A practical noninvasive method to identify sentinel lymph node (SLN) status in breast cancer patients, who had a suspicious axillary lymph node (ALN) at ultrasound (US), but a negative clinical physical examination is needed. To predict SLN metastasis using a nomogram based on US and biopsy‐based pathological features, this retrospective study investigated associations between clinicopathological features and SLN status.n = 472, 175, and 81). Lymph node ultrasound and pathological characteristics were compared using chi‐squared and t‐tests. A nomogram predicting SLN metastasis was constructed using multivariate logistic regression models.Patients treated with SLN dissection at four centers were apportioned to training, internal, or external validation sets (+ were as follows: histology type (p < 0.001); progesterone receptor (PR: p = 0.003); Her‐2 status (p = 0.049); and ALN‐US shape (p = 0.034), corticomedullary demarcation (CMD: p < 0.001), and blood flow (p = 0.001). With multivariate analysis, five independent variables  were integrated into the nomogram (C‐statistic 0.714 [95% CI: 0.688–0.740]) and validated internally (0.816 [95% CI: 0.784–0.849]) and externally (0.942 [95% CI: 0.918–0.966]), with good predictive accuracy and clinical applicability.In the training set, statistically significant factors associated with SLNThis nomogram could be a direct and reliable tool for individual preoperative evaluation of SLN status, and therefore aids decisions concerning ALN dissection and adjuvant treatment. This nomogram is using pathology and ultrasound parameters preoperatively to predict SLN metastasis. Cindex, calibration, discrimination, and clinical use of the nomogram show well in the training, internal and external validation sets. In 2018, there were 2.28 million newly diagnosed cases.+ patients have been found to lack non‐SLN involvement after ALND, indicating that these patients received unnecessary axillary treatment.For assessing the pathological status of ALNs in patients with stages I‐IIIA (T3N1M0) breast cancer, the National Comprehensive Cancer Network (NCCN)Currently, differentiating benign from malignant nodes is aided by mammography, contrast‐enhanced US, positron emission tomography‐computed tomography, magnetic resonance imaging (MRI), and so on.Judging the SLN status of patients who have a suspicious ALN on US, but a negative clinical physical examination, is problematic. To differentiate patients with positive or negative SLNs based on ALN‐US features, this retrospective study compared ALN‐US data with pathological evidence, and a nomogram was constructed for predicting SLN metastasis.2n = 472); an internal validation set of patients at PUMCH from 1 January 2019 to 31 December 2019 (n = 175); and an external validation set (n = 81) of patients treated at 3 centers from 1 January 2019 to 31 December 2019 (described below).This study retrospectively analyzed patients who had a suspicious ALN at US, but a negative clinical physical examination, and were treated with SLN biopsy from 1 January 2014 to 31 December 2019 at the following medical centers: Peking Union Medical College and Hospital (PUMCH); Baoji Maternal and Child Health Hospital; Qinghai Provincial People's Hospital; and Shanxi Traditional Chinese Medical Hospital. Our retrospective study is based on real world data, according to clinical practice and NCCN guidelines.+ rate of the entire population, was analyzed. ALN‐US and pathological characteristics were compared between patients with positive and negative SLNs.Patients' demographics and clinical features were recorded, including age; ALN‐US findings; postoperative mass histological pathology, SLN status; type of breast surgery; and pathological risk factors. The latter consisted of lymphovascular invasion (LVI), nerve invasion, and infiltrative micropapillary carcinoma (IMPC). All the pathology was received at biopsy, that is, ductal carcinoma in situ (DCIS) included in our research was diagnosed on initial biopsy and found with invasive breast cancer at the time of the definitive surgical procedure. The SLN status, and SLNn = 2332) were initially reviewed. Potential subjects were excluded for the following reasons: combined negative clinical physical examination and negative US; received preoperative chemotherapy or endocrine therapy; with metastatic breast cancer or other special types of breast cancer; or were lost to follow‐up. Altogether, 1826 were excluded, and 472 patients at PUMCH were enrolled in the training set.To select the training set, patients who had undergone SLN dissection . The US specialists participating in this study are all experienced specialists who have received standardized training. The ultrasound examinations in all centers are performed by two ultrasound doctors at the same time. In case of disagreement, they will discuss and finally give a consensus. The US images were inverted from the ultrasonic system of each center. The size and morphological factors of Level I, II, and III LNs in the axillary and supraclavicular LNs on the side of breast tumors were recorded and evaluated. The morphological criteria included shape, corticomedullary demarcation (CMD), and blood flow on ALN‐US. Shape was described as regular or irregular. Asymmetric focal or diffuse cortical thickening >3 mm was classified as unclear ALN‐US CMD, and otherwise clear.2.2Lymphatic mapping of biopsied SLNs was performed using dual tracer, methylene blue, and indocyanine green (ICG). Surface localization was done by ICG, and then, we resected the observed blue‐stained LNs and enlarged LNs as the SLNs. ICG was used to reconfirm the resected SLNs. Every SLN was routinely analyzed via intraoperative frozen sections. Patients with negative SLNs were not given any further axillary treatment. ALN dissection was avoided in most of the patients who met the American College of Surgeons Oncology Group (ACOSOG) Z0011 criteria; otherwise, ALN dissection was performed.2.32.3.12) or Fisher's exact test was used to analyze an association between SLN metastasis and clinicopathological features. Comparisons of continuous variables  were performed using Student's t‐test. The Kaplan–Meier model was applied to analyze the survival rate of patients. The statistical analyses were two‐sided. p < 0.05 was considered a statistically significant difference. Statistical analyses were performed using SPSS software  and GraphPad Prism software (version 6.01).The clinicopathological and ALN‐US data of the patients were analyzed, and the patients were followed up. The last date of follow‐up was 1 January 2020. The main endpoint of follow‐up was death or distant metastasis. The mean follow‐up time was 42 months. The chi‐squared test  and corresponding 95% confidence intervals (CIs) were calculated. The Hosmer–Lemeshow test was used for multivariate analyses, and a forest map to reflect the results of multivariate analysis. Based on the results of the multivariate analysis, independent variables were selected for inclusion in the nomograms to predict SLN metastasis, using statistical software .Associations between relevant clinicopathological variables plus ALN‐US with SLN status were estimated by multivariate logistic regression models; variables with 2.3.3The nomogram was constructed using RMS software. The performance of the model was assessed through discrimination and calibration. Discrimination ability was calculated using concordance (C)‐statistics. The C‐statistic was applied to evaluate the concordance between the observed and predicted outcomes of the model, which is roughly equivalent to the receiver operating characteristic (ROC) curve. C‐statistics ranged from 0.5 and 1. To evaluate the calibration of the nomogram, a regression smoothing method was used to produce the calibration plots by bootstrapping with 1000 resamples, representing the association between the observed outcome frequencies and the predicted probabilities. The standard curve is a straight line passing through the origin of the coordinate axis with a slope of 1. The ROC curves were plotted using RROC software.2.3.4Decision curve analysis was performed with remote direct memory access (RDMA) software and was used to evaluate the clinical value of the nomogram. The nomogram was constructed to estimate a net benefit for the prediction model by quantifying the net benefits at different threshold probabilities.33.1− and SLN+. The patients were treated at PUMCH from 1 January 2014 to 1 January 2019. The internal validation cohort consisted of 175 patients, who were treated at PUMCH from 1 January to 31 December 2019. Eighty‐one patients made up the external validation cohort, and these patients were treated from 1 January to 31 December 2019 at Baoji Maternal and Child Health Hospital, Qinghai Provincial People's Hospital, and Shanxi Traditional Chinese Medical Hospital. The clinical, US, and pathological characteristics from the multicenter cohorts are listed in Table The training set comprised the data of 472 patients, with 360 and 112, respectively, SLN+; in the subgroup specifically with DCIS (at biopsy), this rate was 11.63%. The median age at diagnosis was 48 years . The patients' ALN‐US and basic clinical pathological characteristics are listed in Table + differed significantly from the SLN− with regard to histology type (p < 0.001); ALN‐US shape, CMD, and blood flow ; progesterone receptor (PR) status (p = 0.003); and Her‐2 status (p = 0.049). There were no differences for LVI, nerve invasion, and IMPC .In the overall training cohort, 23.73% were SLN3.23.2.1+ status. Based on the univariate analysis, variables with a difference of p < 0.1 were incorporated into the multivariate analysis , PR positivity , irregular ALN‐US shape , unclear ALN‐US CMD , and blood flow on ALN‐US .The multivariate logistic regression model found that histological type, PR status, ALN‐US shape, CMD, and blood flow were associated with an increased likelihood of SLN metastasis. Specifically: IDC histology  and ALN‐US morphology  and 0.942 (95% CI: 0.918–0.966). This further confirmed that the LN prediction model had good predictive accuracy and clinical applicability  of 0.714. The PUMCH nomogram can be used to assist clinicians to predict SLN metastasis preoperatively, and thus may assist decisions regarding surgical strategy for patients with breast cancer.Herein, ALN‐US was evaluated relative to pathological characteristics for differentiating patients with positive or negative SLNs, and a PUMCH nomogram was constructed for predicting SLN metastasis. It was found that patients who were SLNThis study provides preoperative axillary evaluation for all breast cancer patients, and not only for the specific types covered by the Z0011 or SOUND trial.The current rise in SLN dissection has highlighted the need to predict LN metastasis and aid the selection and treatment of these patients. Scientists have reported many clinical characteristics that are related to LN metastasis, including multifocality, LN palpability, histological type, and LN status determined via radiomics.In recent years, more and more nomograms have been built using imaging data to preoperatively evaluate SLN metastasis, including breast MRI,Others have predicted ALN in early breast cancer,Although the Tenon, MSKCC, MDA, Mayo, Cambridge, and Stanford modelsTo our best knowledge, there is no reliable evaluation and reporting standard for ALN, and the overall accuracy of ALN‐US remains controversial.We wonder if ALN criteria in breast cancer might be incorporated into the Breast Imaging‐Reporting and Database System (BI‐RADS). Such criteria could include detailed evaluation of the eccentric placement, complete anechoic or hypoechoic appearance of the LN, axillary and echogenic hilus obliteration, asymmetric cortical thickening, and other possible quantitative evaluations gained from US ALN‐US. Our team is also developing another artificial intelligence‐assisted SLN prediction system. Based on a convolutional neural network, deep learning is gained via US images. In this way, metastatic LNs may be identified by ultrasonography with high sensitivity and positive predictive value, assisting in the stratification of patients with ALN metastasis, aiding in therapy planning and patient staging, and ultimately contributing to improvements in surgery strategy and survival rates.2 = 6.333 and p = 0.032, there was a statistical difference. Hence, about 25% of women with seemingly pure DCIS on initial biopsy will be found with invasive breast cancer at the time of the definitive surgical procedureIn the research of Fidan et al.Besides, this study has other limitations. First, the sample size was limited, especially for external validation data from other institutions. Second, this is a retrospective study rather than prospective study. Thus, selection bias may have existed. Meanwhile, there is still room to improve the diagnostic efficiency of the model. What's more, ultrasound specialists' examination is subjective and inevitably biased. Therefore, we are conducting a prospective study to construct an artificial intelligence model.5+. The PUMCH‐SLN nomogram should be of great benefit to assist clinical decisions. US morphological characteristics are a highly credible reference to supplement pathology for predicting SLN metastasis. This nomogram could be a convenient practical preoperative tool to warn of false‐negative SLN. This nomogram could also assist in decisions about further treatment in some patients with complicated breast cancer.In the present study, 1 in 4 patients with a suspicious ALN on US, but a negative clinical physical examination, had ALN metastasis. The PUMCH‐SLN nomogram constructed in this study shows that patients with IDC, PR‐positive status, and ALN‐US with irregular shape, unclear CMD, and presence of blood flow are more likely to be SLNXuefei Wang: Conceptualization ; writing – original draft ; writing – review and editing . Guo‐chao Zhang: Methodology . Zhi‐chao Zuo: Conceptualization ; formal analysis . Qing‐li Zhu: Data curation . Zhen‐zhen Liu: Data curation . Shafei Wu: Data curation . Jia‐xin Li: Supervision . Jian‐hua Du: Supervision . Cun‐li Yan: Conceptualization ; formal analysis . Xiao‐ying Ma: Conceptualization ; formal analysis . Yue Shi: Conceptualization ; formal analysis . He Shi: Conceptualization ; formal analysis . Yidong Zhou: Project administration . Feng Mao: Project administration . Yan Lin: Project administration . Song‐jie Shen: Project administration . Xiaohui Zhang: Project administration . Qiang Sun: Project administration .This work was supported by Fundamental Research Funds for the Central Universities (No. 3332021012) and CAMS Innovation Fund for Medical Sciences (CIFMS) (No. 2021‐I2M‐C&T‐B‐018), National High Level Hospital Clinical Research Funding(2022‐PUMCH‐A‐018), and Tsinghua University‐Peking Union Medical College Hospital Initiative Scientific Research Program (No. 2019Z).Each author declared that he/she has no conflict of interest.This study was approved by the ethics committee of Peking Union Medical College and Hospital. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.All data published here are under the consent for publication. Written informed consent was obtained from all individual participants included in the study.Figure S1Click here for additional data file.


