 21% shorter compared to 2.59 seconds for e-liquids with a nicotine strength of 3 mg/mL (groups D and E). Similar observations have been reported previously, and it has been shown that the use of lower nicotine strength e-liquids can increase the total puffed e-cigarette aerosol . The aveSecond, the PR-ENDS puff data indicated that the puffing behaviors of vapers are different from those of smokers. For example, vapers tended to have longer puffs than smokers based on the average puff duration . This is consistent with published results , where rThird, nicotine consumption calculated from PR-ENDS puff data indicated that device power and nicotine strength could significantly influence nicotine consumption during ad libitum use, even when users could freely adjust and “self-titrate” their puff behaviors to compensate for the change in nicotine supplies. For example, the overall average nicotine consumption for e-liquids with nicotine strength of 12 mg/mL  was approximately 1.96 mg, which is 188% higher compared to consumption of 0.68 mg for e-liquids with nicotine strength of 3 mg/mL (groups D and E). The overall average nicotine consumption for high device power  was approximately 1.87 mg, which is 131% higher compared to consumption of 0.81 mg for low device power (groups C and D). In other words, higher nicotine strength and device power significantly boosted nicotine consumption within the scope of this study, even when we considered users’ puff compensatory behaviors. Furthermore, the relative impact on nicotine consumption from e-liquid nicotine strength was found to be bigger than device power. Such an observation is important, as it not only sheds light on the necessity of reducing the nicotine addictiveness of tobacco products, including the recently announced proposed rules by the Food and Drug Administration  of estabThe current assessment was limited to 24 participants (12 smokers and 12 vapers). The small sample size caused a relatively large variation in puff measurements and thus hindered any further statistical analyses across different product groups. For example, no significant differences in puff topography parameters were recognized among groups A-E . The 1-hThere are a number of strengths. This is the first study to compare recorded puff topography parameters between an e-cigarette with a built-in puff recording feature and the CReSS device, a benchmark specialized puff-sensing device. Our results make an important contribution to the field of e-cigarette behavior research and provide a new avenue for assessing e-cigarette naturalistic puff topography and further derived analyses, such as nicotine consumption. With puff data readily accessible via a potentially paired smartphone app, we expect that the feasibility of the established PR-ENDS device in this report could inspire more research on the long-term real-time assessments of puff topography and nicotine consumption in real-world conditions, as well as more studies on the impact of empowered product use awareness  on users’ puffing behaviors.In this study, we demonstrated the feasibility of a novel PR-ENDS device for assessing naturalistic puff topography and nicotine consumption during ad libitum use among smokers and vapers. The results proved the viability of using a PR-ENDS device for estimating the nicotine intake in the human body without drawing blood samples. Further, the potential effects of device power, nicotine strength, and e-liquid flavor, as well as differences between smokers and vapers on puff topography/behaviors and nicotine consumption were discussed. Together, the results presented in this study pave the way for future research with a focus on measuring naturalistic puff topography and behaviors in real-time and in real-world settings.


Coronavirus disease-2019 (COVID-19) is a pandemic with a high morbidity rate occurring over recent years. COVID-19 is caused by the severe acute respiratory syndrome causing coronavirus type-2 (SARS-CoV-2). COVID-19 not only challenged mankind but also gave scope to the evolution of various vaccine design technologies. Although these vaccines protected and saved many lives, with the emerging viral strains, some of the strains may pose a threat to the currently existing vaccine design that is primarily based on the wild type spike protein of SARS-CoV-2. To evaluate the risk involved from such mutant viral strains, we performed a systematic in silico amino acid substitution of critical residues in the receptor binding domain (RBD) of the spike protein. Our molecular modeling analysis revealed significant topological changes in the RBD of spike protein suggesting that they could potentially contribute to the loss of antigen specificity for the currently existing therapeutic antibodies/vaccines, thus posing a challenge to the current vaccine strategies that are based on wild type viral spike protein epitopes. The structural deviations discussed in this article should be considered carefully in the future vaccine design. Coronavirus disease-2019 (COVID-19), a contagious viral disease that affects the respiratory tract, is caused by Severe Acute Respiratory Syndrome Coronavirus type-2 (SARS CoV-2) . SARS CoTraditionally, vaccines have been prepared by heat-killing or weakening  of the disease causing microbe. However, agents that resemble the disease causing organism, toxin, or its surface protein have also been used via recombinant DNA technology . DespiteThe mRNA vaccine does not contain the disease causing organism, hence the person receiving an mRNA vaccine neither becomes exposed to the disease -ausing agent through the vaccine, nor can they become infected because of the vaccine components, unlike inactivated or live vaccines, which can have a chance of containing live disease-causing agents and can pose a threat of infection if not prepared properly ,33. In tThe Receptor Binding Domain (RBD) from the S1 subunit of spike protein interacts directly with the hACE-2r. X-ray crystallographic analysis revealed that residues Arg319-Phe541 contribute to the RBD of SARS CoV-2, which has some similar features to the RBD of SARS CoV-1 . It is ihttp://rna.tbi.univie.ac.at/). Once the mutant RNA sequences were generated, their corresponding amino acid sequences were also manually generated separately using the standard codon dictionary. Separate files for mutant amino acid sequences are convenient for copy-pasting them into the SWISS MODEL server (https://swissmodel.expasy.org) in order to generate the mutant protein models.The RBD nucleotide sequence of wild type SARS-CoV-2 spike protein  was takehttp://rna.tbi.univie.ac.at/; accessed on 1 January 2022) in order to calculate the mRNA secondary structure. The secondary structure of the mRNA mutant molecule was downloaded along with the minimum free energy (MFE) values and ensemble diversity (ED) numbers. After analyzing the MFE values it was found that there were variations in the MFE values as well as the ED numbers. To maintain uniformity, the MFE value of each mutant secondary structure was