Panthera tigris altaica) have been reported because these tigers comprise an endangered species; however, it is extremely difficult to obtain blood samples necessary for these analyses. This study presents 14 haematological and 16 biochemical parameters of 133 Siberian tigers, of which 112 and 21 were from Heilongjiang Siberian Tiger Park (HB) and Hailin Siberian Tiger Park (HD), China, respectively. Our study is the first to determine the following parameters in Siberian tigers: red blood cell volume distribution width, platelet count, mean platelet volume, amylase (AMY), sodium/potassium, globulin and albumin/globulin levels. As the data for total bilirubin and AMY were not statistically significant, no statistical analysis was conducted for these parameters. Few parameters were significantly different according to sex and region (p < 0.05). The concentration of alkaline phosphatase decreased with age, whereas the creatinine (CREA) increased with age. The CREA concentration of tigers raised in HB was much lower than that of tigers raised in HD. The data obtained in this study provide a reference for monitoring the health of wild and captive Siberian tigers and will add important information to the standards for haematological and biochemical parameters of wild felines.Haematological and biochemical parameters play important roles in safeguarding animal health and preventing disease, but the blood reference values of many wild animals are still unknown. Recently, few descriptions of the blood parameters of Siberian tigers ( This study collected and analysed the haematological and biochemical parameters of 133 Siberian tigers; these data can provide a reference for monitoring the health of wild and captive Siberian tiger. Currently, there are approximately 400 wild Siberian tigers globally, with only a few of these in China  maintained in captivity at Lahore Zoo (n = 4) and in the seminatural environment of Lahore Wildlife Park (n = 6), Pakistan. They found that captivity has a significant effect on various haematological and biochemical parameters of these tigers. Larsson et al. (P. tigris) and one Siberian tiger. They found the electrophoretic patterns to be similar in all tigers. Moreover, except for the mean concentrations of α2 and β1 globulins, the mean values of albumin and globulin fractions for all tigers were within the range of reference values found in domestic cats. Sample sizes in the existing literature are extremely small to draw firm conclusions from, and these conclusions may lack generalizability.The health of various animal organs can be evaluated using haematological and biochemical parameters. However, reports on the haematological and biochemical parameters of tigers are extremely limited, particularly those of Siberian tigers. Sajjad et al.  and Faron et al.  assessedn et al. . Proverbn et al.  conducteThis study aimed to further assess the baseline haematological and biochemical parameters of Siberian tigers. These values would be useful for monitoring the health of Siberian tigers and may be used as a reference for captive and free‐ranging Siberian tigers.22.1We examined Siberian tigers (hereafter referred to as tigers) from two different Siberian Tiger Parks. Blood samples from 112 tigers  from Heilongjiang Siberian Tiger Park (HB) and 21 tigers  from Hailin Siberian Tiger Park (HD) were collected. All examined animals were 2–9 years old and weighed 98–442.4 kg. Their food consisted mainly of pork and chicken, and they were fasted for 1–2 days per week, with no physical or behavioural abnormalities observed before blood sample collection.2.22 EDTA . The sampled blood was placed at 20℃–25℃ for 30 min and then centrifuged at 489g for 10 min; then, 200‐μl supernatant (serum) was extracted from each sample for biochemical analysis. Following the same collection protocol, an additional 2 ml of whole blood was collected in vacuum blood tubes containing 2.7‐nM Na2 EDTA  for haematological analysis. To minimize stress to the animals, collection procedures were standardized to the maximum extent possible. This included being performed by the same personnel, using the same techniques and at approximately the same time each day (between 9:00 a.m. and 12:00 a.m.).All tigers were fasted for 1 day before blood sample collection. Before examination, tigers were sedated within their own enclosures using anaesthetic darts containing ketamine  at 10 mg/kg. The animals were then weighed, and 5 ml of blood was collected aseptically from their jugular vein using disposable vacuum blood tubes without Na2.3Haematological parameters were assessed using an Exigo H400 automatic haematology analyzer, along with a specialized software , for analysing the haematological parameters of tigers. The haematological parameters evaluated were as follows:White blood cell (WBC), Lymphocytes (LYM), Monocytes (MON), Neutrophils (NEU), Eosinophils (EOS), Red blood cell (RBC), Haematocrit (HCT), Red blood cell volume distribution width (RDW), Haemoglobin (HGB), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet (PLT) and Mean platelet volume (MPV).2.4Biochemical parameters were processed using the automatic veterinary biochemical analyzer Skyla VB1 with a specialized software , and for tiger blood biochemical analysis, 200‐μl serum was placed in Skyla VB1. The evaluated biochemical parameters are as follows:Triglyceride (TG), Cholesterol (CHOL), Glucose (GLU), Total protein (TP), Albumin (ALB), Globulin (GLOB), Albumin/Globulin (A/G), Alkaline phosphatase (ALP), Alanine transaminase (ALT), Ureophil (UREA), Creatinine (CREA), Calcium (Ca), Phosphate (PHOS), Sodium (Na), Potassium (K), Sodium/Potassium (Na/K), Total bilirubin (TBIL) and Amylase (AMY).2.5t test, and non‐normally distributed data were analysed using the Mann–Whitney U‐test. Statistical analyses were performed using IBM SPSS Statistics Software version 26. A value of p < 0.05 was considered statistically significant. As only two samples were obtained from 8‐year‐old tigers, the effect of age on the parameters could not be statistically analysed.Data were expressed as minimum, maximum, mean and standard deviation. The sex and area variables were analysed for normality using the Shapiro–Wilk normality test. Normally distributed data were analysed using an independent‐sample 3−1. As the TBIL and AMY data were not statistically significant, they have not been listed in the included tables.Currently, there are no globally acceptable reference ranges for all haematological and biochemical parameters of Siberian tigers. Except for the reference ranges of TG, CHOL, GLOB, A/G and Na/K levels, those of other haematological and biochemical parameters in this study were assessed using the specialized software developed by Tianliang Medical Equipment Co., Ltd., Taiwan. The two Siberian tiger parks have been using the specialized software to monitor whether the data are within the reference range provided by it, but the basis for establishing the reference range has not been made public. Although this reference range is not a standard data range for the genus, it provided a baseline reference to compare our results against. TBIL values were all lower than 6.8 μmol/L, while AMY values were between 1910 and 3,000 U Lp < 0.05) higher WBC, NEU, RBC counts, MCHC, TG, CHOL, PHOS and Na levels and significantly lower MCV, PLT count, MPV and A/G than female tigers. All other parameters were not significantly different between female and male tigers (Tables p < 0.05) higher CHOL and PHOS levels and significantly lower GLU levels than female tigers  (He et al., 5Haematology is an important tool for assessing the physiological status of individuals and often provides the first and only indicator of a disease. Blood biochemical test is also one of the most important tests for initial diagnosis and treatment. However, many factors, including biological, methodological, dietary and management variables, influence the haematological and biochemical parameters of captive wild animals. The results of this study are similar to those reported previously; however, our study is the first to analyse the haematological and biochemical parameters in a large sample of Siberian tigers and the first to report certain specific parameters.This new report aims to further improve our understanding of the haematological and biochemical parameters of Siberian tigers, and these values will be useful for monitoring the health of captive and free‐ranging Siberian tigers. Future work will involve the collection of more samples from captive and free‐ranging Siberian tigers from various provinces in China for the detection and analysis of other haematological and biochemical parameters.The authors declare that they have no conflict of interest.Enqi Liu: Writing‐original draft. Dan You: Formal analysis. Liying Ma: Data curation. Cen Yang: Investigation. Yawen Hu: Investigation. Haitao Xu: Resources. Dan Liu: Resources. Yajun Wang: Conceptualization; Funding acquisition; Project administration; Writing‐review & editing.The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received.https://publons.com/publon/10.1002/vms3.395.The peer review history for this article is available at