Protozoans are endemic in the Middle East and diarrhea workup post stem cell transplant should always include stool examination for ova and parasites.Blastocystis sp. infection in a multiple myeloma patient who developed diarrhea on day five of post‐autologous stem cell transplantation. Diarrhea is a major complication and a potential cause of mortality in this population. Testing for endemic protozoan is recommended.We present a case of  Blastocystis sp. (red arrow).Stool sample under light microscopy showing  The incorporation of high‐dose chemotherapy and autologous stem cell transplantation (ASCT) more than 40 years ago led to significant improvement in progression‐free survival and overall survival compared to conventional therapies.22 subcutaneously, Lenalidomide 25 mg by mouth daily for 14 days continuously on days 1–14 followed by 7 days without lenalidomide, and weekly dexamethasone 40 mg by mouth) for 6 cycles with minimal side effects. He has achieved a stringent complete response (sCR). After a thorough assessment, including a negative viral screen for hepatitis C, B, and HIV serologies, he was deemed fit for an ASCT. He underwent stem cell mobilization with cyclophosphamide 2000 mg/m2 intravenously with G‐CSF 5 mcg/kg subcutaneously every 12 h. A total of 3.92 × 106/kg CD34+ cells were collected and stored in a fridge at 6°C with no cryopreservation. The next day, melphalan 200 mg/m2 intravenously was administered, and after 2 days after the collection, the patient received his own stem cells back. On the 5th day of the transplant, the patient developed significant diarrhea, watery stool, and low‐grade fever.A 52‐year‐old Middle Eastern male known to have hypertension was diagnosed in June 2021 with IgG kappa multiple myeloma with an unknown cytogenetic profile after presenting with severe anemia. He was started on VRD protocol (Velcade (bortezomib), Revlimid , dexamethasone dosed at weekly bortezomib 1.3 mg/mOn examination, the patient had mild periumbilical discomfort with no signs of peritonitis. His pulse rate was 84 beats/min, his temperature at 37.4°C, and his blood pressure was 130/80 mmHg.9/L, and a total leukocyte count of 0.1 × 109/L. The liver enzymes and kidney functions were within the normal range. A fresh stool sample was sent to the microbiology lab for culture, ova and parasites and Clostridium difficile toxin A/B assay. All test results returned normal except for stool microscopy which identified a round cyst‐like organism with a large central body surrounded by small, multiple nuclei consistent with Blastocystis sp. with no white or red blood cells seen . It requires more than five organisms to be seen in a high‐power field microscope to be considered significant.Clinical presentation is typical for mild abdominal colicky pain, flatulence, watery, greenish diarrhea, and in some cases, arthralgia.Blastocystis sp. includes metronidazole and trimethoprim‐sulphamethoxazole.Blastocystis sp. was limited by using conventional microscopy alone. This method alone can under‐detect pathogens and hence molecular methods are recommended.The treatment of This case highlights the need to capture infectious events in stem cell transplant patients and record them in a registry. It also highlights the importance of investigating parasitic intestinal infections when a stem cell transplant patient develops diarrhea, especially in endemic areas which are currently our standard of care for patients coming from endemic areas. They are easy to treat, and early identification with prompt therapy can help speed their recovery.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.Osman Radhwi: Supervision; writing – original draft. Abdullah Almohammadi: Investigation; writing – original draft. Adel Almarzouki: Data curation; investigation; writing – review and editing. Ahmed Barefah: Conceptualization; data curation; writing – review and editing. Salem Bahashwan: Conceptualization; data curation; writing – review and editing. Hatem Alahwal: Conceptualization; writing – review and editing.None.All authors declare no conflict of interest.


Cardiac amyloidosis (CA) may affect all cardiac structures, including the valves. From 423 patients undergoing a diagnostic workup for CA we selected 2 samples of 20 patients with amyloid transthyretin (ATTR-) or light-chain (AL-) CA, and age- and sex-matched controls. We chose 31 echocardiographic items related to the mitral, aortic and tricuspid valves, giving a value of 1 to each abnormal item. Patients with ATTR–CA displayed more often a shortened/hidden and restricted posterior mitral valve leaflet (PMVL), thickened mitral chordae tendineae and aortic stenosis than those with AL–CA, and less frequent PMVL calcification than matched controls. Score values were 15.8 (13.6–17.4) in ATTR–CA, 11.0 (9.3–14.9) in AL–CA, 12.8 (11.1–14.4) in ATTR–CA controls, and 11.0 (9.1–13.0) in AL–CA controls . Area under the curve values to diagnose ATTR–CA were 0.782 in patients with ATTR–CA or matched controls, and 0.773 in patients with LV hypertrophy. Patients with ATTR–CA have a prominent impairment of mitral valve structure and function, and higher score values. The valve score may help identify patients with ATTR–CA among patients with CA or unexplained hypertrophy.The online version contains supplementary material available at 10.1007/s10554-023-02901-2. Amyloidosis is a systemic disorder characterized by extracellular deposition of insoluble fibrils. The vast majority of cases of cardiac amyloidosis (CA) are caused by the accumulation of immunoglobulin light-chain (AL–CA) or transthyretin (ATTR–CA), a carrier for thyroxine and retinol-binding protein . On echoWe evaluated 423 consecutive patients referred to the Fondazione Toscana Gabriele Monasterio (FTGM), Pisa, Italy from 2015 to 2020 for a diagnostic work-up for suspected CA. Patients were referred because of proven systemic AL amyloidosis n = 60, 14%) or unexplained increased LV wall thickness on echo  , together with clinical and/or laboratory findings compatible with CA % or unex. CA was For the purposes of this study, we randomly selected 2 samples of patients with ATTR–CA or AL–CA (n = 20 each), and we matched them by age and sex with 2 samples of patients with CA excluded. Patients with prosthetic valves or an history of valvuloplasty were excluded both from the CA and control groups. The study protocol conformed to the 1975 Declaration of Helsinki and was approved by the Institutional Human Research Committee. All patients provided written informed consent.2 (men) or ≥ 95 g/m2 (men) . Among all clinical, laboratory and echocardiographic characteristics, only age, interventricular septum and posterior wall thickness, LVMI, and continuous score values or values higher than or equal to 14 emerged as univariable predictors of ATTR–CA. None of these variables independently predicted ATTR–CA . Valve score values had an AUC of 0.739 to distinguish ATTR–CA from matched controls; the best cut-off was again 14, with the same sensitivity, specificity, PPV and NPV than for the differentiation between ATTR- and AL–CA. Absolute valve score values and the 14 cut-off were both univariable predictors, while they did not reach independent prognostic significance .We also compared the valve score and 2 validated diagnostic scores (IWT and AMYLI). AUC values to diagnose ATTR–CA were 0.782, 0.846 and 0.902, respectively, in patients with ATTR–CA or matched controls, and 0.773, 0.679 and 0.706 in patients with LV hypertrophy    died over 2.1 years (1.0–3.7); 6 patients out of 19 with available data (32%) died for cardiovascular causes. 10 Patients out of 18 (55%) were hospitalized for HF over 1.3 years (0.6–2.3). Among patients with AL–CA, 9 died (45%), 7 of whom for cardiovascular causes (35%), over 3.1 years (0.7–7.4); 9 patients out of 16 (56%) were hospitalized for HF over 0.4 years (0.2–1.2). The valve score was not a good predictor of fatal outcomes in ATTR–CA , while it was more predictive in AL–CA . AUC values for HF hospitalization were quite similar in ATTR–CA (0.700) and AL–CA (0.635).The valve score is the first attempt to synthesize an in-depth description of multiple morphological and valve function parameters of CA patients in a score, easily derivable by both expert and novice echocardiographers. We report that patients with ATTR–CA have a greater impairment of MV structure and function, conditioning higher score values in this group. ATTR–CA patients more often displayed a shortened or hidden and retracted PMVL and a thickened mitral chordae tendineae compared with AL–CA patients, and at the same time patients with AL–CA displayed more often a short or hidden PMVL than their matched controls. While the score was designed with primarily descriptive purposes, we showed that it retains diagnostic and potentially prognostic potential.The valve score was designed to allow a comprehensive evaluation of multiple valves, in agreement with the notion of CA as a systemic disorder. The more recognized feature of valvular involvement in CA is increased thickness of atrioventricular valve leaflets and sub-valvular apparatus. The thickness of each mitral and tricuspid leaflet and aortic cusps was explored. Restricted leaflet motion in the MV has been associated with valve dysfunction in CA, even if there is less consensus that it is a characteristic feature of CA compared to leaflets thickening . In our A particularly interesting aspect is the role of calcification. Even if the literature on this subject is scant, we assumed that a typical feature of CA should be thickening of the valvular leaflets without accompanying calcification, which usually represents an ominous sign in degenerative (broadly defined as “senile”) forms. Thus, we gave 1 point to the absence of calcification and 0 to its presence, as we deemed calcium deposits to be less determinant in causing CA-related valve dysfunction. Our results were in line with this assumption, as we report significantly less frequent calcification of the PMVL in ATTR–CA compared to their matched controls. Except for the tricuspid valve, calcification was less present in ATTR–CA cases compared to controls, although differences did not reach statistical significance possibly because of the small number of patients.The valve score shares some similarities with other established echocardiographic score systems, especially with ones designed to assess the mitral valve. The most similar is the Wilkins score, which is still the most used in clinical practice . This scThe greater valve involvement of ATTR–CA patients was mirrored by a significantly higher mean global valve score in this group . This could be explained by the fact that ATTR–CA patients were older than the AL–CA group. However, we found no other significant clinical difference between our groups other than age, and even measures of global functional impairment, such as NYHA class, did not differ. The only notable difference was that patients with ATTR–CA had greater degrees of LV hypertrophy than their matched controls or even than patients with AL–CA. Another possible explanation is that the slower disease progression of ATTR–CA allows for the slow deposition of amyloid and thus the manifestation of valve dysfunction, a phenomenon that with AL–CA does not have time to realize. Lastly, it is possible that ATTR has a greater tropism for heart valves compared to light-chains, even if this hypothesis does not appear to be supported by autoptic histopathological studies assessing the amyloid burden in AL–CA heart valves , 25, 26.ATTR–CA valve involvement was even more marked considering the mitral valve: indeed, ATTR–CA patients more often displayed a shortened or hidden and retracted PMVL and a thickened mitral chordae tendineae compared with AL–CA patients. In particular, considering only partial score results for the mitral valve, we found that ATTR–CA had higher mitral score values than those with AL–CA or than ATTR–CA controls. Additionally, patients with AL–CA displayed more often a short or hidden PMVL