divided by its corresponding ED number to obtain the average MFE (AMFE) values per iteration of the secondary structure ensemble. These AMFE values of each mutant were then compared to that of the wild type to calculate the viral fitness, taking the wild type AMFE as 100%.Each mutant sequence was copy pasted into the RNAfold web server  in order to build the 3D models. For model building, an analysis was made as described previously [y-axis) were then plotted vs. the amino acid positions taken on the x-axis.The mutant protein amino acid sequences as prepared above were submitted to the online SWISS MODEL server  and each of the mutant models of the viral receptor binding domain (RBD) was performed using PyMOL molecular graphics software. Amino acid substitutions in the various mutant models showed either loss/gain of hydrogen bonds or no change in the profile as compared to the wild type model that was used as a reference. Binding affinity values were calculated based on the number of hydrogen bonds between the hACE-2r and RBD for each of the mutant models in this study. This protocol considers the enthalpic contributions of the hydrogen bonds towards the binding affinity thus ignoring the entropic contributions. However, the binding affinity values calculated here are only relative to the wild type RBD-hACE-2r interface with only one amino acid substitution which may not generate significant differences in the relative entropic components of the mutants when compared to the wild type. Furthermore, the molecular surface area calculations for each of the mutant models including the wild type were performed using CCP4 software.Viral fitness in general is defined as the collective viral replication capacity and viral infectivity rates both of which are in vitro and/or in vivo studies. In this in silico study, we considered three factors, such as mRNA stability, number of hydrogen bonds at the RBD-hACE-2r interface and structural deviations in the mutant RBD models generated by the amino acid substitutions as contributors to the successful viral replication and infectivity. Evidently, the wild type virus already created a pandemic thus proving that it has 100% viral fitness. By taking the three factors from wild type, we calculated the relative viral fitness of the mutants that were analyzed in this study.The full length of 600 base pairs RBD mRNA nucleotide sequence that was considered for this study  was diviComparative analysis of the AMFE values revealed three mutant triplets  in particular that have higher values compared to the wild type . The secTypically, the stability of mRNA in the host cytoplasm is directly proportional to its secondary structure; in other words, the AMFE value among several other factors. The AMFE values are sequence dependent and hence they differ for wild type and mutants, suggesting the relative stability of the corresponding mRNA. Additionally, the longevity of mRNA in the host cytoplasm depends on its stability. The longer the mRNA exists in the host cytoplasm, the higher the chances of more rounds of the spike protein translation which in turn helps package and release of the new virions . Hence, the mRNA stability was considered to be one of the important factors in this study. Furthermore, the AMFE values of all the mutants  were useThe mutant RBD-hACE-2r interface hydrogen bond profiles were analyzed to understand any changes in the hydrogen bonding pattern of the mutants compared to the wild type. As shown in The wild type with 13 hydrogen bonds at the interface was taken as 100% fit; accordingly, any increase or decrease in hydrogen bonds at the interface for the mutants was calculated as relatively higher fitness or lower fitness compared to the wild type. Lys417 (K89 in this study) showed an increase in viral fitness only for the K89Y substitution. Gly446 (G118 in this study) showed equal viral fitness with any amino acid substitution at that position. Tyr449 (Y121 in this study), Asn487 (N159 in this study) and Gln493 (Q165 in this study) showed decreased viral fitness with any of the 20 amino acid substitutions. Gly496 (G168 in this study) showed either equal or higher viral fitness compared to the wild type. Especially G168N and G168R showed the highest viral fitness among the 20 amino acid substitutions at that position. While Gln498 (Q170 in this study) and Asn501 (N173 in this study) showed either equal or lesser viral fitness compared to the wild type, Gly502 (G174 in this study) showed either equal or higher viral fitness in comparison to wild type. In particular, the substitutions G174D/H/Q/R/Y showed the highest viral fitness (115.4%) and G174E/N/S/T showed higher viral fitness (107.7%) than wild type but relatively lower fitness than the aforementioned substitutions. In the case of Thr500 (T172 in this study) and Tyr505 (Y177 in this study) all the substitutions showed either equal or lesser viral fitness compared to the wild type. However, the amino acid substitutions T172D and Y177R both showed highest viral fitness values (115.4%) compared to the wild type. Overall, 9 substitutions showed highest viral fitness (115.4%) compared to wild type . As showIn addition to the mRNA stability analysis, we further complimented the study with the three-dimensional structural deviations in the mutant models compared to the wild type model. As shown in


Somatic symptoms with the heterogeneous character that are not fully explained by a medical condition are common in bipolar disorder (BD) which might interfere with the choice of treatment, health care utilization, medical costs as well as functionality.The purpose of this study was to evaluate somatic symptoms in remitted type 1 BD and to examine the association of somatization, functionality, and childhood trauma which is a known mediator of adult somatization.After excluding patients with medical comorbidities, 61 patients diagnosed with BD type-1 according to the Diagnostic and Statistical Manual of Mental Disorders-V (DSM-V) participated in the study. We required at least 8 weeks of remission and confirm it with Hamilton Depression Rating Scale (HDRS) and Young Mania Rating Scale (YMRS). Somatization Scale, Functioning Assessment Short Test (FAST) and Childhood Trauma Questionnaire (CTQ) were administered to the participants.Somatization scores were significantly correlated with CTQ , FAST- total , FAST-financial issues  and FAST-interpersonal relationships  subscale scores while inversely correlated with years in education . When a partial correlation was run to determine the relationship between somatization and functioning whilst controlling for childhood trauma, there was no statistically significant correlation between somatization and functioning (p=.076).Our study suggests childhood trauma may have a major influence on the relation between somatization and functionality in patients with type- 1 BD. When addressing physical symptoms in patients with type-1 BD, an integrated approach including childhood trauma should be considered.No significant relationships.