Recent reports have indicated the beneficial role of strain measurement in COVID‐19 patients.To determine the association between right and left global longitudinal strain  and COVID‐19 patients' outcomes.Hospitalized COVID‐19 patients between June and August 2020 were included. Two‐dimensional echocardiography and biventricular global longitudinal strain measurement were performed. The outcome measure was defined as mortality, ICU admission, and need for intubation. Appropriate statistical tests were used to compare different groups.In this study 207 patients  were enrolled. During 64 ± 4 days of follow‐up, 22 (10.6%) patients died. Mortality, ICU admission, and intubation were significantly associated with LVGLS and RVGLS tertiles. LVGLS tertiles could predict poor outcome with significant odds ratios in the total population . Although odds ratios of LVGLS for the prediction of outcome were statistically significant among hypertensive patients, these odds ratios did not reach significance among non‐hypertensive patients. RVGLS tertiles revealed significant odds ratios for the prediction of mortality , ICU admission , and need for intubation . Odds ratios of RVGLS remained significant even after adjusting for hypertension when considering mortality and ICU admission.RVGLS and LVGLS can be acceptable prognostic factors to predict mortality, ICU admission, and intubation in hospitalized COVID‐19 patients. However, RVGLS seems more reliable, as it is not confounded by hypertension. Patients were consecutively enrolled from a general educational hospital  between June 21 and August 24, 2020. A consecutive series of COVID‐19 patients were included and underwent echocardiography in the first 12–24 h of admission. Furthermore, both deceased and survived cases were selected from a single center to reduce selection bias. The flow chart of patients' enrollment is depicted in Figure 2.2Two‐dimensional echocardiography was performed in all patients by a Siemens ACUSON SC‐2000 device based on the latest guidelines of the American Society of Echocardiography.The two‐dimensional echocardiography findings were left ventricular ejection fraction (LVEF), left ventricular end‐diastolic diameter (LVEDD), left ventricular end‐systolic diameter (LVESD), left atrial volume index (LAVI), interventricular septal diameter (IVSD), right ventricular end‐diastolic diameter (RVEDD), E', fractional area change (FAC), S velocity, tricuspid annular plane systolic excursion (TAPSE), tricuspid regurgitation peak velocity (TRPV), and systolic pulmonary artery pressure (PAP). LVEF was calculated based on the biplane Simpson method. Furthermore, LVEDD, LVESD, IVSD were calculated based on long‐axis parasternal view and M mode method. LV diastolic function was calculated by measuring E velocity, A velocity, LAVI, E', E/E', and E/A ratio.The parameters of RVEDD, S velocity, TAPSE, RV FAC were used to evaluate the right ventricular function, and RVEDD was calculated in the 4‐chamber view. To clarify, RV FAC was obtained from the 4‐chamber view based on the following formula: (RVED area‐RVES area)/(RVED area*100). Tricuspid regurgitation peak gradient (TRPG) was also calculated based on the maximum gradient between RA & RV by the Doppler method. Finally, systolic PAP was obtained using the formula “TRPG+RA pressure” in which RA pressure was measured based on inferior vena cava (IVC) size and respiratory collapse of IVC.LV global longitudinal strain (LVGLS) was calculated using three standard echocardiographic views, including 4‐chamber, 2‐chamber, and apical long‐axis based on the American Society of Echocardiography guideline. The region of interest (ROI) was set to be confined to the myocardium, and LVGLS, as bull's eye, was automatically calculated by the device . RVGLS w2.3p‐value for interaction was used to assess the interaction between hypertension and GLS. Whenever there was an interaction, a subgroup analysis was performed between hypertensive and non‐hypertensive patients. In case of no interaction, adjusted odds ratios were calculated. p‐values less than 0.05 were considered statistically significant.Data analysis were performed using the SPSS version 20.0 software. Kolmogorov–Smirnov test was utilized to test the normality of all data. Means of variables were compared using the appropriate statistical test. Also, outcome analysis was performed based on three tertiles for LVGLS and RVGLS. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for the prediction of mortality, ICU admission, and need for intubation, and they were also adjusted for hypertension, where relevant, as the confounding effect of hypertension on LVGLS was described before.3p = 0.001), those with ICU admission (p = 0.001), and cases needing intubation (p = 0.001).In this study, 119 male (57.5%) and 88 female cases 42.5%) were enrolled. The mean age of patients was 54.5 ± 14.8 years. Patients' baseline characteristics are summarized in Table .5% were p < 0.05). LVEF, E', RV FAC, TAPSE, were higher among patients with desirable outcome, while LAVI and systolic PAP were higher among those with poor outcome. Echocardiographic measurements were compared between three tertiles of LVGLS (p < 0.05). In terms of RVGLS, LVEF, LVEDD, E', LAVI, FAC, S Velocity, and TAPSE indicated significant differences according to the tertiles (p < 0.05).During 64 ± 4 days of follow‐up, 22 (10.6%) patients died. Among cases, 40 (19.3%) and 28 (13.5%) patients were admitted to intensive care unit (ICU) and Post‐ICU wards (patients with the intermediate condition), respectively. Intubation was also performed in 18 (8.7%) patients. Comparative results of echocardiography according to the outcome are shown in of LVGLS  and RVGLof LVGLS . The terp = .001, 0.001), ICU admission , and need for intubation  were significantly associated with LVGLS and RVGLS tertiles, respectively , a subgroup analysis was performed. Among non‐hypertensive patients, odds ratios of LVGLS for the prediction of outcome were not statistically significant . However, these odds ratios were significant among hypertensive patients .As demonstrated in Tables p‐value for interaction = 0.259, 0.345). However, hypertension and RVGLS tertiles interacted for the outcome of intubation . Thus, odds ratios of RVGLS were adjusted for hypertension when considering mortality and ICU admission, and they remained significant . A subgroup analysis was performed for the outcome of intubation; among hypertensive patients, the odds ratio of RVGLS for the prediction of intubation was significant , while it revealed insignificant results among non‐hypertensive patients .Also, RVGLS tertiles revealed significant odds ratios for the prediction of mortality , ICU admission , and need for intubation . In the final model adjusted for hypertension and ischemic heart disease, RVGLS tertiles could predict mortality , ICU admission , and need for intubation . Among patients, there were not any significant interactions between hypertension and RVGLS tertiles for mortality and ICU admission (4This is one of the largest COVID‐19 populations studied by 2D strain echocardiography. In this cohort, it was found that the majority of findings in two‐dimensional echocardiography are related to the outcomes in COVID‐19 patients. Both LVGLS and RVGLS could predict all outcomes including ICU admission, need for intubation, and mortality in admitted COVID‐19 cases, even when adjusted for hypertension and ischemic heart diseases.Our findings for RVGLS were congruent with the study of Krishnamoorthy et al.Although Cau and colleaguesA recent study by Li et al.Fukui et al.Monitoring of cardiovascular involvement is another indispensable part of the clinical management of COVID‐19 patients. Cameli et al.We also found no association between the troponin level and any of RVGLS, LVGLS, or patients' outcomes. Consistent with this finding, Kocas et al.Finally, there is still a question: Why to use GLS measurements over routine echocardiographic parameters such as LVEF? First, GLS is considered to be a more reproducible method regardless of echocardiographic training as it has better inter‐ and intra‐observer reproducibility.This study was limited by the fact that only hospitalized COVID‐19 patients were included. Also, patients' pharmacologic therapy was not included in our analysis. Thus, further prospective studies are needed to take all confounding factors into account.In conclusion, RVGLS and LVGLS can be both acceptable prognostic factors to predict mortality, ICU admission, and intubation in hospitalized COVID‐19 patients. However, RVGLS seems more reliable, as it is not confounded by hypertension. Also, other echocardiographic findings, such as LVEF, E', LAVI, FAC, TAPSE, and systolic PAP may be used as prognostic factors, if speckle tracking echocardiography is not available.The authors declare no potential conflict of interest.Appendix S1. Supporting informationThe method of strain assessment and detailed measurements of echocardiographic parameters are available online.Click here for additional data file.