than their matched controls.As for the aortic valve, we found a higher prevalence of valve stenosis in the ATTR–CA group than the AL–CA group, and a trend towards less frequent calcification in patients with ATTR–CA compared with those with AL–CA (p = 0.091). Differences between groups in the tricuspid valve scores did not reach statistical significance, perhaps reflecting a less severe involvement of the tricuspid valve in the disease process.Beyond the description of individual valves, we reported significant differences also in terms of global score values: the total mean valve score was 15.8 in patients with ATTR–CA, 11.0 in those with AL–CA, 12.8 in ATTR–CA controls, and 11.0 in AL–CA controls . The only instance where our score was not able to discriminate between groups was between AL–CA and matched controls. These results were driven by differences in mitral valve features.Valve score values allowed to distinguish between ATTR- and AL–CA and between ATTR–CA and their matched control, with 14 as the best cut-off, and a fair diagnostic performance. The valve score, possibly integrated in a more comprehensive echocardiographic evaluation, may at least orient towards one of the two forms of CA, although it cannot replace other more specific tests such as the search for a monoclonal plasma component or diphosphonates scintigraphy. Interestingly, the performance of the valve score to diagnose ATTR–CA did not differ significantly from two echocardiographic diagnostic scores, i.e., the IWT and AMYLI scores.The valve score was not a good predictor of fatal outcomes in ATTR–CA, while it was slightly more predictive in AL–CA . AUC values for HF hospitalization were similar in ATTR–CA (0.70) and AL–CA (0.63). The prognostic value of the score is therefore quite limited but not entirely negligible, also considering the small number of patients considered and the lack of composite endpoints.This hypothesis-generating study evaluated a small number of patients, none of whom with ATTRv.We also focused on a single echocardiogram at the time of diagnosis, while follow-up echocardiograms might have provided valuable insights on valvular disease progression. The IWT and AMYLI scores were employed in a different way than originally proposed . Possible developments of this study are the identification of the diagnostic and prognostic yield of each score item, and the creation of two versions of the score  to be used for diagnosis or risk stratification.Also, weighting of each item should be assessed in larger cohorts, as well as modified AP diameter.The valve score is a reproducible, easily derivable, assessment tool for valve disease in CA. Although valve morphology and function alone are not sufficient to diagnose CA or ATTR–CA, patients with ATTR–CA have a prominent impairment of mitral valve structure and function. Higher score values may then help identify patients with ATTR–CA among those with CA or with unexplained hypertrophy.Supplementary file1 (DOCX 115 KB)Below is the link to the electronic supplementary material.


Manganese (Mn) is an important trace element for laying hen's nutrition, which is required in small amounts in the diet. Its deficiency results in lowered production performance and eggshell quality.This experiment was conducted to investigate the influence of different sources and levels of Mn on egg production performance and eggshell quality in aged laying hens.A total of 720, 83‐week‐old Hy‐Line W‐36 laying hens were fed a non‐Mn supplemented basal diet for 4‐week (to ime Mn‐exhaustion of body) and then were allocated to a completely randomized design with 10 treatments, six replicates and 12 birds each. Concentration of Mn in the non‐Mn supplemented basal diet was 10.34 mg/kg (treatment 1), the added doses of dietary Mn were included 30, 60, and 90 mg/kg of three different sources  for treatments 2‐10, respectively. The experiment lasted for 12 week.Dietary supplementation with either organic or inorganic Mn sources significantly enhanced egg production (EP), egg mass (EM), feed conversion ratio (FCR), and relative eggshell weight (RESW) compared with the non‐Mn supplemented diet. However, the experimental diets did not influence feed intake (FI), egg weight, and other eggshell quality traits. Based on the broken line regression models, the performance traits were optimized at 30–40 mg/kg Mn concentration when supplemented by Mn‐sulphate or Mn‐organic. Although, it was 80–90 mg/kg when supplemented by Mn‐oxide. The relative bio‐efficacy of inorganic Mn sources include Mn‐oxide and Mn‐sulphate in compare with Mn‐organic were estimated 45% and 87% (for EP trait), 30% and 94% (for EM trait), 36% and 99% (for FCR trait), and 37% and 78% (for RESW trait), respectively.In the aged laying hens, Mn requirement is higher than the NRC's recommendation. Sulphate and organic sources of Mn are more effective than Mn‐oxide. In the aged laying hens: Mn requirement is higher than the NRC (1994) recommendation. Mn‐organic and Mn‐sulphate sources of manganese are more effective than Mn‐oxide. Mn is aHowever, the National Research Council had recommended a dietary Mn concentration of 20–25 mg/kg for laying hens NRC, . This suMinerals are added to the poultry diets in inorganic or organic forms. In organic form, minerals linked with amino acids and become stable and more bioavailable  and soybean meal  were analyzed for composition by the methods described AOAC, , and cor2.22/hen’. The experimental period included a 4‐week fed a non‐Mn supplemented ration, Mn‐exhaustion phase, followed by a 12‐week fed experimental diet and data collection phase. The birds were individually weighed and randomly assigned to 10 treatments in a completely randomized design with six replicates/treatment and 12 birds/replicate. The basal diet was formulated to meet energy and nutrients requirements, except Mn, as recommended by the strain Management Guide  or organic supplement ‘Availa Mn; Manganese Amino Acid complex 10% Mn, Zinpro Amino Acid Nutrition Inc., Eden Prairie Mn’ at the rate of 300, 600, and 900 mg/kg was added on top of each portion and mixed to make one non‐Mn supplemented diet that contained 10.3 mg/kg Mn concentration and nine experimental diets with 40, 70, and 100 mg/kg Mn concentration supplemented by each source, respectively  and mortality were recorded daily, egg weight was recorded weekly, and feed intake and egg quality traits were recorded monthly. Feed intake was calculated by weighing the residual feed in feeders for the respective period. Feed consumption was adjusted for mortality. The feed conversion ratio (FCR) was adjusted for mortality and calculated as follows: total feed intake divided by total egg mass.2.4For evaluating egg quality traits, six eggs/replicate (36 eggs/treatment) from eggs laid throughout the 3 consecutive days by the end of every period (26–28 days) were randomly selected and transported to an egg quality laboratory. On the sampled eggs, maximum width and length were measured by passing the eggs through the digital Caliper  to obtain the maximum points and then calculating the egg shape index by the following formula , the egg components, including yolk and albumen, were separated with a commercially hand‐held egg separator. A moist cloth napkin was used to get rid of the adhering albumen residues from the yolk, and then it absolutely was weighed. The eggshells were washed with water, dried for 48 h, and weighed . After filtration, the volume was increased to 100 mL using demineralized water , adjusted R2 (adj. R2), root means square error (RMSE), and Akaike's information criterion (AIC) values were calculated using the following formulas  or I = 0 (if X > R), U is the rate constant, yt is the observed values, ŷt is the predicted values, N is the number of observations, P = k + 1, and k is the number of parameters.where A multi‐linear regression model was applied to estimate the relative bio‐efficacy (RBE) value of inorganic Mn (Mn‐oxide and Mn‐sulphate) to Mn‐organic. The nonlinear models procedure (PROC NLIN) of the SAS system was applied, and the models were fit using the following equation:Y is the dependent variable, a is the intercept , b is the asymptote response, b1 is the slope ratio for Mn‐oxide, b2 is the slope ratio for inorganic Mn‐sulphate, b3 is the slope ratio for organic Mn, and X1, X2, and X3 are dietary supplemented levels of Mn‐oxide, Mn‐sulphate, and organic Mn, respectively.where The RBE value is the ratio between the standard amount and testing source that is required to generate equivalent responses in which a nutrient source at different levels is used and then it is compared to a reference standard based on a biological response such as growth and bone mineralization  as an effect of increased dietary Mn concentration with Mn‐oxide. The birds fed supplemented diet at 90 mg/kg Mn‐oxide reached the highest EP, EM, and lowest FCR. However, differences were not significant. An improvement was noticed in egg production performance traits  by using diet supplementation with Mn‐organic and Mn‐sulphate up to 60 mg/kg Mn, but by increasing dietary supplementation to higher level (90 mg/kg Mn), birds showed undesirable performance .The effect of different treatments on egg production performance indices is shown in Table 3.2p < 0.05) and Mn‐oxide , while egg shape index, the other eggshell quality traits, such as egg special gravity and shell thickness showed non‐significant response (p > 0.05) to dietary Mn supplementation level. Similarly, the internal and external egg quality traits such as shape index, Hugh unit, and egg composition showed a non‐significant response (p > 0.05) to dietary Mn supplementation (data not shown).The effects of dietary supplementation with Mn on egg quality characteristics are presented in Table 3.3p > 0.05). We did not observe differences in tibia Mn, calcium, and phosphorus content (data not shown) between the dietary treatments (p > 0.05).The influence of dietary sources and levels of Mn on the tibia bone characteristic of laying hens at 98 weeks of age is shown in Table 3.4R2 and the lowest RMSE and AIC, the linear broken‐line (LBL) fit model was selected to estimate Mn requirements for the diets supplemented with Mn‐oxide and the quadratic broken‐line fit model was achieved to estimate the Mn requirement for the diet supplemented with Mn‐sulphate and or Mn‐organic. The best balance of performance traits and dietary Mn concentration was found at 82–92 mg/kg of diet as supplemented by Mn‐oxide, 38–40 mg/kg of diet as supplemented by Mn‐sulphate, and 33–40 mg/kg of diet as supplemented by Mn‐organic for egg production performance traits .In order to investigate the effect of dietary Mn supplementation on egg performance traits, a crucial goal of the current study was to estimate Mn requirement of Hy‐Line W‐36 aged laying hens. The optimization model was solved by using the NLIN program SAS 9.1 procedure. Fitted broken‐line models for the egg performance traits as a function of dietary Mn concentration are shown in Figures 3.5p < 0.05), and it is reasonable to apply these sensitive parameters to evaluate the RBE of organic Mn compared with inorganic Mn in aged layer eggshell quality. The RBE of inorganic Mn‐oxide was estimated 45%, 30%, 36%, and 37% as efficacious as Mn‐organic at equimolar Mn levels for EP, EM, FCR, and RESW traits, respectively. Similarly, the RBE of Mn‐sulphate was estimated 87%, 94%, 99%, and 78% as efficacious as Mn‐organic at equimolar Mn levels for EP, EM, FCR, and RESW traits, respectively.The slope ratio method for the variables of performance and eggshell quality response was employed to measure the RBE of inorganic Mn (Mn‐oxide and Mn‐sulphate) compared with Mn‐organic Table . In the 44.1p > 0.05). Sazzad et al.  concentration results in an optimum egg productive performance. Various diet Mn levels for laying hens have been reported by researchers. The NRC  proposed4.5In the current experiment, the RBE of Mn‐oxide and Mn‐sulphate was estimated to be 30%–45% and 76%–99%, respectively, as efficacious as Mn‐organic at equimolar Mn levels. However, limited reports are available on the RBE of different Mn sources for laying hens, and certain published results are inconsistent  were estimated 45% and 87% (for EP trait), 30% and 94% (for EM trait), 36% and 99% (for FCR trait), and 37% and 78% (for RESW trait). Overall, in the aged laying hens, Mn requirement is higher than the NRC's  recommenHeydar Zarghi and Nafise Barzegar designed and carried out the experimental trail. Nafise Barzegar performed lab analysis. Heydar Zarghi performed the statistics, tabulated the data, and wrote the draft paper. Ahmad Hassanabadi revised and reviewed the manuscript.The authors declare no conflict of interest. The authors are faculty member at Ferdowsi University of Mashhad, Mashhad, Iran. They are employed by an institutional that has a primary function research and/or education. The authors did not submit this manuscript as an official representative or on behalf of the government.https://publons.com/publon/10.1002/vms3.1116.The peer review history for this article is available at The authors confirm that the ethical policies of the journal, as noted in the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors also confirm that they have followed EU standards for the protection of animals used for scientific purposes and feed legislation.