Yearly average mileage per month was taken from each participant. Results: There were two distinguished clusters: Less-healthy-more-active-low-Z-score (LessHA) (n = 33) and More-healthy-less-active-high-Z-score (MoreHLA) (n = 20). LessHA had a lower frequency intake of pro-healthy food groups, a lower number of meals during a typical day, and a higher mileage training than the group of athletes who followed the MoreHLA. Athletes following the LessHA pattern also had a lower Z-score in the lumbar spine and femoral bone and a lower PINP. Conclusion: The current study suggests that pro-healthy dietary patterns and lower mileage may favour higher bone mineral density in male amateur marathoners.Background: Excessive mileage can be detrimental to bone mineral density among long-distance runners. The negative effects of mileage could be alleviated by appropriate nutrition. The purpose of this study was to analyse the dietary-lifestyle patterns in relation to bone mineral density and bone turnover markers among amateur marathoners. Methods: A total of 53 amateur male distance runners were divided into two clusters by k-means cluster analysis. Bone mineral density was measured by dual X-ray absorptiometry (DXA). Blood was drawn to analyse bone resorption marker C-terminal telopeptide (cTX) and bone formation marker amino-terminal propeptide of type I collagen (PINP). Food frequency intake and lifestyle information were measured by multicomponent questionnaire KomPAN Running is one of the most popular and practised sports around the world. The number of participants in running, jogging, and trail running in the U.S. between 2006 and 2017 was almost 60 million [Despite the health benefits associated with practising amateur jogging, it should be emphasised that practising it without adjusting training loads, supplementation, and diet may be associated with health risks. Amateur marathon runners bear a certain degree of health risks such as sports injuries or nutritional deficiencies. Additionally, experts have warned non-athletic amateurs about the “exercise paradox” and the probable deleterious effects of prolonged high-intensity exercise on the musculoskeletal system . EpidemiBearing in mind this data, specialists should have a balanced discussion with amateur runners training for marathons about the risks and benefits of high intensity exercise and should evaluate them to rule out the health risk.Low bone mineral density increases the risk of osteoporosis and stress fractures . RunningAn effect of energy deficiency similar to female athlete triad can be seen in male athletes, which has been described as Relative Energy Deficiency in Sports Syndrome (RED-S) . The incEndurance running is a growing discipline among amateurs who often race multiple marathons during the year and are at an older age in comparison to professional runners who usually race marathons twice a year. Amateur marathon runners, except for the strenuous training regime, also work full time very often, which may leave less time for rest and meal planning. Furthermore, running a marathon is often one of the main goals of a person who wishes to start a healthy lifestyle, often with the addition of a restrictive diet for weight loss purposes. Some studies show that amateur runners have healthier eating habits than non-exercising individuals , which iTo date, there have been no studies that evaluated the factors that influence the bone density of amateur runners, and there are no specific guidelines on nutritional recommendations to avoid the incidence of stress fractures and low bone mineral density.The purpose of this study is to analyse the impact of diet quality and mileage on bone turnover markers and bone mineral density among amateur marathon runners.n = 53) were recruited into the study between October and March 2017–2018 from local social media groups and marathon races  and one activity marker (in km/month) component of DLPs. All input variables were standardised to achieve a mean equal to 0 and a standard deviation equal to 1. To identify the optimal number of clusters, the analysis was performed several times. The K-means clustering algorithm was used, and participants were grouped according to Euclidean distances. Finally, two clusters representing two dietary-lifestyle patterns were selected (Dietary-lifestyle patterns (DLPs) were derived using cluster analysis. The input variables were three dietary and four bone mineral density and turnover markers  was used [To assess the nutritional habits, the validated Dietary Habits and Nutrition Beliefs Questionnaire KomPANwas used . A totalwas used ,20,21,22All study participants had a dual-energy X-ray absorptiometry (DXA) scan at the Department of Human Nutrition and Dietetics by the same technician on a GE Lunar Prodigy machine .2), and body composition .The DXA measured bone mineral content , bone mineral density  to compare BMD to the average for a healthy person of the same age and sex. The standards of the International Society for Clinical Densitometry (ISCD) recommend using Z-scores to assess BMD in paediatric participants, premenopausal women, and men under 50 years of age  (ISCD 203) and blood morphology. The collection took place in the morning in the fasted state, and the participants were advised to not exercise vigorously before the test. All of the material was handled by a certified diagnostic laboratory Synevo sp. z.o.o. Poland. Bone turnover markers were assessed by using the chemiluminescence method. The two kits were used according to the manufacturers’ instructions: IDS-iSYS Intact PINP and IDS-iSYS CTX-I (CrossLaps®) . 25-OHD3 was measured by the commercially available ELISA kit .Blood was taken to assess the serum bone turnover markers C-terminal telopeptide (cTX) and amino-terminal propeptide of type I collagen (PINP), as well as vitamin D status  software. Bone mineral density Z-scores and T-scores were calculated by the DXA Lunar Prodigy software  for each participant.Descriptive statistics  were used to characterise the sample. To determine the distribution, the Shapiro–Wilk normality test was used. For the categorised data, the chin = 33) and More-healthy-less-active-high-Z-score   . The anaThe participants were amateur marathon runners who ran an average 227 ± 92 km a month, and 75% of them had been practising long-distance running for more than 5 years . A totalExcept for the overall lower pro-healthy diet index (p-HDI) among LessHA, this group had a statistically lower frequency intake of butter and fresh cheese products but a higher intake of vegetables than the MoreHLA group .Besides the low lumbar and femoral Z-scores and PINP which were used to distinguish clusters, the LessHA group had lower bone mineral density in the femoral neck, ward’s triangle, and trochanter area of the femoral bone as well as TBS score . There wThis study reached its aim by distinguishing the group of amateur marathon runners at a higher risk for low bone mineral density and potential future bone fracture. The low bone mineral density was related to the dietary behaviours and training volume, which confirms the results of the previous studies conducted on professional marathoners ,13. TherThe cluster of runners with lower bone mineral density was characterised by a low total hip Z-score and total lumbar spine Z-score. Lower bone mineral density is related to future fracture risks among adult populations and trained individuals . The TBSThe PINP was also statistically lower in the LessHA cluster than in the high BMD cluster, and its levels reflect the number of newly formed collagen molecules . It has The