This set of cases provides important evidence of re‐infection and recurrence of SARS‐CoV‐2 even for the third time. Consequently, this possibility should be considered more in recurrent patients with Covid‐19 symptoms. This set of cases provides important evidence of re‐infection and recurrence of SARS‐CoV‐2 even for the third time. Consequently, this possibility should be considered more in recurrent patients with Covid‐19 symptoms. Several studies on re‐positive cases after complete recovery caught our attention. Here, we reported the re‐positive RT‐qPCR for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) after complete recovery. Our results are just a report and need to be confirmed by a larger cohort.Less than a year ago, coronavirus disease 2019 (COVID‐19) was discovered in a Chinese province, spread rapidly across continents, which generating a pandemic with serious consequences for the health of the world.Most people who get COVID‐19 have mild (with no sign of pneumonia) or moderate symptoms , then severe symptom , and finally critical symptom .Most recent studies show that immunity after acute respiratory syndrome infection can develop in infected people.1.1Show at least one clinical sign of COVID‐19 including flu‐like symptoms such as fever, chills, shortness of breath, anosmia, or dyspepsiaa positive SARS‐CoV‐2 RT‐PCR test result.Clarifying the characteristics and frequency of re‐infection is important because it can affect our understanding of acquired immunity after natural infection. A COVID‐19 positive was defined by two criteria:Here, we present three re‐infected cases who went to the laboratory for re‐testing, whose clinical and laboratory characteristics are shown in Table 2A 58‐year‐old female, with no underlying disease just occasionally mild flu‐like symptoms  was referred on March 18, 2020. The first chest x‐ray revealed ground‐glass opacity At this stage, the diagnosis is made solely on the basis of clinical signs and was subjected to treatment with chloroquine and Oseltamivir.Blood test results were normal regarding both inflammation and respiratory parameters. Qualitative IgG/IgM was negative for both antibodies at the end of the symptoms. The patient was isolated at home and three weeks later the SARS‐CoV‐2 PCR assay was negative.Approximately 93 days after her initial symptoms, she presented new clinical symptoms with dry cough, fever, shortness of breath, weakness, excessive sweating, hypotension, and acute depression. Resting heart rate (75/min) and oxygen saturation (less than 85%) and other symptoms led to hospitalization. During the hospitalization, blood test results showed leukocytosis 13000 with lymphopenia, increased D‐dimer (240 ng/ml), and ferritin (267 mg/ml). On May 26, 2020, the SARS‐CoV2 RT‐PCR assay was positive with a Ct value of 21 and qualitative IgG/IgM was negative for both antibodies at the end of symptom. About 65 days later, recurrent symptoms of cough, severe weakness, myalgia and bone pain, fever, and mild chills appeared. The SARS‐CoV2 RT‐PCR assay was positive, but a CT scan was not performed. Cough and chest pain lasted up to two months. The cough has continued and led to the use of various inhalation sprays.3A 48‐year‐old male was presented with headache, fever, cough, chills, Proteinuria, diarrhea on March 9, 2020. A nasopharyngeal swab was positive for SARS‐CoV‐2 with a Ct value of 30.3. The patient was self‐isolated at home and most symptoms vanished after a few days except coughing. Four months  later, the patient complained of a mild headache for three nights. More symptoms appeared with a runny nose and body aches, cough, paresthesia, and fatigue. The symptoms lasted for 10 days and were resolved without hospitalization. His nasopharyngeal swab was again positive for SARS‐CoV‐2 . Chest X‐ray was not performed and complete blood count was normal.After 98 days, in November 2020, symptoms like cough, severe weakness, myalgia, paresthesia, fever, diarrhea, and nausea reappeared. There were no severe respiratory symptoms but mild fever and gastrointestinal symptoms. The SARS‐CoV‐2 PCR assay was positive (Ct=24.3). The resting heart rate was 96/min with an oxygen saturation of 94%. In the first period of infection with Covid‐19, his heart rate was consistently 94–96/min, whereas, during the second time, it was consistently 92–99/min. During all periods, viral serology revealed negative SARS‐Covid‐2 IgM and IgG antibodies.4In April 2020, a 46‐year‐old male presented with three days of intense headache and drowsiness. After four days, he complained about fever, shortness of breath, cough, headache, dizziness, weight loss, severe weakness, and loss of appetite. His pulse was 95/min during rest with oxygen saturation of 89% and a rapid antibody test was positive for IgM and IgG. The RT‐PCR test was positive (Ct=12.85). Subsequently, the patient's symptoms improved, and after three weeks, the SARS‐CoV‐2 PCR assay was negative. In July 2020, the patient presented again with mild symptoms like fever and chills, weakness. The SARS‐CoV‐2 PCR test showed a positive result (24.3) and oxygen saturation was 90%. Despite moderate symptoms and dyspnea, the patient was not hospitalized and his clinical condition improved after 15 days.Four months later, the symptoms were more severe including fever (>40 degrees), chills and severe weakness, severe diarrhea, weight loss, shortness of breath, and loss of appetite. The SARS‐CoV‐2 PCR assay was positive with a Ct value of 21. Finally, a quantitative antibody test showed positive for both IgG and IgM antibodies.5About 12 months after the emergence of the first cases of COVID19, there is still clinical suspicion of reinfection by SARS‐CoV‐2, although some reports have been evaluated by different scientists globally.In our study, the clinical manifestations of reinfection differed across all three cases: one person showed mild symptoms, two showed moderate symptoms, required oxygen support. Serological test results were variable in all patients over three periods.According to the World Health Organization, there is no sufficient evidence to verify antibody‐mediated immunity in this epidemic to ensure the safety of the individuals.Two of these are more likely: One of them is the possibility of viral reactivation which is related to the host immunity status, virological factors, and type and immunosuppression level.In our cases, patients who recovered at least two months from COVID‐19, later developed Covid‐19 for the third time. Due to high suspicion of COVID‐19 based on clinical signs, nasopharyngeal swabs were sent for RT‐PCR, which were positive. Unfortunately, because of the technical limitations of the SARS‐CoV‐2 serological tests, variable results were obtained in all of our three cases.6This set of cases provides important evidence of re‐infection and recurrence of SARS‐CoV‐2 even for the third time. Consequently, this possibility should be considered more in recurrent patients with Covid‐19 symptoms.The authors declare that there is no conflict of interest.Sepideh Hasanzadeh and Saeid Amel Jamehdar designed the work and interpreted the data. Sara Ganjloo, Mahsa Rahmani, and Mahbubeh Jangi revised the manuscript critically for important content. Somayeh Sadat Shariatmaghani, Atefeh Vakilian, and Alireza Javan critically revised the article for important intellectual content. All authors reviewed and approved the final version of the manuscript.Appropriately written informed consent was obtained for publication of this case report and accompanying images.