Gastropteron rubrum, feeding on hard Foraminifera, diatoms and Porifera. Tooth morphologies and wear were documented by scanning electron microscopy and their mechanical properties were tested by nanoindentation. We determined that gradients of hard‐ and stiffness run along each tooth, decreasing from cusp to basis. We also found that inner lateral teeth were harder and stiffer than the outer ones. These findings allowed us to propose hypotheses about the radula‐ingesta interaction. In search for the origins of the gradients, teeth were visualized using confocal laser scanning microscopy, to determine the degree of tanning, and analyzed with energy‐dispersive X‐ray spectroscopy, to test the elemental composition. We found that the mechanical gradients did not have their origins in the elemental content, as the teeth did not contain high proportions of metals or other minerals. This indicates that their origin might be the degree of tanning. However, in the tooth surfaces that interact with the ingesta high Si and Ca contents were determined, which is likely an adaptation to reduce wear.The molluscan feeding structure is the radula, a chitinous membrane with teeth, which are highly adapted to the food and the substrate to which the food is attached. In Polyplacophora and Patellogastropoda, the handling of hard ingesta can be facilitated by high content of chemical compounds containing Fe or Si in the tooth cusps. Other taxa, however, possess teeth that are less mineralized, even though animals have to avoid structural failure or high wear during feeding as well. Here, we investigated the gastropod  Mollusks possess highly adapted radular teeth that allows them to forage on their preferred food. We here investigated the teeth of a colorful sea slug that feeds on hard Foraminifera and Porifera. Teeth contained high content of silica to reduce abrasion. Mechanical property tests revealed that this radula seems to function as a clamp, which can adjust to all kinds of irregular objects. Radulae can show adaptations to the preferred ingesta  in morphology and the arrangement of teeth on the membrane  by over‐ and underlain epithelia  resulting in hard tooth cusps as adaptation to feeding from algae growing on stones , which forages on Foraminifera . For visualization, we used a SEM Zeiss LEO 1525 (One Zeiss Drive). Only mature teeth from the working zone, which can be identified by lack of covering epithelia, were studied. To document the wear on the teeth, radulae were rewetted by 70% EtOH afterwards, cleaned by a short ultrasonic bath, rearranged on SEM sample holders and visualized again in the SEM. Nomenclature of teeth was adapted from Ong et al. .For the 3D visualization, mature radular teeth of the working zone of two radulae were extracted manually with forceps. Each tooth was mounted on SEM specimen holders by double‐sided adhesive carbon tape, sputter‐coated with platinum (5 nm layer), and visualized under the SEM from all sides. Using the 3D software Blender v2.83 (Blender Foundation), the teeth were then modeled by hand constantly comparing the 3D visualization with the SEM images taken from different sides  were attached to glass object slides by double‐sided adhesive tape, following our previous protocol  were analyzed to receive the data. Following elements were detected and their proportions measured: H (hydrogen), C (carbon), N (nitrogen), O (oxygen), Pt (platinum), Al , Ca , Cl (chlorine), Cu (copper), Fe (iron), K (potassium), Mg (magnesium), Na (sodium), P (phosphorus), S (sulfur), Si (silicon), and Zn (zinc). Some elements were not discussed as they are either the elemental basis of chitin and proteins , the coating (Pt), or the polishing powder . For test purposes, we also performed 10 EDX tests on the epoxy to identify putative pollution due to the mechanical application, embedding or polishing. We could not detect Si (which is part of the sandpaper), or any other elements, that we further discuss as Ae , in the resin. Their presence is therefore considered part of the teeth.The single peak of P overlaps with one of Pt. Due of this, the software could not discriminate between these two elements and P content could not be reliably determined. Therefore, P and Pt are discussed together (P + Pt). We, however, measured 20 areas of pure epoxy to receive values on their Pt content  to further estimate the proportions of P in the teeth.We tested the inner tooth structure by EDX and the thin outer layer (“surface”) of the teeth (500–1000 nm thickness), which covers the inner tooth structure. We did not detect high content of elements in the inner structure and no differences in the distribution there. We thus decided to summarize the point measurements of the inner structure. With regard to the surfaces, we could determine variations in the distribution of elements and thus differentiated between the tooth basis, the bulges, the basal region of the stylus , the terminal region of the stylus , the cusps and the sides  and Young's modulus (E) were determined from force‐distance curves by applying the continuous stiffness mode. All tests were performed under normal room conditions  and each indent and corresponding curve were both manually controlled. After this, samples were smoothened and polished until the next target localities were on display.To test the mechanical properties, nanoindentation experiments were performed on three additional radulae . Mean values and standard deviations were calculated and Shapiro–Wilk‐33.1Gastropteron rubrum possessed one prominent inner lateral tooth, followed by five outer laterals to each side of the radula, per row . The cusps' lateral surfaces, the tooth styli and bases appeared rather red‐ to yellow‐brown  .In the inner tooth structure, the following proportions were found (sorted from high to low mean content): P + Pt (mean ± standard deviation: 0.56 ± 0.26 atomic %), Fe (0.53 ± 0.35), Ca (0.47 ± 0.23), S (0.34 ± 0.12), Mg (0.26 ± 0.09), Zn (0.06 ± 0.03), Cu (0.05 ± 0.04), Si (0.04 ± 0.13), Cl (0.03 ± 0.03), and K (0.03 ± 0.02). All lateral teeth showed a rather small proportion of these elements in the inner tooth structure, compared to the surface : Si (mean ± standard deviation: 5.79 ± 6.40 atomic %), Ca (2.28 ± 2.28), P + Pt (0.85 ± 0.60), S (0.67 ± 0.30), Fe (0.60 ± 0.72), Mg (0.34 ± 0.20), Cu (0.14 ± 0.11), Zn (0.12 ± 0.08), Cl (0.08 ± 0.08), and K (0.05 ± 0.03). In each tooth, especially Si and Ca were present in larger proportions in comparison to the other elements . For the distribution of Cl, Cu, K, Mg, P + Pt, S, and Zn, no clear gradient could be detected, even though most regions differed highly significantly as determined by pairwise comparison . These elements were present in rather small proportions in each tooth .When the results from the tooth surfaces were sorted according to the tooth region  describes the resistance to local plastic deformation induced by abrasion or indentation. The Young's modulus (E) is the measure of the stiffness of a solid material and describes the relationship between tensile stress and axial strain.The hardness  was always the stiffest and hardest region, followed by the terminal stylus , the basis of the stylus , the basis , and finally the bulge as the softest and most flexible region . The parts of the inner laterals were harder and stiffer, followed by the outer laterals A, B, C, D, and finally E with the softest and most flexible parts . Most regions were also highly significantly different, when they were compared between the teeth .Pairwise comparison by Wilcoxon method revealed, that the regions within each tooth showed highly significant differences .Despite the differences of 3.6The regions of the teeth appearing green under CLSM  contained high proportions of Si and Ca. The sides of the stylus and the bases appeared red‐ to yellow‐brown ; here no high contents of Ca and Si were detected. The bulges appeared blue ; these regions contained no high content of the targeted elements and were the softest and most flexible tooth parts. We could not determine relationships between the elemental content of the inner tooth structure and the mechanical property values.44.1E) relates to the ability of a structure to transmit force  is the measure of the resistance to local plastic deformation induced by indentation or abrasion.The mechanical properties of materials directly contribute to the mechanical behavior of structures. The Young's modulus  without clear and pronounced gradients in mechanical properties from the tooth basis across the stylus to the cusp  feeding on soft ingesta  possess soft and more flexibles teeth (Felimare picta and Doris pseudoargus feeding on Porifera with hard spiculae) possess harder and stiffer teeth reducing wear and structural failure. Each tooth shows pronounced gradients in both H and E: the cusp  is the hardest and stiffest part, followed by the stylus, and finally the basis as the softest and most flexible part  or have some interactions with hard ingesta (the nudibranch gastropods Patella vulgata (Patellogastropoda) are characterized by a very high inorganic content and high E and H values (see Table Megathura crenulata (Fissurellidae), E values of 16 GPa were determined  and stiffer (Emax = 45 GPa) than the inner structure, due to high proportions of Si or Ca. The unmineralized teeth of paludomid gastropods foraging from solid surfaces were even softer and more flexible in comparison to the inner structure of the nudibranch taxa , the different teeth of each row had similar mechanical properties. Here, teeth probably also had similar functions , which are hard and protected against wear , high proportions of iron, silicon, and calcium are incorporated into some tooth cusps, which directly relate to mechanical property differences in the various tooth regions ; data curation (lead); funding acquisition (lead); investigation ; methodology ; project administration (lead); supervision (lead); validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Charlotte Neumann: Investigation ; methodology ; validation (supporting); visualization (supporting). Stanislav N. Gorb: Conceptualization (supporting); methodology (supporting); project administration (supporting); resources (lead); writing – review and editing (supporting). Alexander Koehnsen: Investigation (supporting); methodology (supporting). Heike Wägele: Conceptualization (supporting); project administration (supporting); resources (supporting); supervision (supporting); writing – review and editing .This research was financed by the Deutsche Forschungsgemeinschaft (DFG) grant 470833544 to WK.The authors declare that they have no competing interests.Not applicable.