cTX was not statistically different between the two clusters. This bone turnover marker is related to bone resorption, and some studies suggest that it is one of the least influenced by intensive physical exercise bone turnover markers ,33. On tThe analysis of a marathon runner’s lifestyle factors influencing the BMD and TBS will enable them to take appropriate preventative action.The runners from the LessHA cluster consumed less frequent meals during a typical day than the runners from the MoreHLA cluster. The high frequency of meals has been associated with a lower body fat and a high fat-free mass among the adult population . AdditioThe pro-healthy dietary index (pHDI-10), which was higher among men in the MoreHLA group, includes groups such as milk, fermented drinks, and cottage cheese, among others. High pHDI-10 could contain a substantial number of dairy products. There was a significant difference in the intake of cottage cheese, which is not only a source of calcium but also protein. The LessHA group also has a lower intake of butter. Low protein and fat intake could be one of the causes of the previously mentioned Relative Energy Deficiency in Sports Syndrome (RED-S) . Our resAlso surprising is the fact that the runners from the LessHA cluster had a higher frequency intake of vegetables. A high intake of vegetables could indicate that runners could also restrict their energy intake . This is3 was not associated with LessHA, which supports previous studies on bone health in professional athletes [The LessHA cluster is also characterised by high mileage and running history. This supports the fact that intensive long-distance running compromises the bone mineral density among both professional athletes and amateurs . In turnathletes .There is a significant age difference among the analysed clusters, which could be a weakness of the study. It is already known that BMD decreases with age. However, in our study, we used a Z score instead, which is adjusted for age . The higThis study addresses for the first time the bone health concern of amateur distance runners and compares it with dietary lifestyle patterns. Our results indicate that a high training load and low frequency intake of healthy foods could be a problem for amateurs who also have different everyday activities such as work. This study supports the experts’ advice about the “exercise paradox,” by which prolonged and intensive physical activity could have a detrimental influence on bone health in amateur marathoners. Additionally, it points out that lifestyle and nutrition are also important factors in male athletes’ bone health, and this matter should be further studied.


Iliotibial band traction syndrome (ITBTS) after total knee arthroplasty (TKA) has been well documented following first-generation guided motion bicruciate substituting (BCS) TKA. The incidence of ITBTS following second-generation BCS has been found to be rare, and surgical release of the IT band has not been reported. A 64-year old male was diagnosed with ITBTS following second-generation guided motion BCS TKA. After a three-month trial of non-surgical treatment, he underwent selective open release of the iliotibial band (ITB), which successfully relieved his symptoms. Orthopedic surgeons should keep ITBTS as a possible differential diagnosis when evaluating the lateral-sided knee pain following guided motion BCS TKA. Iliotibial band traction syndrome (ITBTS) after total knee arthroplasty (TKA) is a rare cause of painful TKA. This condition has been reported secondary to guided motion bicruciate substituting (BCS) TKA  . HoweverA 64-year-old male presented with end-stage medial compartment osteoarthritis (OA) of the left knee Figure . After fAt the two-week follow-up visit, the patient complained of sharp pain along the lateral aspect of the knee, which persisted for three months despite NSAIDS and physical therapy. Going up and downstairs caused significant lateral-sided sharp pain. A clinical examination demonstrated no evidence of laxity with varus and valgus stress, and the patella was found to be tracking well. The patient noticed most of the pain in the mid-arc range of motion (20° to 70°). The ultrasound evaluation confirmed the fact that the point of tenderness was correlated to the anterior portion of the ITB  at the level of the lateral epicondyle. No prominent hardware was noted, and no probe tenderness was noted at the tibial and femoral implants. A diagnostic injection with lidocaine combined with cortisone  was given using ultrasound guidance at the most tender spot. This spot was located (defined using probe tenderness) along the anterior border of the ITB and the origin of the iliopatellar band (IPB) at the level of the lateral femoral epicondyle (LFEC). This procedure resulted in immediate 100% relief from the pain. The patient could climb a flight of stairs without discomfort immediately after the diagnostic block. However, the pain recurred after a week, and the patient elected to go for surgical release of the ITB with decompression of the lateral gutter.Selective Iliotibial band releaseUnder general anesthesia, with the patient in a supine position, a 3 cm longitudinal incision was placed along the anterior border of the ITB at the level of the lateral epicondyle. An IPB was identified running from the anterior border of the ITB and lateral border patella. We noted that IPB was taut when the patella was engaged in the trochlear groove. Surgical release of the IPB was performed with a lazy S-shaped incision Figure . The wouThe patient's pre-operative Oxford Knee Score (OKS) was 23 (before primary TKA) and was reduced to 11 after six weeks of post-TKA and before the selective ITB release. The OKS improved significantly to 37 after the selective ITB release at a one-year follow-up.Isolated iliotibial band-related pain after TKA is a very uncommon presentation. This pain could be either from the rolling of the ITB against unresected osteophytes or cementophytes, sharp edges of the implant, or implant overhang, which is collectively termed iliotibial band friction syndrome (ITBFS) . HoweverThe incidence of unsatisfied TKA after surgery is around 15-20% . AbnormaThe surgical treatment and results of selective ITB release have not been reported in terms of patient-reported outcome measures for this unique condition. We ruled out other differential diagnoses for isolated causes of lateral-sided knee pain after TKA Table . InitialEven though the modifications in second-generation BCSKS have significantly decreased the incidence of ITBTS, this unique complication can still occur with motion-guiding second-generation BCSKS. We caution surgeons to specifically look for this problem to evaluate its real incidence. Several anatomical variations and thicknesses of ITB have been described in the literature. Future research should be aimed at evaluating if any anatomical variant of ITB is associated with an increased risk of ITBTS with guided motion BCSKS. Ultrasound significantly increases the diagnostic accuracy, and ultrasound-guided injections with local anesthesia (with or without steroids) are confirmatory and guide the surgical procedure. The surgical procedure (ITB release) is relatively benign without residual instability.This is an isolated case report of ITBTS following second-generation BCSKS and successfully treated with limited surgical release of the anterior portion of the ITB and IPB with decompression of the lateral gutter. This procedure resulted in immediate symptom relief, no instability was noted, and no further complications were reported after 18 months of follow-up.