Eliminating primary tumor (“roots”) and inhibiting associated‐circulating tumor cells  are vital issues that need to be urgently addressed in cancer therapy. Associated‐CTCs, which include single CTCs, CTC clusters, and CTC–neutrophil clusters, are essential executors in metastasis and the cause of metastasis‐related death in cancer patients. Herein, a “roots and seeds” multipoint costriking nanodevice (GV‐Lipo/sorafenib (SF)/digitoxin (DT)) is developed to eliminate primary tumors and inhibit the spread of associated‐CTCs for enhancing metastasis inhibition and the therapeutic effect on hepatocellular carcinoma (HCC). GV‐Lipo/SF/DT eliminates primary tumor cells by the action of SF, thus reducing CTC production at the roots and improving the therapeutic effect on HCC. GV‐Lipo/SF/DT inhibits associated‐CTCs effectively via the enhanced identification and capture effects of glypican‐3 and/or vascular cell adhesion molecule 1 (VCAM1) targeting, dissociating CTC clusters using DT, blocking the formation of CTC–neutrophil clusters using anti‐VCAM1 monoclonal antibody, and killing CTCs with SF. It is successfully verified that GV‐Lipo/SF/DT increases the CTC elimination efficiency in vivo, thus effectively preventing metastasis, and shows enhanced antitumor efficacy in both an H22‐bearing tumor model and orthotopic HCC models. Overall, the “roots and seeds” multipoint costriking strategy may open a new cancer treatment model for the clinic. A multipoint costriking nanodevice (GV‐Lipo/SF/DT) with the capability of eliminates primary tumor cells and associated‐circulating tumor cells (CTCs) is obtained for enhancing metastasis inhibition and therapeutic effect on HCC. GV‐Lipo/SF/DT can specifically target tumor cells and capture CTCs, dissociate CTC clusters, block the formation of CTC–neutrophil clusters, finally kill CTCs and primary tumor cells. Then, CTCs extravasate into surrounding organs and form metastasis initiating cells or metastatic nodes. Primary tumor cells can be considered the “roots” of metastasis, while CTCs can be considered the “seeds” of metastasis, and together they confer a poor prognosis for cancer patients. Therefore, both the elimination of “roots” and the inhibition of the spread of “seeds” are vital issues that urgently need to be addressed in cancer treatment. Conventional treatments, such as surgical treatment, chemotherapy, and immunotherapy, have made great progress in eliminating primary tumors, but metastasis and recurrence after treatment still lead to a low survival rate. Currently, some strategies have focused on the importance of CTCs. For example, an exosome‐like sequential‐bioactivating prodrug nanoplatform was designed for breast cancer therapy and found to suppress metastasis through CTC clearance.Metastasis is the leading cause of death in cancer patients.1 Meta CTC clusters are groupings of multiple cells derived from primary tumor cells and are more efficient in forming metastases than single CTCs. CTC clusters form strong cell‐cell adhesion through desmosomes and adherens junctions, which could help overcome anoikis and promote metastasis. The dissociation of CTC clusters remodels key binding sites and suppresses metastasis. CTCs can also associate with neutrophils to form CTC–neutrophil clusters and maintain high levels of proliferation in the circulation, and the adhesion of CTCs to neutrophils promotes extravasation and metastasis. Increasing evidence has shown that neutrophils interact with CTCs via binding to highly expressed adhesion molecules on tumor cells, such as vascular cell adhesion molecule 1 (VCAM1). Inhibition of the VCAM1 binding prevents the formation of CTC–neutrophil clusters and thus inhibits tumor metastasis. CTC clusters and CTC–neutrophil clusters show increased metastatic potential compared with single CTCs and are as important as single CTCs in the process of tumor metastasis. Here, single CTCs, CTC clusters, and CTC–neutrophil clusters are collectively considered associated‐CTCs. Although CTCs have become one of the critical targets for cancer therapy, an effective therapeutic system focusing on primary tumor cells and associated‐CTCs simultaneously has not been developed to date. Therefore, designing a “roots and seeds” multipoint costriking strategy that could simultaneously eliminate primary tumor cells and prevent metastasis by targeting associated‐CTCs would be a promising strategy to improve the treatment effect on cancer.With the in‐depth study of CTCs, it has been found that in addition to single CTCs, CTC clusters and CTC–neutrophil clusters are present in the bloodstream of cancer patients and have a high potential to promote tumor metastasis.12 CTC To implement the “roots and seeds” multipoint costriking strategy to treat HCC, it is necessary to improve the efficiency of CTC identification and capture. CTCs are extremely rare in the bloodstream, and a more specific and sensitive CTC‐targeting capture system is urgently needed. Glypican‐3 (GPC3) is an oncofetal proteoglycan with high specific expression in HCC that possesses great potential for early diagnosis and precise therapy of HCC. In our previous work, it was indicated that GPC3 targeting could realize early and high‐sensitivity theranostics in HCC. Thus, it is hypothesized that GPC3 is a reliable target for the identification and capture of associated‐CTCs in HCC. It was proven that Na+/K+ ATPase inhibitors could induce loss of cell–cell junctions in cancer cells by increasing intracellular Ca2+ levels, which dissociated CTC clusters into single CTCs and suppressed metastasis formation. Digitoxin (DT) is an effective Na+/K+ ATPase inhibitor approved by the FDA and has a great capacity to maximize the dissociation of CTC clusters. Thus, DT possesses relatively good clinical prospects for CTC cluster dissociation. Using an anti‐VCAM1 monoclonal antibody (mAb) to bind VCAM1, which is overexpressed on the membrane of CTCs, would prevent the formation of CTC–neutrophil clusters. Moreover, anti‐VCAM1 mAb and anti‐GPC3 mAb could specifically recognize and combine with their receptors overexpressed on the membrane of CTC respectively improving the targeting sensitivity of therapies directed against CTCs and primary tumor cells.Hepatocellular carcinoma (HCC) has a high metastatic potential and the third highest fatality rate among cancers worldwide; thus, effective therapeutic strategies are urgently needed.21 To SchemeHerein, we designed a primary tumor and associated‐CTCs multipoint costriking nanodevice (GV‐Lipo/SF/DT) for enhancing metastasis inhibition and therapeutic effect on HCC. GV‐Lipo/SF/DT was developed by coloading sorafenib (SF) and DT into liposomes, and modifying anti‐GPC3 mAb and anti‐VCAM1 mAb on the surface of the coloaded liposomes Scheme1a. SF, 22.12000. First, the DSPE‐PEG2000‐anti‐GPC3 mAb and DSPE‐PEG2000‐anti‐VCAM1 mAb were successfully synthesized and characterized by 1H nuclear magnetic resonance (1H‐NMR) , V‐Lipo/C6 group (p < 0.05) and GV‐Lipo/C6 group (p < 0.01) were increased than that of the Lipo/C6 group  and V‐Lipo/IR780 (p < 0.01) groups, demonstrating the more specific in vivo dual‐targeting ability of GV‐Lipo/IR780.Both the Hepa1‐6 cell line and the H22 cell line were selected as mouse‐derived HCC cell models. We evaluated the expression of GPC3 and VCAM1 in the Hepa1‐6 cell line and H22 cell line through CLSM and flow cytometry (FCM), respectively. GPC3 and VCAM1 were highly expressed in both cell lines . The mean fluorescence intensity of GV‐Lipo/C6‐incubated Hepa1‐6 cells was increased compared to G‐Lipo/C6‐treated Hepa1‐6 cells (p < 0.05) and V‐Lipo/C6‐treated Hepa1‐6 cells (p < 0.05) after shearing at 37 °C for 0.5 h .31 The−6m, with no detectable reduction in cell viability , GFP Hepa1‐6‐luc clusters (H‐CL), or H‐CL preincubated with free DT or Lipo/DT  for 8 h. We found that the intracellular Ca2+ concentration increased significantly upon treatment with DT (p < 0.01), Lipo/DT (p < 0.05) or GV‐Lipo/DT (p < 0.01) compared with no treatment  or V‐Lipo/SF/DT , which confirmed that dual‐targeted liposomes could recognize and capture CTCs more specifically in vivo, thus inhibiting CTCs more efficiently.Furthermore, the final effect, namely, GV‐Lipo/SF/DT inhibiting the spread of “seeds” in the blood circulatory system in vivo, was examined. GFP Hepa1‐6‐luc cells were intravenously injected into mice as a CTC simulant, followed by intravenous administration of different formulations. At 24 h postadministration, the CTCs in the blood were separated and purified and quantified by FCM. As shown in Figure 2+ concentration. The competitive binding of the anti‐VCAM1 mAb to VCAM1, which is highly expressed on the surface of CTCs, could prevent the formation of CTC–neutrophil clusters. SF eliminated associated‐CTCs after cellular uptake.Overall, GV‐Lipo/SF/DT effectively inhibited the spread of “seeds”. When GV‐Lipo/SF/DT circulated in the bloodstream, the anti‐GPC3 mAb and anti‐VCAM1 mAb could specifically recognize and capture CTCs and CTC clusters, and DT could dissociate CTC clusters by increasing the intracellular Ca2.33 at day 21). Compared with that of the NS group, the tumor volumes of the free DT, Lipo/DT, and V‐lipo groups were not significantly different. Notably, the GV‐Lipo/SF/DT group