TP53, BAX, FOXO1, and RB1 are related genes in cell survival, proliferation, and inflammation which could be important in RA development and disease severity. Here we investigated their expression in peripheral blood mononuclear cells (PBMCs) from RA patients in comparison to healthy controls.Impaired apoptosis and proliferation resulted in autoreactive lymphocyte development and inflammation in Rheumatoid arthritis (RA). Fifty healthy controls and 50 RA patients were selected. The quantitative real‐time polymerase chain reaction was used to assess the gene expression level in PBMCs.TP53 , BAX , FOXO1  and RB1  were significantly reduced in RA PBMCs. TP53 expression was negatively correlated with miR‐16‐5p (p = .032) and FOXO1 expression was negatively correlated with miR‐335‐5p (p = .005) and miR‐34a‐5p (p = .014). A positive correlation was seen between TP53 expression and its downstream gene, BAX (p = .001). FOXO1 expression was also negatively correlated with disease activity, DAS28 (p = .021).The mRNA expression of TP53 and FOXO1 expression in RA PBMCs, they could be used as future therapeutic targets.All selected genes have downregulated expression in RA PBMCs which could be correlated with RA pathogenesis by regulating apoptosis, cell survival, inflammatory mediator production, and proliferation. Due to the correlation of miR‐16‐5p, miR‐34a‐5p, and miR‐335‐5p with  Impaired apoptosis and proliferation resulted in autoreactive lymphocyte development and inflammation in rheumatoid arthritis (RA). Our results demonstrated that the expression of TP53, BAX, FOXO1, and RB1 genes are downregulated in RA PBMCs which could be correlated with RA pathogenesis by regulating apoptosis, cell survival, inflammatory mediator production, and proliferation. Due to the correlation of miR‐16‐5p, miR‐34a‐5p, and miR‐335‐5p with TP53 and FOXO1 expression in RA PBMCs, they could be one of the regulatory mechanisms and RA treatment targets. The miR‐335‐5p targeted RB1, and FOXO1, the miR‐150‐5p targeted TP53, the miR‐34a‐5p directly targeted BAX, and TP53, and indirectly targeted FOXO1, and the miR‐16‐5p targeted TP53.Based on our last experiment miR‐335‐5p, miR‐150‐5p, miR‐34a‐5p, and miR‐16‐5p were upregulated in peripheral blood mononuclear cells (PBMCs) of RA patients. In the miRTaRBase database . The Real Q Plus Master Mix Green High ROX (AMPLIQON) was used for quantitative real‐time polymerase chain reaction (PCR) . The test reaction included 10 μL 2× SYBR Green PCR master mix, 8 μL cDNA (1/8 dilution), and 1 μL each primer for reaction. The primer details are listed in Table 2.3T test or Mann–Whitney were performed. The GraphPad Prism v.8 software was used for plotting.SPSS v.28 was used to calculate data. The Kolmogorov–Smirnov test was applied for For normality evaluation. Spearman's rho test or Pearson's correlation coefficient was carried out for correlation analysis. For gene expression analysis Unpaired 33.1Clinical details of healthy control and RA patients are listed in Table 3.2TP53 , BAX , FOXO1 , and RB1 were significantly reduced in PBMCs of RA patients in comparison to controls  and RB1 expression downregulated (p = .032). however, the mRNA expression of TP53 and RB1 was also significantly decreased  in RA patients who did not receive alendronate treatment compared to controls.The mRNA expression of 3.3TP53 expression was negatively correlated with miR‐16‐5p expression (p = .032) and FOXO1 expression was also negatively correlated with miR‐335‐5p expression (p = .005). The expression of miR‐34a‐5p was negatively correlated with FOXO1 (p = .014). A positive correlation was seen between the expression of TP53 and its downstream gene, BAX (p = .001). FOXO1 expression was also negatively correlated with disease activity DAS28 (p = .021). All of the miRNAs' data were listed in Table Based on our recent experiment,4RA is a systemic inflammatory autoimmune disease marked by the deterioration of joints.TP53, BAX, FOXO1, and RB1 which contribute to the cell‐cycle regulation was investigated in the same RA patients' samples. As we discussed later, the P53 and its downstream proteins including BAX, FOXO1, and RB1 were necessary for cellular apoptosis and proliferation which are essential in autoimmune disorders including RA. P53 was reported to be downregulated in mRNA and protein levels in different cell types including PBMCsTP53 which finally leads to abnormal cell function could enhance the aggressive nature of RAFLS or autoreactivity in lymphocytes and leads to the inflammatory phenotype in RA joints.TP53 knockdown in RAFLS resulted in the production of inflammatory cytokines and T‐cell differentiation which is important in RA pathogenesis.TP53 knockout mice were also reported to have higher arthritis scores compared with TP53+ mice.TP53 downregulation can cause dendritic cells (DCs) maturation and activation, and reduce the expression of FOXP3 in T cells, which is a master regulator of regulatory T cells (Tregs) and Th17 polarization. All these changes were important in RA pathogenesis.In our previous report,TP53 transcription in RA patients' samples that might be correlated with RA pathogenesis. One of the important regulators of this protein is miR‐16‐5p which significantly increasedTP53 expression in RA PBMCs. As much as we know, these findings are the first report in RA PBMCs. P53 also has the ability to induce the expression of proapoptotic genes such as BAX.TP53 and BAX in RA patients as one of the BAX regulatory mechanisms. In this study, a significantly lower expression of BAX was reported in RA PBMCs. It was previously reported that T lymphocytes in RA patients were resistant to apoptosis which could be correlated with reduced expression of some related genes including BAX in these cells.TP53 and BAX expression was reported in another study which could help control normal apoptosis as a new strategy for the treatment of RA.Our study showed a significant reduction in FOXO1 in PBMCs of RA patients which could be correlated with disease pathogenesis and severity.FOXO1 in patients' peripheral blood and synovial tissue is correlated with disease pathogenesis, clinical parameters, and inflammatory mediators such as IL‐6 expression.FOXO1‐silenced RAFLSs have increased survival with reduced apoptosis due to a reduction in caspase 3/7 activity.FOXO1‐silenced RAFLSs also have reduced inflammatory cytokines including IL‐6, IL‐8, TNF‐α, and IL‐1β which were essential in RA pathogenesis.FOXO1 in PBMCs of RA patients which also negatively correlated with disease activity, DAS28 in patients. Our previous study reported the significant upregulation of miR‐34a‐5p and miR‐335‐5p.FOXO1 expression in the RA background.FOXO1 is an important mediator in apoptosis, autophagy, antioxidative stress, cell cycle arrest, metabolism, and immune system regulation, which was important in the pathogenesis of different diseases.TP53 induction in RAFLS resulted in P21 overexpression, a potent inhibitor of CDK, and could activate RB1 and cell cycle progression in these cells.RB1 was reported in PBMCs of RA patients which might be important in the pathogenesis of this disorder.In general, the cell cycle is progressed with cyclin‐cyclin‐dependent kinase (cyclin‐CDK). RB1 is inactivated with CDK kinase activity. Cyclin‐dependent kinase inhibitors (CDKIs) activated the RB1 by inhibiting CDK activity. It was reported that CDKI gene delivery into arthritic joints could arrest RAFLS proliferation and reduce the expression of pro‐inflammatory cytokine.TP53 and RB1 gene expression to some extent in PBMCs of RA patients that did not influence the statistical analysis.Based on our analysis the reduction in the expression of studied genes in patients was not associated with their received treatment and we did not find any significant association between patients' treatment and the expression of selected genes, except for alendronate. Alendronate was able to alter TP53, BAX, FOXO1, and RB1, was reduced in RA patients' PBMCs. This reduction in gene expressions may result in cell survival, resistance to apoptosis, and expression of inflammatory mediators, which were crucial for RA joint destruction or autoreactive lymphocyte selection. Here, the association between the expression of these genes and the miRNAs previously identified to target them was explored in RA PBMCs. Based on our results, miR‐16‐5p may negatively influence the expression of TP53, while miR‐335‐5p and miR‐34a‐5p negatively regulate the expression of FOXO1 in RA PBMCs. Due to the fact that there is no specific and effective treatment for RA patients, these regulatory miRNAs and their target genes or molecular pathways might be used as future therapeutic targets which require additional research.In conclusion, the expression of apoptosis and cell survival‐related genes, such as Acquisition of data, drafting the article, analysis and interpretation of data, and final approval of the article: Hamidreza Ebrahimian, Maryam Akhtari, Maassoumeh Akhlaghi, and Gholam Hossein Alishiri. The conception and design of the study, revising the article critically, interpretation of data, and final approval of the article: Elham Farhadi, Ahmadreza Jamshidi, Mahdi Mahmoudi, and Mahmood Tavallaie.The authors declare no conflict of interest.This study was performed based on the Declaration of Helsinki guidelines and was approved by the Ethics Committee of Tehran University of Medical Sciences . The written informed consent was signed by all participants before enrolling in the study.