The key players and processes of the MHC class I and class II pathways were described decades ago and are essential to develop an efficient T cell response. However, they are not confined to canonical pathways only. Indeed, cross-presentation and autophagy are non-canonical processes that allow the presentation of peptides by MHC-I and MHC-II molecules that originate from proteins located in the endocytic or endogenous compartments, respectively. In addition, the structural features of the MHC-peptide interaction were described decades ago. Nevertheless, MHC molecules can bind thousands of different peptides and the complexity of these immunopeptidomes open the door to the study of “non-typical” peptides. Thus, some of them are post-translationally modified peptides, spliced peptides generated by the proteasome, or peptides derived from non-canonical open reading frames. In this special Research Topic issue, we wanted to include an array of manuscripts that provides an extensive view of the alternative processing and presentation by MHC molecules and its involvement in immune disorders. A brief synopsis or each  is included below.Mansurkhodzhaev et al. investigate the formation of proteasome-generated spliced zwitter peptides originating from self (human) and non-self  antigens. Although such sequences can be envisioned to induce clonal deletion or other tolerance processes and restrain responses against viruses, their analysis suggests that cis-spliced peptides only marginally impinge upon functional anti-viral CD8+ cytotoxic T cell responses.Building on their previously reported work, Münz highlights basic knowledge about how macroautophagy machinery contributes to antigen processing and presentation, strengthening its noncanonical functions, such as its role in nonconventional phagocytosis and secretion. Therefore, as argued in the manuscript, the multiple tasks played by this machinery makes it a fascinating object of study and, potentially, a powerful target to modulate antigen presentation in pathological situations.In his Mini Review Nanaware et al. addressed this issue aiming at the MHC classs II (MHC-II) immunopeptidome in the mouse thymus. They developed a strategy to discard non-binding peptides and to validate bona fide binders, allowing them to identify distinct structural features of thymus MHC-II ligands compared to those from spleen, pointing to differences in antigen processing in these tissues.One of the crucial issues in defining the immunopeptidome is the discrimination of signal from noise, which becomes critical when peptide abundance is limiting. Mei et al. presented original research work focused in the need to use computer-based immunoinformatic analysis to predict T cell targets from different strains of SARS-Cov-2 that may be used in future vaccine design. The authors put emphasis in HLA-A*11:01, the most frequent HLA allele in the Chinese population. They experimentally validated three epitopes from Spike protein.Dhatchinamoorthy et al.., where they cover different angles from where this issue can be observed: mechanisms of downregulation of MHC-I expression, mutations affecting MHC-I itself or essential components of the antigen processing pathway, the impact of these immunoevasion mechanisms on cancer cell recognition, as well as potential strategies to restore MHC-I expression.The role that CD8+ T lymphocytes play in immunosurveillance of cancer cells is highlighted by the evasion mechanisms that they develop that affect MHC-I antigen presentation. This is the topic of the exhaustive Review by Carré and Mallone elaborate the physiology and function of beta cells and antigen processing and presentation (APP) pathways. Thus, the authors generated some hypotheses on APPs relevant to beta cells, and directed this analysis to T1D pathogenesis. Finally, the authors raised some gaps regarding the HLA-I immunopeptidomes that need to be filled, as their composition in basal or stress conditions or the differences in the antigenic peptides presented by beta cells or dendritic cells during the T cell priming in draining lymph nodes.Ng et al. in this issue explores how this natural product may weaken anti-α-synuclein T cell responses through alterations in the vesicular trafficking of this antigen in dendritic cells. The authors propose that this altered trafficking may preclude its efficient antigen processing, thus thwarting a damaging T cell response.The contribution of neuroinflammatory processes to the development of Parkinson’s Disease (PD) has been gaining momentum in recent years. Celastrol, a compound that modulates autophagy and mitophagy, has been proposed to mitigate PD and other neurodegenerative diseases. The work of Ito et al. explore the effects of palladium metal, a well-known allergen, on the surface dynamics of MHC class I protein. Using complementary assays, they demonstrated distinct temporal effects elicited by palladium treatment of murine DC2.4 dendritic cells leading to altered peptide presentation. Inhibition of membrane movement blocked this effect, leading to suppressed Pd-induced T cell-mediated responses.Zhao et al. explore the functional impact of a spontaneous H2-Aa point mutation on MHC II expression and function. They document an atypical type II bare lymphocyte syndrome mediated through a cascade of erroneous mRNA splicing, deletion of eight bases in the mRNA, and a protein frameshift, all leading to a general loss of expression and significantly impaired CD4+ T cell numbers.The impact of sequence defects or variants on the expression and function of MHC molecules provides an important means of understanding the potential impact of natural polymorphisms and acquired mutations. Thakur and Luthra-Guptasarma elaborate mechanisms through which some HLA-B27 subtypes are associated with ankylosing spondylitis (AS) and others are not. In their work, they conducted a proteomics-based study to analyze the protein clearance mechanisms in two AS differentially associated alleles . They show that B*27:04 misfolded chains are mainly cleared through activation of the unfolded protein response (UPR), and autophagy. Instead, the B*27:09 misfolded chains are mainly cleared through the endosome-lysosome pathway.In an original research article, Welsh et al. review the often-overlooked role of B cell antigen presentation on the differentiation and longevity of protective memory CD4 T cells. Specifically, they highlight the impact of antigen density and the contributions of MHC Class II accessory molecules (HLA-DM and HLA-DO) to epitope selection and possible impacts on the formation of memory. They propose that low levels of antigen presentation might be to regulate long-term survival of CD4 memory T cells and to prevent cross-reactivity to