exhibited the strongest tumor inhibition compared with the Lipo/SF/DT (p < 0.01), G‐Lipo/SF/DT (p < 0.01) and V‐Lipo/SF/DT (p < 0.01) groups. Furthermore, the tumor weight in the GV‐Lipo/SF/DT group was the lowest, with an inhibition rate of 90.28% . We hypothesized that SF and DT at experimental concentrations might have combined antitumor effects. Thus, possible reasons for the antitumor effect of DT and SF combination were further explored. Studies have shown that DT combined with SF could inhibit HCC by cell cycle arrest. The effects of SF and DT on the cell cycle in vitro were examined. Cell cycle arrest was induced in the G1 phase after treatment with Lipo/SF/DT , which may be because DT induced the cancer cells to lose cell–cell junctions to some extent, and thus conducive to the deep penetration of liposomes  . The bioluminescence intensities in the free DT, Lipo/DT, and V‐lipo groups were enhanced on the last day and showed no significant differences in comparison with that in the NS group, which indicated that free DT, Lipo/DT, and V‐lipo could not inhibit tumor growth effectively. However, the bioluminescence intensity in the free SF group did not significantly increase from day 1 to day 11, indicating that free SF could inhibit tumor growth. Compared to that in the NS group, the bioluminescence intensities in all other groups were significantly decreased on day 11 (p < 0.01). Notably, the bioluminescence intensity in the GV‐Lipo/SF/DT group was the lowest compared with that in the Lipo/SF (p < 0.01), Lipo/SF/DT (p < 0.05), G‐Lipo/SF/DT (p < 0.05), and V‐Lipo/SF/DT (p < 0.01) groups, which also indicated that GPC3 and VCAM1 dual targeting of GV‐Lipo/SF/DT could enhance the antitumor effects compared with the single targeting of G‐Lipo/SF/DT and V‐Lipo/SF/DT. These results suggested that GV‐Lipo/SF/DT exhibited superior antitumor efficiency in orthotopic HCC model mice. Body weight showed no significant change after various treatments  , Lipo/DT (p < 0.01), and V‐Lipo (p < 0.01) groups compared with the NS group, which indicated that DT and the anti‐VCAM1 mAb could prevent metastasis to a certain extent. Additionally, the number of pulmonary metastasis nodes observed after treatment with different formulations (free SF (p < 0.01), Lipo/SF (p < 0.01), Lipo/SF/DT (p < 0.01), G‐Lipo/SF/DT (p < 0.01), V‐Lipo/SF/DT (p < 0.01), and GV‐Lipo/SF/DT (p < 0.01)) was significantly decreased in these groups compared with the NS group. Significantly, the number of pulmonary metastasis nodes after GV‐Lipo/SF/DT treatment was less than that after treatment with Lipo/SF/DT (p < 0.01), G‐Lipo/SF/DT (p < 0.05) or V‐Lipo/SF/DT (p < 0.05)  Figure . The num) Figure , with an) Figure . Represe) Figure , with thGV‐Lipo/SF/DT could effectively inhibit the recurrence and metastasis of HCC, clarifying that dual targeting of GPC3 and VCAM1 could enhance the recognition of and uptake by tumor cells, thus killing primary tumor cells by SF, DT could dissociate CTC clusters and the anti‐VCAM1 mAb could prevent the formation of CTC–neutrophil clusters, all of which inhibited tumor metastasis together.3 which indicated that GV‐Lipo/SF/DT liposomes with SF and DT coloaded might have great translational potentials. Overall, the “roots and seeds” multipoint costriking strategy has opened a new possibility for omnidirectional inhibition of tumor metastasis, which could be translated into a new treatment concept for various cancers in the clinic.In summary, primary tumor and associated‐CTCs multipoint costriking nanodevice (GV‐Lipo/SF/DT) enhanced metastasis inhibition and therapeutic effect on HCC. GV‐Lipo/SF/DT inhibited the recurrence and metastasis effectively (inhibition rate 95.42%) by specific targeting of associated‐CTCs, dissociating CTC clusters, preventing CTC–neutrophil cluster formation and eliminating CTCs. Importantly, GV‐Lipo/SF/DT showed superior antitumor effects on the H22‐bearing tumor model (inhibition rate 90.28%) and orthotopic HCC models  in vivo with remarkable tumor suppression. The FDA approved the liposome Vyxeos (coloading of daunorubicin and cytarabine for injection) to treat acute myeloid leukaemia in 2017,38 whi+/K+ ATPase inhibitors digoxin has been used to dissociate CTC clusters in phase I clinical study (NCT03928210). However, associated‐CTCs, such as CTC clusters, CTC–neutrophil clusters, and other CTC forms, promote metastasis through various immune escape mechanisms. For example, apart from single CTCs, CTC clusters, and CTC–neutrophil clusters, platelets promote tumor metastasis by aggregating with CTCs, thus helping CTCs avoid immune attack and metastasis. In order to target associated‐CTCs, additional studies are needed to explain how CTCs escape from immune surveillance. More further research on the relationships between associated‐CTC formation and immune escape, and targeting associated‐CTCs strategies should be carried out. In consequence, targeting associated‐CTCs strategy is a promising avenue to inhibit tumor metastasis and has great clinical application prospects.CTCs are closely related to cancer metastasis and recurrence. Along with the development of CTC isolation technologies and in‐depth study of CTCs, CTCs have begun to be considered an important therapeutic target, and adopting therapeutic strategies to eliminate CTCs would be a new and feasible way to inhibit cancer metastasis or recurrence. At present, Natastasis.39 In The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Deep breathing (DB) and handgrip (HG) exercise -with and without circulatory occlusion (OC) in muscle-, have been shown to have beneficial effects on cardiovascular function; however, the combination of these maneuvers on heart rate (HR) and cardiac sympathovagal balance have not been previously investigated. Therefore, the aim of the present study was to evaluate the effect of simultaneous DB, HG, and OC maneuvers on the sympathovagal balance in healthy women and men subjects.Electrocardiogram and ventilation were measured in 20 healthy subjects  at baseline and during DB, DB + HG, or DB + HG + OC protocols. Heart rate (HR) and respiratory rate were continuously recorded, and spectral analysis of heart rate variability (HRV) were calculated to indirectly estimate cardiac autonomic function. Men and women showed similar HR responses to DB, DB + HG and DB + HG + OC. Men exhibited a significant HR decrease following DB + HG + OC protocol which was accompanied by an improvement in cardiac autonomic control evidenced by spectral changes in HRV towards parasympathetic predominance . In women, there was a marked decrease in HR after completion of both DB + HG and DB + HG + OC tests which was accompanied by a significant increase in cardiac vagal tone . No adverse effects or discomfort were reported by men or women during experimental procedures. Independent of sex, combination of DB, HG, and OC was tolerable and resulted in decreases in resting HR and elevations in cardiac parasympathetic tone.These data indicate that combined DB, HG and OC are effective in altering cardiac sympathovagal balance and reducing resting HR in healthy men and women. Sympathovagal imbalance is a common pathophysiological feature of many cardiovascular diseases, and thus an important target of therapeutic strategies . StandarVoluntary slow breathing exercises have been proposed as a potential non-pharmacological approach to improving outcomes in cardiovascular disease due to their effect on sympathovagal tone . VoluntaIsometric exercise involves sustained contraction against an immovable load or resistance with no or minimal change in length of the involved muscle group. Importantly, it has been shown that this training modality produces significant and clinically meaningful reductions in heart rate (HR) and blood pressure (BP) , and betDespite the fact that several studies indicate DB, HG, and OC individually may exert similar salutary effects on autonomic control of cardiovascular function in physiological and pathophysiological states, there are currently no studies that have addressed the potential additive or synergistic effect of these approaches in combination. Therefore, the aim of the present study was to identify suitability and feasibility of the combination of DB, HG and OC as an affordable strategy to improve HR control through cardiac sympathovagal modulation. Gender differences on the effects of the combined maneuvers on cardiac sympathovagal balance were also determined.Body mass and body height were significantly different between men and women p < 0.01); however, body mass index (BMI) was similar between groups  analysis, during baseline and the recovery period after DB + HG + OC in men. We found that low frequency to high frequency (LF/HF) ratio was significantly decreased during recovery compared to baseline values  Fig. . Our datCompared to men, DB + HG (recovery 2) was sufficient to elicit a significant reduction in chronotropy in women. After evaluating autonomic responses during recovery 2, we found that following DB + HG the LF/HF ratio was significantly reduced when compared to previous resting values  Fig. .Fig. 3EfIn the present study we observed that: (i) women had a significant reduction in resting HR after DB + HG, which was more pronounced after DB + HG + OC, (ii) men