Evidence suggests that choroid is thinner in myopes as compared to nonmyopes. However, choroidal thickness varies with the refractive error, age, axial length, and ethnicity. The purpose of this study was to determine the subfoveal choroidal thickness (SFCT) in high myopic Nepalese subjects and to investigate its association with the mean spherical equivalent refractive error (MSE), axial length, and age.  Ninety-two eyes of 92 high myopic subjects (MSE ≤ −6 diopters) and 83 eyes of 83 emmetropic subjects (MSE: 0.00 Diopters) were included in the study. SFCT was assessed using spectral domain optical coherence tomography, and the axial length was measured using partial coherence interferometry. SFCT was measured manually using the inbuilt tool within the imaging software. μm) as compared to the emmetropic subjects (353.24 ± 65.63 μm) . In high myopic subjects, there was a significant negative correlation of choroidal thickness with the axial length  and MSE . Regression analysis demonstrated a decrease of choroidal thickness by 40.32 μm (p < 0.001) for every 1 millimeter increase in the axial length and by 11.65 μm (p < 0.001) for every 1 diopter increase in the MSE.  SFCT in the high myopic subjects was significantly thinner (mean ± SD: 224.17 ± 68.91  High myopic Nepalese subjects had significantly thinner choroid as compared to emmetropes. The MSE and axial length were inversely correlated with the SFCT. Age had no effect on SFCT in this study. These findings may have implications in interpreting choroidal thickness values in clinical and epidemiological studies in myopes, especially in the south Asian population. The global prevalence of myopia has increased so rapidly in the recent decades that it has now become a significant public health concern , from EaDespite the measurement of choroidal thickness (CT) being a topic of interest in the scientific community, particularly in relation to myopia progression and myopia control, there is still no consensus on the influence of genetics  and ethnFurthermore, recent research has suggested that CT measurement is influenced by the density of choroidal and retinal pigment epithelium (RPE) pigmentation , especiaGiven the ethnic/racial and genetic attributes which impact CT, it is imperative to measure the CT data in particular myopic racial groups to account for racial differences and use CT data specific to the group in myopia control. As such, it is essential to measure CT in high myopic subjects from unique racial groups such as Nepalese myopic subjects, where these attributes are specific to them. Data on CT from other ethnic groups may not be relevant to study CT-related changes in myopia in Nepalese subjects.Several cross-sectional studies on subfoveal choroidal thickness (SFCT) in myopic and nonmyopic subjects are available in the literature , 21, 22.A cross-sectional, case-control study was conducted at B. P. Koirala Lions Center for Ophthalmic Studies (BPKLCOS), a tertiary eye hospital in Kathmandu, Nepal, for a period of 1 year. The study was approved by the Institutional Review Committee of the Institute of Medicine, Tribhuvan University, and adhered to the Declaration of Helsinki. Written informed consent was obtained from all the participants prior to the study, which was recruited from retina and refraction OPD (out-patient department) of BPKLCOS. A detailed medical and ophthalmic history including systemic disease, ocular trauma, and ocular surgery was taken. Ninety-two healthy high myopic subjects with no systemic and ocular disease  were included. Inclusion criteria for high myopic subjects were the mean spherical equivalent refractive error of ≤−6 diopters with the best corrected visual acuity of 6/9 or better. Furthermore, the subjects with ocular conditions such as macular hole, epiretinal membrane, vitreomacular traction syndrome, foveoschisis, uveitis, angioid streaks, central serous chorioretinopathy, choroidal neovascularization, keratoconus, glaucoma, and amblyopia that might alter choroidal thickness were excluded from the study. Eighty-three age and gender matched emmetropes were enrolled as control subjects. Routine ophthalmic examinations including visual acuity, objective/subjective refraction, and dilated fundus evaluation were conducted in all the participants. Both groups had normal slit-lamp biomicroscopy and funduscopy and well-defined chorioscleral junction in the OCT scan. Control subjects had a refractive error of 0.00 diopters with the visual acuity of 6/6 or better. The refractive status of the subjects was determined using dry retinoscopy as cycloplegic drugs are shown to affect choroidal thickness , 24.The axial length of high myopic subjects was measured using noncontact partial coherence interferometry . For each eye, three consecutive measurements were obtained, and the average value was taken for further calculation.Choroidal thickness was assessed using spectral domain optical coherence tomography . The enhanced depth imaging mode, which allows for a better visualization of posterior structures such as choroid and sclera was used. Volume scans consisting of 7 lines within 30° × 5° rectangle, centered on the fovea were captured from both eyes. For optimal image quality and noise reduction, averaging was kept to 100 frames per image. The horizontal section passing directly through the center of fovea was chosen for choroidal thickness measurement. SFCT in the captured enhanced image was determined as the perpendicular distance between the outer portion of hyper-reflective layer corresponding to the retinal pigment epithelium  and the hyporeflective layer corresponding to the chorioscleral junction . The meaAll images were obtained between 9 am and 12 noon to avoid possible effects of diurnal variations in choroidal thickness . For eacp value <0.05 was considered statistically significant.Statistical analyses were performed using IBM SPSS statistics version 21.0. . The data were tested for normality using the Kolmogorov–Smirnov test. Normally distributed data are expressed as mean ± standard deviation (SD), and non-normal data are expressed as median and interquartile range (IQR). Spearman's correlation test and regression analysis were used to observe the association of SFCT with the axial length and the MSE. The independent t-test was used to compare SFCT between high myopic and emmetropic subjects. The μm and 353.24 ± 65.63 μm in high myopic and emmetropic subjects, respectively . Regression analysis showed a decrease in choroidal thickness by 40.32 μm  for every 1 mm increase in the axial length. Similarly, for every 1 diopter increase in the myopic refractive error, choroidal thickness decreased by 11.65 μm   and emmetropic subjects . In high myopic subjects, mean SFCT in males and females were 230.40 ± 60 μm and 217.95 ± 77 μm, respectively. Similarly, in emmetropic subjects, mean SFCT in males and females were 348.83 ± 66.54 μm and 357.97 ± 65 μm, respectively. There was no significant difference in SFCT between males and females in both high myopic subjects  and emmetropic subjects .The median age of both high myopic and emmetropic subjects was 23 years (IQR = 8 years). The median refractive error was −7.38 D (IQR = 3.75D) for high myopic subjects and 0.00 D for emmetropic subjects. The median axial length of high myopic subjects was 26.33 mm (IQR = 1.07 mm) . The meaectively . High my< 0.001) . In highl length  and p=−0ve error ). There In the two extant studies examining choroidal thickness in healthy Nepalese population , 27, disμm for every millimeter increase in the axial length. Similar negative correlation between the choroidal thickness and axial length has been reported by several previous studies [μm for every 1 mm increase in the axial length [μm for every 1 mm increase in the axial length [μm for every 1 diopter increase in the refractive error. This rate of choroidal thinning per diopter falls within the range reported in the previous studies (5.00–25.00 μm) [μm for each diopter increase in myopia [μm for each diopter of myopia [The findings show that SFCT and the axial length are inversely correlated. SFCT decreased by 40.32  studies –32. Xionl length . Gupta el length . Such ra5.00 μm) . A studyn myopia . In anotf myopia .There are significant variations in SFCT measurements across studies. A few such studies are summarized in μm thinner choroid than age-matched emmetropes in our study. This finding is in line with the previous studies that used EDI OCT imaging for measuring choroidal thickness in high myopes (see μm vs. 300.3 ± 62.1 μm) [μm, which was significantly thinner than that of the control subjects (276.21 ± 64.67) μm [μm and 375.15 ± 6.58 μm, respectively, which are very similar to the findings in our study [μm vs. 310.31 ± 75.70 μm), which is likely due to the inclusion of relatively older patients, age ranging from 17 to 79 years [High myopic subjects had, on average, 127 opes see . Duan et62.1 μm) . Similar62.1 μm) . Wang etur study . The SFCThese findings suggest a significant thinning of SFCT in axially elongated eyes. It is speculated that the thinning of choroid with increasing axial length could be a result of progressive degenerative changes associated with myopia and peripapillary atrophy, and increased mechanical stretching of the sclera , 43, 44,This study did not find any association between age and choroidal thickness, contrasting with several previous studies where age has been found to be a significant predictor of SFCT , 44–47. A few limitations in this study must be acknowledged. First, the choroidal thickness was measured at a subfoveal location only. A complete choroidal thickness profile at various eccentricities of retina could have provided a more detailed understanding of the role of choroid in myopia. However, we were limited by the availability of resources (time and instrument) to capture such data. Nevertheless, choroidal thickness is most commonly studied at the subfoveal regions in myopic studies, and it has been shown that the effects on choroid due to axial elongation are more pronounced in the subfoveal region , 42, 39.High myopic Nepalese subjects had significantly thinner choroid compared to age- and gender-matched emmetropic subjects. The axial length and refractive error were inversely correlated with SFCT, with axial length being the greatest predictor of SFCT. The findings of this study support and corroborate the existing literature regarding the relationship between AL, MSE, and SFCT. The study provided new data on SFCT of myopic eyes from a South Asian country . Given the fact that there is a considerable ethnic and regional variability in choroidal thickness, these new data can provide important insights into the pathophysiology of myopia in the south Asian population and aid in the development of tailored myopia management strategies.


This article has been corrected: In the Materials and Methods section, under the ‘MRI Acquisition,’ heading, the model of MRI scanner has been incorrectly spelled as “All MRI examinations were performed with a 3.0 T MR scanner  in the Second Hospital of Anhui Medical University.” However, the correct model is “All MRI examinations were performed with a 3.0 T MR scanner  in the Second Hospital of Anhui Medical University.” The authors declare that these corrections do not change the results or conclusions of this paper.60568-60575. https://doi.org/10.18632/oncotarget.19104Original article: Oncotarget. 2017; 8:60568–60575.