autoantigens.Another topic of great interest is the idea that specific populations of professional antigen presenting cells can play distinct roles in health and disease. Kang et al. investigate the number and function of Dendritic cells (DCs) in scrub typhus patients. Examining peripheral blood, they observed that plasmacytoid and conventional DCs were numerically deficient and functionally impaired in scrub typhus patients. Alterations in the number and surface phenotype of DCs were absent during disease remission, but phenotypic alterations could be recapitulated through treatment with pro-inflammatory cytokines, suggesting a direct relationship between these alterations and disease severity.In their article, Wang et al. suggest in their contribution that IVIG treatment reverts the immature (or tolerant) phenotype of circulating myeloid and plasmacytoid dendritic cells, restoring the numbers of peripheral CD4+ T cells, which circulate at reduced levels prior to the treatment.Kawasaki disease (KD) is a severe illness that primarily affects children under the age of five. The condition is characterized by a vasculitis that, if untreated, can cause coronary artery aneurism and adult cardiovascular disease. Intravenous immunoglobulin (IVIG) is an effective treatment for acute KD, but its therapeutic mechanism of action is not completely understood. We would like to dedicate this ResearchTopic to the memory of two pioneers that the Antigen Presentation field lost in the past few years, Enzo Cerundolo and Nilabh Shastri. Their contributions laid the foundations of a knowledge that pervades many concepts used by the papers published in this ResearchTopic.EAJ, IA, and LCA each wrote individual sections of the editorial, corresponding to the respective articles that they edited. All three authors organized, revised, and edited the manuscript and agree to its style and content in its final form.IA is supported by Grant RTI2018-097414-B-I00 from the Ministerio de Ciencia e Innovación. LCA is supported by Grant PID2019-110407RB-I00 from the Ministerio de Ciencia e Innovación; the Centro de Biología Molecular Severo Ochoa is funded by institutional grants from Fundación Ramón Areces and Banco de Santander. EAJ is supported by NIH/NIDDK grant no U24 DK104162-08 (Niland) NIAID contract no 75N93019C00068 (Kwok), NIAID grant no 2R01DK081166-11 (Haskins), and an award from the Heidner Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this paper we present a structurally-complex biomimetic scattering structure, fabricated with two-photon polymerization, and utilize this object in order to benchmark a computational imaging system. The phantom allows to tailor the scattering by modifying its degrees of freedom i.e. refractive index contrast and scattering layer dimensions and incorporates a 3D imaging quality test, representing a single cell within tissue. While the sample may be used with multiple 3D microscopy techniques, we demonstrate the impact of scattering on three tomographic phase microscopy (TPM) reconstruction methods. One of these methods assumes the sample to be weak-scattering, while the other two take multiple scattering into account. The study is performed at two wavelengths (visible and near-infrared), which serve as a scaling factor for the scattering phenomenon. We find that changing the wavelength from visible into near-infrared impacts the applicability of TPM reconstruction methods. As a result of reduced scattering in near-infrared region, the multiple-scattering-oriented techniques perform in fact worse than a method aimed for weak-scattering samples. This implies a necessity of selecting proper approach depending on sample’s scattering characteristics even in case of subtle changes in the object-light interaction. This can be attributed to the fact that complex biological structures such as spheroids or organoids tend to be more relevant models than 2D cell cultures e.g. for drug discovery2. Also, most in vivo imaging techniques require the probing light to pass through the complex structure of a tissue which highly limits imaging depths due to multiple scattering. This demand stimulates the development of new methods5, however, it is difficult to select an appropriate one based on the scattering strength of the analyzed specimen. For this reason a versatile, repeatable and quantitative method for the evaluation of different imaging systems and algorithms is essential to determine their limits of applicability depending on the object’s scattering properties. One possibility is to use calibrated microphantoms as imaging targets. Unfortunately, existing microphantoms are typically either weak-scattering (e.g. index-matched microspheres) or overly simplistic (e.g. index-mismatched microspheres)7 compared to the types of heterogenously scattering multicellular samples that multiple-scattering methods are intended for. This is a critical limitation when characterizing computational imaging methods that utilize nonconvex solvers, where iterative convergence depends on the complexity of the energy landscape and directly associates with a sample’s 3D complexity8.One of the modern challenges in computational optics is to image scattering samples with high resolution12. Among multiple available 3D printing techniques18, we chose a two-photon polymerization that enables 3D printing of microphantom samples with known geometry and calibrated RI. When compared to other implementations of direct laser writing, it allows (1) to control the RI with relatively high modulation range, (2) to adjust the RI contrast or scattering strength post-fabrication using different immersion liquid and (3) to handle and measure the microphantom in the same way as biological specimens. Next, we present the application of the phantom in the field of tomographic phase microscopy (TPM), a technique which has demonstrated impressive biological imaging results in prior works. However, it is important to note that all computational imaging methods can be evaluated with the proposed procedure.In this work we present a 3D-printed microphantom with multiple-scattering refractive index (RI) distribution. To do so, we leverage recent developments in 3D printing via direct laser writing19, cell structure21, photobiochemical effects on cells22, influence of external factors on cellular parameters24 and many others. Given the wealth of information provided by the dry mass at the single-cell level, there is significant demand to extend the capability to analyze dry mass to large multicellular clusters, thick tissue slices, or whole microorganisms. However, to reconstruct 3D RI, traditional TPM methods utilize critical assumptions in their computational reconstruction methodologies that rely on the sample being weakly