had a reduction in resting HR only after the combination of DB + HG + OC, (iii) reductions in resting HR following intervention were accompanied by spectral shifts in HRV and, (iv) men and women both showed a predominance of vagal autonomic modulation of heart rate following DB + HG and DB + HG + OC. Considering the limited literature in the area, these results support the notion that the combination of these maneuvers may elicit beneficial reductions in resting HR and changes in cardiac sympathovagal balance, offering an readily available non-pharmacological strategy to help in cardiovascular improvements following disease-related autonomic failure.The present study showed that the combination of two interventions was effective in reducing resting HR (study primary outcome) in a healthy population. Previous work has shown that slow breathing techniques decreases both resting HR and BP in humans with cardiovascular disease , 7. ImpoOne of the principal mechanisms related to the beneficial cardiovascular effects of DB is activation of the baroreflex by stimulation of stretch receptors located in the aortic arch and carotid sinuses . ArteriaOur study provide evidence showing gender-related differences in the cardiac/autonomic responses to DB, HG and OC. Although we assessed the potential of these interventions in the short-term, long-term studies are needed to gain a clearer picture of their efficacy and tolerability in humans. Another limitation is that we used indirect techniques to assess cardiac autonomic function. With that said, it is important to mention that this technique has been widely used in the clinical setting due to its low cost and non-invasive nature , 19. FutOur results indicate that concomitant DB + HG + OC reduces resting HR in men while DB + HG with and without OC were both effective to reduce resting HR in women. Also, DB + HG + OC in men and DB + HG in women induced marked shifts in HRV towards parasympathetic predominance. Together, these findings support the use of DB, HG and OC for cardiac autonomic modulation in healthy young adults.This study was approved by the ethics committee of the corporation of rehabilitation Club de Leones Cruz del Sur  (#20190719). Participants were carefully informed about the experimental procedures and the possible risks and benefits associated with their participation in the study. An appropriate signed informed consent document was obtained from all participants in accordance with the Declaration of Helsinki.Inclusion criteria for this study were as follows: (i) age between 20 and 35 years old; (ii) non-smoker; (iii) free of comorbidities including hypertension, diabetes, and heart failure; and (iv) free of arm lesions and/or arm surgeries in the 6 months prior to the study. Exclusion criteria were: (i) use of recreational drugs; (ii) taking any of the following medications: beta blockers, statins and/or any blood pressure reducing agents, anticonvulsant drug therapy, or any another drug that could influence the autonomic nervous system ; (iii) pregnancy; and (iv) abnormal cardiac autonomic function at rest as determined by HRV analysis (low to high frequency ratio of HRV < 2.3) [All participants were familiarized with the experimental procedures one week before measurements were taken. All experimental procedures were done between 09:00 and 17:00 h in a noise-isolated room at 21–23 °C. Experimental procedures were always administered by the same qualified investigators in the same order to all participants . The day before experimental procedures, participants were instructed to: (i) sleep ~ 8 h and (ii) not consume coffee or other caffeine-containing beverages.2.Prior to all experimental procedures, height, body mass, SBP, DBP, and 3-lead ECG were recorded. In all sessions and prior to exercises BP was determined using a sphygmomanometer  and a stethoscope  by the same experienced physical therapist. MABP, PP, HR, and HRV were calculated. Height was measured using a wall-mounted stadiometer  and recorded to the nearest 0.1 cm. Body mass was measured to the nearest 0.1 kg using a digital scale . BMI was calculated and expressed as kg/mPhysiological experiments consisted of 5 min of seated baseline data, followed by 5 min of DB (0.1 Hz) , 21, folf) were recorded during the experiment using an analog–digital acquisition interface .DB exercise was performed as previously described . BrieflyHG exercise and OC protocols were performed as described previously . Subjectf were analyzed using Lab Chart Pro 8.0 software .During all experimental procedures, ventilatory movements and a 3-lead ECG  were continuously recorded . ECG elePrior to experimental maneuvers, SBP and DBP were determined. From SBP and DBP, MABP (1/3 of SBP + 2/3 of DBP) and PP (SBP-DBP) were calculated. Measurements were determined using a sphygmomanometer  and a stethoscope  by the same experienced investigator.HRV: 0.04–0.08 Hz and HFHRV: 0.08–0.45 Hz [HRV ratio was calculated as an indicator of sympathovagal balance. LFHRV and HFHRV were expressed as normalized units (n.u.). Analysis was performed within a 35 min window using Kubios HRV Premium Software v3.1 .HRV was used as an indirect measure of cardiac autonomic function . From EC–0.45 Hz . AdditioThe independent variables were the different interventions  and the dependent variables were HR and HRV. Data is expressed as mean ± standard deviation (SD). All data were tested for normality (Shapiro–Wilk) and homoscedasticity (Levene). Data were evaluated as dictated by data structure using either an unpaired t-test or a repeated measures analysis of variance (ANOVA) (rest – DB – rest – DB + HG – rest – DB + HG + OC − recovery) followed by Holm-Sidak post-hoc analysis. Non-parametric variables were evaluated using Kruskal–Wallis analysis followed by Dunn´s post-test. The statistical power  for each significant comparison was also calculated considering arithmetic mean, standard deviation and number of subjects with an α < 0.05  . A p-val


Melanoma is a highly aggressive malignant skin tumor as well as the primary reason for skin cancer‐specific deaths. We first identified immune‐related long noncoding RNA (lncRNA) prognostic signature and found potential immunotherapeutic targets for melanoma cancer.RNA‐seq data and clinical features of melanoma samples were obtained from The Cancer Genome Atlas. Samples of melanoma were randomly assigned to the training and testing cohort. The immune‐related lncRNA signature was then obtained via using univariate, LASSO, and multivariate Cox analysis of patients in the training cohort. Eight significant immune‐related lncRNA signature was then subsequently obtained through correlation analysis between immune‐related genes and lncRNAs. The association between risk score and immune cell infiltration was finally assessed using TIMER and CIBERSORT.Three hundred and fifty‐six immune‐related lncRNAs were obtained. Among them, eight immune‐related lncRNAs were identified to build a prognostic risk signature model. The model's performance was then confirmed using the Kaplan–Meier curves, risk plots, and time‐dependent receiver‐operating characteristic curves in the training cohort. The risk score was identified and confirmed as an independent prognostic factor through univariate and multivariate Cox regression analyses. These results were further verified in the testing and whole cohorts. CIBERSORT algorithm showed that the infiltration levels of T cells CD8, M1 macrophages, plasma cells, T cells CD4 memory activated, T cells gamma delta, and mast cells activated were significantly lower in the high‐risk group while the infiltration level of macrophages M0 was significantly lower in the low‐risk group.The immune‐related lncRNA signature offers prognostic markers and potential immunotherapeutic targets for melanoma. We applied univariate Cox, LASSO, and multivariate Cox regression analysis to identify immune‐related lncRNAs with remarkable prognostic potential and constructed a multiple immune‐related lncRNAs signature to predict melanoma' prognosis. lncRNA signatures of immune‐related offers prognostic markers and potential immunotherapeutic targets for melanoma. It is the most malignant skin cancer.Long noncoding RNAs (lncRNAs) are RNAs with nonprotein coding that are usually over 200 nucleotides long. However, they have an indispensable role in tumor progression.Herein, a signature model comprising eight immune‐related lncRNAs for overall survival (OS) was first constructed utilizing uni‐ and multivariate, and LASSO Cox regression analysis to assess the value of immune‐related lncRNAs in melanoma prognosis. The samples were further assigned into two groups (low‐risk vs. high‐risk) based on the median risk score. Besides, we calculated the proportion of 22 immune cells in the training cohort using CIBERSORT algorithms to analyze the association between the differential proportion of immune cells and the risk score.22.1RNA sequencing fragments per kilobase per million (FPKM) for melanoma and the relevant clinical features were download from The Cancer Genome Atlas (TCGA) program. The merge script of perl and Ensembl database were used to combine RNA‐seq results into a matrix of gene symbols. The data set contained 471 melanoma tissues and one adjacent normal