Cancer is currently the second leading cause of death globally. There is much uncertainty regarding the comparative risks of new‐onset overall cancer and pre‐specified cancer for Type 2 diabetes mellitus (T2DM) patients on sodium‐glucose cotransporter 2 inhibitors (SGLT2I) versus DPP4I.This population‐based cohort study patients included patients who were diagnosed with T2DM and administered either SGLT2 or DPP4 inhibitors between 1 January 2015 and 31 December 2020 in public hospitals of Hong Kong.p= 0.04), cancer‐related mortality  and new diagnoses of any cancer . SGLT2I use was associated with a lower risk of new‐onset breast cancer , but not of other malignancies. Subgroup analysis on the type of SGLT2I, dapagliflozin  and ertugliflozin  use was associated with lower risks of new cancer diagnosis. Dapagliflozin use was also linked to lower risks of breast cancer .This study included 60,112 T2DM patients , of which 18,167 patients were SGLT2I users and 41,945 patients were dipeptidyl peptidase 4 inhibitor (DPP4I) users. Multivariable Cox regression found that SGLT2I use was associated with lower risks of all‐cause mortality (HR: 0.92; 95% CI: 0.84–0.99; Sodium‐glucose cotransporter 2 inhibitor use was associated with lower risks of all‐cause mortality, cancer‐related mortality and new‐onset overall cancer compared to DPP4I use after propensity score matching and multivariable adjustment. SGLT2I was associated with lower risk of all‐cause mortality, cancer‐related mortality and new‐onset overall cancer compared to DPP4I after propensity score matching and multivariable adjustment. In 2020, the Global Cancer Observatory estimated a total of 19.3 million new cancer cases and 10 million cancer deaths.Metformin, in multiple pre‐clinical studies, has been described to be useful in the treatment of various types of malignancies.Despite the aforementioned findings, there is still much uncertainty regarding the comparative associations between SGLT2I and DPP4I with different types of new‐onset overall cancer.22.1This population‐based, retrospective study has assessed integrated medical records of patients through the Clinical Data Analysis and Reporting System (CDARS), including disease diagnosis, laboratory results, past comorbidities, medication prescription details and clinical characteristics. The system has also been used by our team in previous epidemiological research in Hong Kong.2.2Biochemical and clinical data were extracted for this cohort. Patients' demographic information includes sex, baseline age and date of initial drug use. Past comorbidities include diabetes mellitus disease duration, hyperlipidaemia, obesity, hypertension, alcoholism, liver diseases, autoimmune diseases, HIV, carcinogen pathogens, previous irradiation, chronic obstructive pulmonary disease, gastrointestinal diseases, cardiovascular diseases, ischemic stroke, diabetic eye diseases and renal diseases. Moreover, Charlson's standard comorbidity index was also calculated. Renal function was calculated using the CKD‐EPI equation.Moreover, anti‐diabetic and non‐SGLT2I/DPP4 medications and baseline laboratory data results were also extracted. Data on the following medications were extracted: sulphonylurea, insulin, metformin, thiazolidinedione, acarbose, glucagon‐like peptide‐1 receptor agonists, statins and fibrates, Angiotensin‐converting enzyme inhibitors, Angiotensin receptor blockers, anti‐depressant drugs, antihypertensive drugs, anti‐hepatitis drugs, anticoagulants, diuretics, nitrates, beta‐blockers, calcium channel blockers and non‐steroidal anti‐inflammatory drugs. The extracted laboratory data include lipid profiles, complete blood count, renal function test, biochemical test and glycaemic profiles.2.3The primary outcome of this study was new‐onset all‐cause cancer incidence, all‐cause cancer‐related mortality and all‐cause mortality. Mortality data were extracted from the Hong Kong Death Registry, an official government registry linked with CDARS that registers death records of all Hong Kong citizens. Study outcomes and comorbidities were documented using the ICD‐9 codes, whilst mortality outcomes were recorded using the ICD‐10 coding system. ICD‐10 codes C00‐C97 were used to identify all‐cause cancer mortality. The ICD‐9 and ICD‐10 codes are summarised in Table p < 0.05 was considered statistically significant. Statistical analyses and propensity score matching was performed with RStudio software (version: 1.1.456) and Stata software (version 13.0), respectively.Descriptive statistics were used to summarise baseline characteristics for this cohort. Mean and standard deviation (SD) was used to represent continuous variables, while a number and percentage were used to represent categorical variables. Propensity score matching with a 1:1 ratio between SGLT2I and DPP4I users and patients with and without new‐onset overall cancer risk based on demographics, prior comorbidities, laboratory data, medication usage, Charlson comorbidity index and abbreviated modification of diet in renal disease were performed using the nearest neighbour strategy with the Calliper set at 0.1. Univariable and multivariable Cox proportional regressions were performed for both before and after matching to identify significant predictors of new‐onset all‐cause cancer occurrence and mortality. This is further corroborated by the inverse probability of treatment weighting using propensity scores and calculating incidence rate ratios. Cumulative incidence curves were also calculated to visually depict the difference in the time‐to‐adverse event by comparing the SGLT2I and DPP4I groups. 33.1This study included 60,112 T2DM patients , of which 18,167 patients were SGLT2I users and 41,945 patients were DPP4I users. In the SGLT2I subgroup, the corresponding number of patients on individual SGLT2Is is as follows: 4523 (24.89%) on canagliflozin, 10,556 (58.10%) on dapagliflozin, 3780 (20.80%) on empagliflozin and 2527 (13.90%) on ertugliflozin. During the follow‐up period, 1533 patients developed new‐onset overall cancer, 3033 patients died from any cause, of which 506 patients died due to cancer‐related causes. Data on specific types of new‐onset overall cancers were also extracted: 249 patients developed new‐onset lung cancer, 817 patients developed new‐onset gastrointestinal cancer, 201 patients developed new‐onset breast cancer, 261 patients developed new‐onset genitourinary cancer and 97 patients developed new‐onset bladder cancer , cancer‐related mortality , as well as a 30% reduction in the risk of new‐onset overall cancer . When stratified by cancer subtype, SGLT2I were related to a lower risk of new‐onset breast cancer , but not with other malignancies. With subgroup analysis comparing DPP4I to different subtypes of SGLT2I, dapagliflozin  and ertugliflozin  both demonstrated superiority in relation to new‐onset overall cancer development, with the former also presenting with a relatively lower risk of breast cancer . There were no observable differences when comparing the use of either canagliflozin or empagliflozin with DPP4I in terms of overall or specific cancer risk.The results of univariable Cox regression analysis for predicting general and pre‐specified cancer risk are displayed in Table 3.3To assess the predictivity of the models, sensitivity analysis was conducted to evaluate the effect of matching on the results, namely with inverse probability of treatment weighting Table . The fin4To the best of our knowledge, this is the first territory‐wide study that does a direct comparison of the effect of SGLT2I and DPP4I on overall and pre‐specified cancer risk in a cohort of Asian patients. The main findings of this study are as follows: In comparison with DPP4I, (i) SGLT2I were associated with a lower risk of all‐cause mortality, cancer‐related mortality and new‐onset overall cancer; (ii) SGLT2I were related to a lower risk of new‐onset breast cancer; (iii) when stratified according to the medication subtype, dapagliflozin and ertugliflozin both demonstrated a reduced risk of new‐onset malignancy, with the former also presenting with a lower risk of breast cancer.Anti‐diabetic medications are amongst the most commonly prescribed drugs in the world, with the indications of some expanding beyond T2DM to other non‐diabetic cardiovascular and chronic kidney conditions.The majority of the comparative studies available in the existing literature have evaluated cancer risk across a wide range of anti‐diabetic drugs. Liu et al. performed a retrospective case‐controlled prognostic assessment for different anti‐diabetic medications, including metformin, thiazolidinediones, sulfonylureas, meglitinides, acarbose as well as insulin and its analogues, in turn revealing that apart from pioglitazone and insulin, the other therapies failed to show an association with cancer incidence. This relationship was maintained when stratifying outcomes by cancer type, namely for pancreatic, liver and lung cancer.Despite this, it should be noted that there is much more uncertainty about the malignancy risk of the somewhat newer anti‐diabetic medications. Regarding DPP4I, a meta‐analysis compiled by Zhao et al. did not report any association between these medications and malignancy, even when stratified by different subtypes of DPP4I.Likewise, very much akin to that of DPP4I, the data centred around SGLT2I are also controversial. Most recently, a meta‐analysis performed by Benedetti et al. proposed a reduced cancer risk of SGLT2I when compared to placebo, with particular efficacy for dapagliflozin and ertugliflozin.Given the relatively newer status of DPP4I and SGLT2I, there is a paucity of literature comparing the non‐diabetic outcomes associated with these medications. Au et al. showcased a reduced incidence of pneumonia and pneumonia‐related mortality with SGLT2I relative to DPP4I in patients from Hong Kong.4.1There are certain limitations present in this population‐based study. First, due to the observational nature of this study, acquired results may be susceptible to information bias due to missing data, coding errors or under coding. Second, the retrospective nature of the study suggests that all derived findings regarding the relationship between SGLT2I, DPP4I and new‐onset overall cancer were correlational in nature. Third, information on drug exposure could not be directly obtained, and was instead determined indirectly through prescription refills, which may pose a liability concern. Fourth, as the drug exposure duration could not be standardised, this may have influenced the primary and secondary outcomes of the study. Finally, due to the lack of codes in CDARS, information regarding medical history, such as smoking status, were unattainable and could have been a confounding variable to cancer risk.4.2SGLT2I use was associated with lower risks of all‐cause mortality, cancer‐related mortality and new‐onset overall cancer compared to DPP4I use after propensity score matching and multivariable adjustment.Cheuk To Chung: Conceptualization ; writing – original draft ; writing – review and editing . Oscar Hou In Chou: Methodology ; validation ; visualization . Teddy Tai Loy Lee: Investigation ; resources ; writing – review and editing . Edward Christopher Dee: Conceptualization ; data curation . Kenrick Ng: Resources . Wing Tak Wong: Supervision . Tong Liu: Investigation ; supervision . Sharen Lee: Supervision ; writing – review and editing . Bernard Cheung: Investigation ; supervision . Qingpeng Zhang: Resources . Jiandong Zhou: Formal analysis ; investigation ; methodology ; software ; visualization .The authors declare no potential conflicts of interest.Data S1. Supporting Information.Click here for additional data file.