scattering25. These assumptions limit samples to having thicknesses on the order of only tens of microns. For thick, complex samples, reconstruction frameworks that accommodate for multiple scattering must be utilized. To this end, numerous TPM approaches have been proposed in recent years that introduce new frameworks to accommodate multiple scattering31. Notably, these approaches utilize nonlinear and nonconvex solvers to iteratively solve for a sample’s 3D RI. Though these methods have demonstrated impressive results in reconstructing RI in multiple-scattering samples, their quantitative accuracy has not yet been robustly characterized experimentally, and the presented results usually do not allow comparison of different methods in order to select proper approach for a given scattering level in a sample. The general strategy to experimentally evaluate quantitative accuracy in TPM methods is to reconstruct 3D RI in samples with known RI distributions32. A multiple-scattering TPM method that outputs accurate 3D RI reconstructions of a weakly scattering or overly simplistic microphantom cannot be expected to output similarly accurate RI reconstructions for more complex multiple-scattering samples, where the probability of converging to local minima is drastically higher. To robustly characterize the quantitative accuracy of multiple-scattering TPM methods, it is imperative to use microphantoms with known 3D RI that mimic the structural complexity of the types of samples that the TPM methods are intended to image. To the best of our knowledge, these types of gold-standard multiple-scattering microphantoms do not exist.TPM is a quantitative, label-free imaging method that utilizes optical projections through a semi-transparent sample along various illumination angles to reconstruct the sample’s 3D RI. This method has found several applications in biological imaging, where RI is directly related to the dry mass distribution at the cellular and subcellular levels. Refractive index and dry mass are known to be crucial factors in analyzing the current stage of cell cycleIn this section we present the design of the 3D printed microphantom and its application in evaluation of three TPM reconstruction methods.33 embedded within a pseudo-random distribution of rods that switch their orientation across various layers  and multiple scattering35. The final scattering region is a 60 The scattering microphantom we designed and 3D printed consists of a cell-like target with internal test structuresers Fig. a. The wi36. Information about 3D printing procedure are given in Sect. “37 up to 1667 lp/mm. By assessing the maximum spatial frequency of lines that can be distinguished within the cell test target, the imaging resolution of a TPM method of choice can be characterized and potentially compared to different methods of assessing resolution described by other research groups39. The pseudo-random distribution of rods that compose the scattering portion of the whole microphantom are suppressed within 0.5 40.The cell-like target embedded within the layers of randomly distributed rods mimic a single biological cell encased within a scattering volume, which represents e.g. tissue. The cell-target comprises of substructures that enable assessment of quantitative accuracy in TPM imaging systems41, (2) multi-slice beam-propagation with electric-field measurements (MSBP-E)42 and (3) multi-slice beam-propagation with intensity-only measurements (MSBP-I)31. In order to perform the comparison, the phantom has been measured with the TPM device and two datasets were composed from the complex-valued scattered field measurements of the microphantom being illuminated at different angles, using both 633 nm and 835nm wavelengths. For GPSC and MSBP-E, these datasets were used directly. For MSBP-I, only the amplitude components were used. The measurements and their corresponding 3D RI reconstructions are available in Dataset 140.Three TPM reconstruction methods were implemented and evaluated through comparison of tomographic reconstructions of the proposed microphantom. The methods are: (1) Gerchberg-Paopulis with support constraint (GPSC)x and y) resolution of the TPM reconstructions by visualizing the resolution test lines inside the microphantom. To quantitatively compare the three TPM reconstruction methods, horizontal and vertical cross-sectional plots across these resolution tests were generated by computing the average and standard-deviation of the pixel-values across rows or columns adjacent to the dashed white a-a and b-b lines, by Figure 44. The magnified views of the resolution test region after reconstructing the microphantom using the GPSC algorithm with 633 nm wavelength light reveals significant grainy artifacts which occlude the line features within the microphantom’s test region. We note that these artifacts are heavily decreased when reconstructing with 835 nm wavelength light. This suggests that, at 633 nm wavelength, the microphantom is too highly-scattering for GPSC to be applicable. The decreased GPSC reconstruction artifacts for 835 nm matches conventional knowledge that longer wavelengths of light are more resistant to scattering than shorter wavelengths. In order to confirm that this observation is due to reduced scattering and not different noise characteristics between the two light sources, we analyzed the standard deviation of phase-noise in an object-free region within the input datasets. For 633 nm wavelength light, a phase-noise standard deviation of The known complex geometry and RI distribution of the developed microphantom allows to show that changing the wavelength from visible into near-infrared impacts the applicability of TPM reconstruction methods45. Especially in the case of using 633 nm wavelength light, TV regularization results in 3D RI reconstructions with less noise compared to the 3D reconstructions computed via GPSC, which does not use TV regularization. This can be directly visualized in the 2D cross-sections in Fig. Notably, both MSBP-E and MSBP-I use total-variation (TV) regularization in order to stabilize the convergence of the nonconvex iterative solver in the presence of noise47). Other works have shown that MSBP-I demonstrates higher accuracy when using partially-coherent illumination, which drastically reduces coherent noise31. Future work may include repeating this analysis across a larger range of TPM reconstruction techniques with more complex scattering microphantoms.In terms of the average RI, the knowledge about the ground truth RI distribution of the phantom makes it possible to quantitatively compare reconstruction results with GPSC, MSBP-E, and MSBP-I. We see that all methods successfully capture the bulk characteristics of the microphantom. As described