tissue. Samples without complete survival data as well as those whose OS was less than or equal to 30 days were removed from the study. Finally, 446 samples were included in the subsequent analysis.2.2p < .001). We randomly divided the melanoma samples into the training and testing cohorts using the R package “caret.” The expression levels of immune‐related lncRNAs were further applied to build the prognostic model in the training cohort.Immune‐related genes (IRGs) were downloaded from the Import Shared Data. Pearson's correlation analysis was then used to differentiate between expressed IRGs and lncRNA to identify immune‐related lncRNAs in melanoma samples  × expression (lncRNAi). The “survival,” “glmnet,” and “survminer” package in R were applied for univariate, LASSO and multivariate Cox regression analysis, respectively. The training and testing cohorts were further classified into two groups (high‐risk vs. low‐risk) based on the median risk score. Kaplan–Meier survival curves were drawn utilizing the “survival” package. Finally, time‐dependent receiver‐operating characteristic (ROC) analysis was used to explore the prognostic value of immune‐related lncRNA signature utilizing the “survivalROC” package.Univariate Cox regression analysis was employed to opt immune‐related lncRNAs with significant correlation to overall survival of melanoma patients in training cohort. The LASSO regression analysis was then applied to determine the significance of the results obtained from univariate Cox analysis. Multivariate Cox regression analysis was subsequently applied to optimize the prognostic model of the results of LASSO analysis. The melanoma prognostic signature was finally constructed based on the immune‐related lncRNAs and their relevant coefficients result from multivariate Cox analysis. The formula used was: risk score = ∑2.4Both uni‐ and multivariate Cox regression analyses were employed to explore the clinical information in three cohorts. The information included the risk score, gender, age, and stage of disease.2.5+ T cells, CD8+ T cells, dendritic cells, macrophages, and neutrophils were downloaded from the tumor immune estimation resource (TIMER) database, and the association between risk scores and immune infiltration was assessed by Pearson's correlation analysis.Immune infiltration data of B cells, CD42.6p < .05) were used for further analysis. The differential content of immune cells in the two groups (low‐risk and high‐risk) was further compared utilizing the Wilcoxon rank‐sum test. A heatmap was then applied to show the differential proportion of immune cells in two risk groups. The color green indicated that the infiltrating levels were high while red indicated that the infiltrating levels were low.CIBERSORT was applied to count the proportion of 22 immune cells in all samples of the training cohort given the importance of tumor immune cells infiltration in the tumor microenvironment. Those with significant differential contents of immune cells (33.1The 446 melanoma patients were randomly assigned into the training (224) and testing cohort (222) by using the R package “caret.” In both cohorts, 356 immune‐related lncRNAs were identified.3.2The univariate Cox regression analysis was used to determine if there were any associations between the 356 immune‐related lncRNAs and OS of melanoma samples. From this analysis, 36 immune‐related lncRNAs were found to have a significant association with OS Figure . FurtherPatients in both cohorts were further divided into two groups (high‐risk vs. low‐risk) on the foundation of median risk score Figure . Patient3.3Both univariate Cox and multivariate Cox regression analysis showed that the risk score and stage of disease were the independent prognostic factors for OS of melanoma in the training cohort Figure . The ris3.4The melanoma samples in the training, testing, and whole cohort were first assigned into two groups (high‐risk vs. low‐risk) based on the median risk score Figure . The cli3.5+ T cells, CD8+ T cells, macrophages, and neutrophils with risk score were: −0.219, −0.067, −0.077, −0.251, −0.115, and −0.337, respectively . These results further demonstrated that the eight lncRNA signatures of melanoma were associated with infiltration of the six immune cells.The association between the eight lncRNA signatures associated with tumor immunity and six immune cells downloaded from the TIMER database was further analyzed to determine the correlation between eight immune‐related lncRNA signatures and infiltration of immune cells. The association values of dendritic cells, B cell, CD4y Figure . These v3.6p = .01), T cells CD8 (p = .019), T cells CD4 memory activated (p = .026), T cells gamma delta (p = .027), mast cells activated (p = .039), and M1 macrophages (p = .015)  and T cells CD4 memory resting (p = −.53) indicated a good negative correlation. Besides, several immune cells showed good positive correlation, such as B cells naive and T cell regulatory (p = .42) and B cells memory (p = .44)  Figure . Subsequ) Figure .4Melanoma is a highly aggressive, melanocytic tumor characterized by a bad prognosis in the metastatic stage. It is the most malignant skin cancer.Multiple studies have proved that cell immune responses play vital roles in the progression of cancer and thus are potential factors that influence cancer prognosis.Herein, 356 immune‐related lncRNAs were identified through correlation analysis of lncRNAs and IRGs. Eight immune‐related lncRNAs were correlated with OS in the training cohort through univariate, LASSO, and multivariate Cox regression analyses. The eight immune‐related lncRNAs were then applied to build the prognostic model to predict the OS of melanoma in the training cohort. The patients were further assigned into two groups on the foundation of the median risk score. Significant differences in OS among the two groups were only being found in the training cohort. Moreover, the predictive capability of the immune‐related signature was favorable at 3 years (AUC = 0.715), 5 years (AUC = 0.720), and 10 years (AUC = 0.752) post diagnosis in the training cohort, which is better than the results of previous study 2. The eight immune‐related lncRNA also had a similar predictive capability in the testing and whole cohort. This indicated that they had favorable practicability and repeatability for predicting OS.Among the eight immune‐related lncRNAs, AC091729.3, AC245595.1, LINC02560, and PCED1B‐AS1 were risk‐associated, whereas AC242842.1, AL133371.2, HLA‐DQB1‐AS1, and LINC01871 were protective. Functions of some of the eight immune‐related lncRNAs have been probed and clarified. For example, PCED1B‐AS1 regulates proliferation and apoptosis of glioma by targeting the miR‐194‐5p/PCED1B axis.Both uni‐ and multivariate Cox regression analysis revealed that the risk score and tumor stage were independent prognostic factors in melanoma patients. The number of samples in two groups (high‐risk vs. low‐risk) based on gender, age, tumor stage, T, N, and M in the three cohorts were further studied to determine the association between the immune‐related lncRNA signatures and clinical features of melanoma. Combined results revealed that there was a significant correlation between immune‐related lncRNA signatures and tumor stage. This correlation was highly significant in AC091729.3, AL133371.2, PCED1B‐AS1, LINC02560, and AC242842.1. This strongly indicated that the expression level of the five lncRNAs could be associated with the development of melanoma. As such, the underlying mechanism of action of the five lncRNAs in melanoma development should be further explored.+ T cells, Mast cells, and CD4+ T cells in melanoma patients at high risk.When the association of the infiltration of immune cells and risk score was conducted, plasma cells, T cells CD8, Mast cells activated, M1 macrophages, T cells CD4 memory activated, and T cells gamma delta were found to have significantly low densities in high‐risk groups. This result was similar to that of previous studies that also reported a decrease in infiltrating of CD8Inevitably, there some unavoidable limitations that need to be addressed. First, it was a retrospective study based on the public database. Second, the amount of data available from the public database is still limited, and there is no validation from other databases. So, multicenter studies and actual experiments are needed to verify our results before the immune‐related lncRNA signature can be applied in the clinic.5We first identified and confirmed an immune‐related lncRNA signature targeting IRGs in melanoma. The immune‐related lncRNA signature was validated to be an independent prognostic indicator for melanoma. Moreover, these eight lncRNAs maybe also provide novel diagnostic methods and potential immunotherapeutic targets for melanoma.The authors declare that there are no conflict of interests.Bo Xiao designed the research study; Bo Xiao, Liyan Liu, and Aoyu Li performed the literature search and statistical analysis; and Bo Xiao, Aoyu Li, Pingxiao Wang, and Cheng Xiang interpreted the data and drafted the manuscript. Both Hui Li and Tao Xiao are the corresponding authors. Bo Xiao, Hui Li, and Tao Xiao critically revised the manuscript. All authors read and approved the final manuscript.