The clinical applications of transcatheter arterial embolization (TAE) conversion therapy combined with hepatectomy have been severely restricted by ill‐defined tumoral boundaries and miniscule hidden lesions. Fluorescent surgical navigation is a promising method for overcoming these barriers. However, sufficient delivery of the fluorescent probe into the tumor region after long‐term TAE is challenging due to blockade of the tumor‐supplying artery. Here, a super‐stable homogeneous intermix formulating technology (SHIFT) to physically mix lipiodol and indocyanine green (ICG) formulation (SHIFT and ICG) for fluorescent surgical navigation after long‐term TAE conversion therapy is provided. Through the retrospective study of 45 clinical liver cancer patients, it is found that SHIFT and ICG formulation have excellent tumor deposition effect and safety. During surgical resection after long‐term TAE conversion therapy, SHIFT and ICG could clearly identify in real time the full tumor regions and boundaries and had a high signal‐to‐normal tissues ratio—even the indistinguishable satellite lesions could be identified with a strong fluorescence intensity. Meanwhile, SHIFT and ICG could improve operative, anesthetic, and postoperative variables associated with postoperative complications. This simple and effective SHIFT could provide precise fluorescent navigation for surgical resection following long‐term embolization therapy in clinical practice and has great potential for a translational pipeline. ICG after tumor embolization prevents the infiltration and distribution of ICG in the tumor tissues that results from the blocked supply artery and tumor necrosis.Indocyanine green (ICG) is a near‐infrared fluorescent dye. Given its excellent optical properties and biosafety in vivo, ICG has been used in fluorescence‐guided surgical resection and intraoperative tumor visualization.To address this issue, we developed a super‐stable homogeneous intermix formulating technology (SHIFT) to physically mix lipiodol and ICG formulations (SHIFT and ICG) homogeneously for fluorescence‐navigated hepatectomy after long‐term TAE conversion therapy, which realized the interventional embolization with one‐time administration and follow‐up surgical fluorescence navigation. Previous work has confirmed that SHIFT formulation was prepared in a green physical mixture via a carrier‐free manner, which possesses controlled morphology, long‐term stability, and improved optical characteristics of ICG. Furthermore, the viscosity of the SHIFT and ICG was comparable to lipiodol and exhibited long‐lasting excellent fluorescence navigation in VX2 orthotopic hepatocarcinoma models.22.1This study was approved by the Institutional Review Board and Ethics Committee of the Affiliated Zhong'shan Hospital of Xiamen University, Affiliated Hospital of Southwest Medical University, and Affiliated Hospital of North Sichuan Medical College in China. It is registered in the Chinese Clinical Trial Registry (Register No. ChiCTR2000035055).A clinical application study was conducted to evaluate the usefulness and safety of SHIFT and ICG formulation prepared via SHIFT for surgical navigation after long‐term TAE. Between July of 2019 and April of 2022, 25 liver cancer patients who cannot be radically resected underwent TAE with SHIFT and ICG as the main embolic agent at the Affiliated Zhong'shan Hospital of Xiamen University, Affiliated Hospital of Southwest Medical University, and Affiliated Hospital of North Sichuan Medical College. We obtained consent from all patients, regardless of gender or age. The clinical data of 20 liver cancer patients who received TAE with conventional lipiodol as the main embolic agent were used as a control group. We conducted a retrospective analysis of the clinicopathological data to compare outcomes between the two groups.2.2Inclusion criteria: (1) patients with liver cancer and high surgical potential, confirmed by multiple imaging examinations  or histological or cytological diagnosis, regardless of gender or age with an expected lifespan of ≧3 months; (2) intrahepatic tumors with single or multiple lesions located in half of the liver or confined to three adjacent liver lobes, with at least one lobe of the liver having a primary portal vein branch that is unobstructed, and barcelona clinic liver cancer (BCLC) stage A or BExclusion criteria: (1) patients with recurrent disease or who were previously treated by surgery or drugs; (2) patients with other confirmed malignant tumors or serious diseases of the heart, lung, kidney, brain, and other important organs; and (3) those with an indocyanine green allergy or iodine allergy.2.3Lipiodol was purchased from Jiangsu Hengrui Pharmaceutical Co, Ltd, Jiangsu, China. Indocyanine green was purchased from Dandong Medical and Pharmaceutical Co, Ltd, Dandong, China. An anti‐Ki‐67 antibody, 4′,6‐diamidino‐2‐phenylindole (DAPI), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reagent box were purchased from Servicebio.2.4The fluorescent surgical navigation system (DPM‐01) was provided by the Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing, and Beijing Digital Precision Medicine Technology Co. Ltd. The SHIFT equipment Figure  was deveThe SHIFT and ICG formulation prepared by this method is only a mixture of lipiodol and ICG without chemical reactions or molecular changes in drugs.2.52.5.1i.v.) at a dose of 0.5 mg/kg 3–5 days before surgery after successful TAE in the control group.We punctured the femoral artery and performed celiac trunk and superior mesenteric artery angiography. After the tumor‐supplying artery was identified, a microcatheter was used to super‐selectively enter the target artery. First, local perfusion of the tumor was performed with lobaplatin for injection, and next an appropriate amount of SHIFT and ICG formulation (ICG concentration 1 mg/ml) (SHIFT and ICG group) or conventional lipiodol (control group) were injected respectively. The dosage of SHIFT and ICG formulation was injected according to the size of the tumor, generally within 10–20 ml,2.5.2After successful tumor conversion therapy was confirmed by reexamination of CT or MRI and reached the standard of radical surgical resection, the resection of the liver tumor was performed under real‐time fluorescence navigation. In this study, all liver tumors and benign lesions were observed by intraoperative ultrasonography (IOUS) and fluorescent surgical navigation system, respectively. First, the lesion location, number, characteristics, and relationship to the surrounding structure were determined on the surface of the whole liver with the preoperative imaging examination and IOUS system. Then the fluorescence surgery navigation system was applied to lighten the liver to observe lesions from a distance of 5–20 cm above the liver surface. The contrast mode of the equipment was used to ensure a boundary between tumor and normal liver tissue. Afterward, the tumor lesions could be resected under the guidance of the lit area on the screen. Subsequently, the resected tissues were investigated by a fluorescence surgery navigation system to verify the ICG signal and distribution. Finally, H&E and immunohistochemistry staining were utilized to evaluate the resection and tumor tissues. Meanwhile to evaluate the SHIFT and ICG fluorescent navigation effect and resection quality, fluorescent signal area and intensity of the tumor sites and the signal‐to‐normal tissues ratio (SNR) were calculated using Image J and regions of interest (ROIs).2.6p values < 0.05 were considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.Data analyzed by Prism 7  and SPSS 17.0 were presented as mean ± SD. The Mann–Whitney U test was used to evaluate the differences between the experimental group and the control group. 33.1In this study, 45 patients with unresectable liver cancer, including 29 men and 16 women, with a mean (±standard deviation) age of 57.05 ± 10.27 and 54.8 ± 12.14 years in the control and SHIFT and ICG group, respectively, were successfully treated with TAE, using conventional lipiodol or SHIFT and ICG formulation as the main embolization agents. An irregular liver surface due to cirrhosis was found in 16 patients. The mean ICG‐R15 was 7.17% ± 2.58% and 7.28% ± 2.24% in the control and SHIFT and ICG group, respectively. No intraoperative or postoperative adverse events were observed related to the administration of ICG. The mean tumor size was 9.63 ± 2.35 cm and 9.18 ± 2.06 cm in the control and SHIFT and ICG group, respectively. The basic characteristics and demographic information of both experimental groups are shown in Table 3.2p = 0.486; Figure Maintaining viscosity performance and safety are crucial for effective embolization. Through the TAE treatment of 45 liver cancer patients who could be radically resected, we found that both the SHIFT and ICG formulation and conventional lipiodol group all deposited well in the liver cancer lesions during and after embolization Figure . No signNo obvious abnormality was found in the peripheral blood cells and blood coagulation indices of the two groups Figure . Althoug3.33.3.1In the control group, conversion therapy was successful in three patients and reached the standard of radical surgical resection. ICG was then injected i.v. 3–5 days before the operation. Although the tumor could be located before resection, its location was incomplete, and only a few weak fluorescence signals were seen at the tumor lesions Figure . The boui.v. injection of ICG, we performed fluorescence imaging of the lesions and transected the surfaces of resected tumor lesions. The results showed that the whole resected tumor lesions showed only minimal fluorescence signals, and the tumor boundary could not be distinguished  in the quantitative results indicated that SHIFT and ICG can guide liver tumor resection more effectively than previously available methods.The quantitative evaluation results of tumor resection quality showed that, prior to resection, the fluorescence intensity and SNR of each tumor site was 237.55 ± 14.14 and 3.63 ± 0.69, respectively. After resection, the fluorescence signal intensity of the tumor sites decreased to 73.76 ± 10.33, and the decreased SNR (1.11 ± 0.10) neared 2.52 Figure . QualityTo further verify the fluorescence imaging and embolization effect of SHIFT and ICG, we performed an immunohistochemical examination of resected tumor specimens with Ki‐67, TUNEL, and DAPI. The results showed many ICG fluorescence signals in the tumor tissues, a low expression level of Ki‐67, and a high expression level of TUNEL Figure , indicat3.42), ASA score (3), operative duration, and blood loss ≧300 ml; Figure p = 0.001) was a key independent predictor of postoperative morbidity  in the SHIFT and ICG group were shorter  was lower  was significantly smaller than that in the control group ; formal analysis (lead); funding acquisition (supporting); methodology ; visualization ; writing – original draft (lead). Yongfu Xiong: Data curation ; formal analysis ; methodology ; visualization ; writing – original draft . Bin Luo: Data curation ; formal analysis ; methodology ; visualization ; writing – original draft . Jianming Liu: Data curation ; formal analysis ; funding acquisition ; methodology ; visualization ; writing – original draft . Yang Zhang: Conceptualization ; formal analysis ; methodology ; visualization . Yu Xiong: Data curation ; formal analysis ; methodology ; visualization . Song Su: Data curation ; methodology ; visualization . Cheng Fang: Data curation ; methodology ; visualization . Yisheng Peng: Data curation ; methodology ; visualization . Hongwei Cheng: Formal analysis ; methodology ; visualization . Chengchao Chu: Formal analysis ; methodology . Jingsong Mao: Formal analysis ; methodology . Jingdong Li: Conceptualization ; data curation ; formal analysis ; methodology ; visualization ; writing – review and editing . Gang Liu: Conceptualization (lead); data curation ; formal analysis (lead); funding acquisition (lead); methodology (lead); project administration (lead); writing – review and editing (lead). Bo Li: Data curation ; formal analysis ; methodology ; visualization ; writing – review and editing . Zhenyu Yin: Conceptualization ; data curation ; methodology ; visualization ; writing – review and editing . Jie Tian: Conceptualization ; data curation ; methodology ; project administration ; visualization ; writing – review and editing .The authors declare no competing interests.Appendix S1 Supporting InformationClick here for additional data file.