above, the 3D reconstruction via GPSC exhibits grainy artifacts when using 633 nm wavelength light, likely due to the microphantom being multiple-scattering at that wavelength. Furthermore, MSBP-I outputs slightly overestimated RI values, and also suffers from low-frequency spatial artifacts  or modify the resin with functional particles18 .Future works will focus on developing methods to quantify scattering strengths of phantoms and relate these quantities with the scattering properties of different tissue types. If successful, this would enable us to design and fabricate (using the presented methods) 3D microphantom structures to mimic a wide range of biological specimens ranging from multicellular clusters to bulk tissues and small organisms. Another possible direction is to tune scattering parameters in the phantom to characterize imaging performance for various techniques used to image into scattering tissue, such as optical coherence tomography or confocal reflectance microscopy. Finally, we envision that one can exploit the flexibility of two-photon polymerization to fabricate microphantoms on different substrates , using biocompatible resins (to combine test targets with the living cells in the single measurement volumeWe present below the (1) methodology with which we 3D-print multiple scattering microphantoms; (2) the optical design of the TPM imaging systems that we use to experimentally collect scattering electric-field measurements of the microphantom; and (3) short theoretical descriptions of three tomographic algorithms that were used to reconstruct 3D RI from the measured data.54). After fabrication the structure was developed in PGMEA , followed by isopropyl alcohol (10 min) and then blow-dried. The full methodology for fabrication and validation of the features can be found in our previous work33.The phantom is fabricated using two-photon laser lithography, in which a focused laser beam is scanned within liquid resin. The resin within the laser’s focal volume is locally polymerized. Adjusting the scanning trajectory and the exposure time of the laser beam enables simultaneous control over the 3D printed geometry (accuracy at the order of 100 nm) and RI 56. The research was performed with two wavelengths and thus there were two modified versions of the presented tomographic microscope. First version, 57 is In this work, an optical system, as shown in Fig. There were three different TPM reconstruction methods for computing 3D RI of the microphantom used for comparison. The data for each method was acquired through the multi-angle scattering measurements captured as described in Sect. “41. Complex-valued electric-fields measured by our TPM systems are used as inputs. The procedure is performed in two steps. First, an initial tomographic 3D RI distribution is reconstructed from the electric-field measurements with strong total-variation regularization45. This is performed through the Chambolle-Pock58 optimization method and implemented with ASTRA tomography toolbox59. The result undergoes binarization and a finite object support is generated. Secondly, a classic Gerchberg-Papoulis algorithm is used which is an iterative version of Direct Inversion method 60. This iterative procedure is based on the Fourier Diffraction Theorem61 and utilizes first-order scattering approximation. Here, the reconstruction and its Fourier transform are calculated alternately and constraints are applied: nonnegativity and finite object support in the signal domain, and replenishment of original projections in the frequency domain.To provide a baseline standard to compare to TPM reconstruction algorithms utilizing multiple-scattering models, we first reconstruct the microphantom’s 3D RI using a weak-scattering TPM method. We specifically use the Gerchberg-Papoulis algorithm enhanced with additional finite object support constraint (GPSC)62, which has recently shown promising results for biological imaging63. In our first implementation of MSBP, we use the same exact electric-field dataset utilized by GPSC from above. An initial guess of the sample’s 3D RI is selected to start off the iterative procedure. Afterwards, the MSBP method is used to simulate scattering measurements resulting from plane waves propagating through the sample’s initial estimated 3D RI. The scattered fields resulting from this simulation are compared with those obtained experimentally with our TPM systems. The error computed between the simulated and experimental measurements is back-propagated through each layer of the 3D sample estimate to incrementally modify the RI value of each voxel. Continued iterations repeating these steps eventually result in the 3D sample estimate converging to a stable steady-state solution. We implemented the MSBP with electric-field measurements (MSBP-E) through the Learning Tomography algorithm (LT)42. The LT procedure is an iterative optimization algorithm with additional weak total-variation (TV) regularization applied in each iteration to ensure convergence. We found that the method works best when an initial-guess is chosen as a starting point for the iterative process. In this paper, we use the Direct Inversion method to provide the initial guess.Our main method to model multiple-scattering is the multi-slice beam-propagation method (MSBP)non-interferometric intensity measurements31. The key advantages of this method include the use of a non-interferometric imaging system, which are resistant to mechanical instabilities that often limit long-term use of dual-arm interferometers without realignment. Furthermore, the light source can be partially coherent, to avoid coherent speckle artifacts in the measurements, while retaining sufficient coherence necessary for RI reconstruction. For the purposes of demonstrating 3D RI reconstruction using this intensity-only variant of MSBP (which we refer to here as MSBP-I), we simply use the amplitude component of the electric-field measurements used for the GPSC and MSBP-E reconstructions, described above. Similarly to MSBP-E, total-variation regularization is applied at every iteration. The starting point for MSBP-I is a matrix of zeros.Recent works have demonstrated that the gradient-update step within the MSBP method can be reformulated to reconstruct 3D RI from only 64, and is described in Alg. 1 below. The general intuition behind this procedure is to terminate the iterative computation process when the dynamics of the change between 3D sample estimates outputted from consecutive iterations drops below a certain saturation level All described TPM reconstruction methods are iterative procedures that use the same stopping criterion to automatically terminate the computations. This criterion is a modification of a method presented earlier