To evaluate the differences in macular choroidal thickness and volume between patients with primary open-angle glaucoma (POAG) and healthy controls to assess the correlation between macular choroidal thickness and visual field mean defect (MD).  Case-control study. A total of 101 patients (101 eyes) with POAG who were hospitalized in our hospital and 102 healthy subjects (102 eyes) matched by age, sex, and axial length were consecutively selected as the POAG group and the control group, respectively. The macular choroidal thickness and volume in nine regions were measured in all subjects by enhanced-depth imaging optical coherence tomography (EDI-OCT). µm and 208.24 ± 47.97 µm, and the mean volumes were 0.63 ± 0.19 µm3 and 0.64 ± 0.14 µm3. There were no significant differences in macular choroidal thickness, volumes of various macular regions, or mean choroidal thickness or volume between the POAG and healthy groups . The macular choroidal thickness of various macular regions was not correlated with visual field MD in the POAG group .  The mean thicknesses of the choroid in the macular area in the POAG group and healthy group were 207.97 ± 62.83  The macular choroidal thicknesses and volumes in POAG patients were not significantly different from those in healthy individuals. The macular choroidal thickness was not correlated with MD in POAG patients. Therefore, macular choroidal thickness is not an appropriate parameter to evaluate damage caused by POAG, and the role of the macular choroid thickness in POAG needs to be further investigated. Glaucoma is the leading cause of irreversible vision loss worldwide , 2. QuigThe factors affecting glaucoma progression are not fully understood, but intraocular pressure (IOP) is considered the most important risk factor for glaucoma. Although reducing the IOP can slow or prevent disease progression, in some cases, glaucomatous optic nerve damage can be further aggravated even if the IOP is controlled, indicating that factors other than IOP may play important roles in glaucoma progression –10.A study of choroidal hemodynamics in glaucoma suggested that abnormal choroidal blood flow may be one of the pathogenic mechanisms of POAG . The choPrevious studies of the relationship between POAG and choroidal thickness have generated conflicting results. Some studies  have fouA total of 101 patients (101 eyes) with POAG who were hospitalized in our hospital between October 2016 and October 2019 were consecutively selected as the POAG group, and another 102 healthy subjects (102 eyes) matched by sex, age, and axial length were selected as the healthy control group. There was no significant difference in age, sex, or axial length between the two groups .D or cylinder > ± 3.0 D; and systemic diseases such as diabetes and hypertension. This study was conducted according to the principles set forth in the Declaration of Helsinki and was approved by the ethics committee of Shijiazhuang People's Hospital. All subjects or their guardians signed informed consent forms.The inclusion criteria were a diagnosis of POAG, an IOP > 21 mmHg, and age older than 40 years. The exclusion criteria were other types of glaucoma (such as closed-angle glaucoma or secondary glaucoma); previous history of ocular surgery or ocular trauma; other ophthalmic diseases, such as corneal opacity, lens opacity, or other ocular diseases affecting the examination; retinal or macular diseases of the fundus ; a diopter of spherical equivalent > ± 6.0 All subjects underwent SD-OCT . The macular thickness and volume were scanned using the EDI-OCT macular thickness map examination procedure. The specific measurement methods were as described previously . The meaThe visual fields of all subjects were examined using the SITA-Fast 30–2 examination procedure and a Humphrey 750i visual field analyzer . The reliability criteria included a fixation loss rate of <20%, a false negative rate of <15%, and a false positive rate of <15%. Individuals who did not meet the criteria were excluded. The study included patients who had at least two reliable field tests.t-test. The correlation between macular choroidal thickness and field mean defect (MD) was analyzed by Pearson's correlation. A value of p < 0.05 was considered statistically significant.The data were analyzed using SPSS 21.0 statistical software . The measurement data were normally distributed; therefore, the choroidal thickness and volume values of POAG eyes and control eyes were compared using an independent µm and 208.24 ± 47.97 µm, respectively, and the mean volume was 0.63 ± 0.19 µm3 and 0.64 ± 0.14 µm3, respectively. There were no significant differences in the mean choroidal thickness and volume of the macular area between the two groups . There were no significant differences in the thickness and volume of the central subfield macula (CSM), nasal inner macular (NIM), superior inner macula (SIM), temporal inner macula (TIM), inferior inner macula (IIM), nasal outer macula (NOM), superior outer macula (SOM), temporal outer macula (TOM), and inferior outer macula (IOM) between the two groups  choroidal thicknesses were 207.97 ± 62.83 r = 0.026, p=0.796; r = 0.026, p=0.796; r = 0.009, p=0.926; r = 0.024, p=0.813; r = 0.070, p=0.490; r = 0.041, p=0.687; r = 0.038, p=0.702; r = 0.034, p=0.734; r = 0.055, p=0.583; r = 0.036, p=0.720).The choroidal thicknesses in the CSM, NIM, SIM, TIM, IIM, NOM, SOM, TOM, and IOM areas and the mean choroidal thickness in the macular area were not correlated with visual field defects in the POAG group (μm/year) and increases with increasing axis and myopia (15 μm/diopter) [The uveal tissues of the eye  contain abundant large and small blood vessels, and their blood flow accounts for more than 80% of ocular blood flow. Many systemic factors can affect ocular blood flow, and glaucoma patients with a history of low systolic perfusion pressure, low systolic blood pressure, and cardiovascular diseases may present glaucoma progression , 22. Chodiopter) , 24–26.Despite extensive study of the choroidal thickness in POAG patients, whether choroidal thickness is associated with glaucoma etiology or progression remains unclear, and the relationship between POAG and choroidal thickness is still controversial. Nakakura et al.  suggesteIn this study, the macula was divided into nine regions, and EDI-OCT was used to measure the macular choroidal thickness and volume in POAG patients and healthy subjects. No significant differences in choroidal thickness and volume in the nine regions of the macula were observed between the two groups. In both groups, the macular choroidal thickness was greater in the inner macula than in the outer macula. In the POAG group, the distribution of choroidal thickness in the inner macula followed the order SIM > TIM > IIM > NIM, and the distribution in the outer macula followed the order SOM > IOM > TOM > NOM. In the healthy subject group, the distribution of choroidal thickness in the inner macula followed the order SIM > TIM > IIM > NIM, and the distribution in the outer macula followed the order SOM > TOM > IOM > NOM. Sacconi et al.  found thSince the choroid is a highly dynamic vascular tissue, measurement of the choroidal thickness alone cannot fully reflect the physiological changes in the hemodynamics of the choroid in glaucoma patients. Thus, this study has some limitations. First, because there are diurnal variations in choroidal thickness , the thiIn summary, based on anatomical measurements, the choroid does not seem to be significantly altered in POAG patients compared to healthy subjects. The choroidal thickness in each region of the macula was not correlated with the visual field MD in POAG patients, and thus, choroidal thinning may not be an important component of glaucomatous optic neuropathy. Macular choroidal thickness is not an appropriate parameter to evaluate the damage caused by POAG. The pathogenesis of POAG is probably multifactorial, with important contributions from altered ocular blood flow and vascular regulation . Future


Meta‐analysis identified small but significant associations between IA and number of stool samples positive for all enteroviruses , consecutive positivity for enteroviruses  and number of stool samples positive specifically for enterovirus B . Virome analyses to date have demonstrated associations between enteroviruses and IA that may be clinically significant. However, larger prospective mNGS studies with more frequent sampling and follow‐up from pregnancy are required to further elucidate associations between early virus exposure and IA/T1D.Viruses are postulated as primary candidate triggers of islet autoimmunity (IA) and type 1 diabetes (T1D), based on considerable epidemiological and experimental evidence. Recent studies have investigated the association between all viruses (the ‘virome’) and IA/T1D using metagenomic next‐generation sequencing (mNGS). Current associations between the early life virome and the development of IA/T1D were analysed in a systematic review and meta‐analysis of human observational studies from Medline and EMBASE (published 2000–June 2020), without language restriction. Inclusion criteria were as follows: cohort and case–control studies examining the virome using mNGS in clinical specimens of children ≤18 years who developed IA/T1D. The National Health and Medical Research Council level of evidence scale and Newcastle–Ottawa scale were used for study appraisal. Meta‐analysis for exposure to specific viruses was performed using random‐effects models, and the strength of association was measured using odds ratios (ORs) and 95% confidence intervals (CIs). Eligible studies  included 1,425 participants  and examined IA ( Here, we report the first systematic review and meta‐analysis of observational studies using mNGS to investigate vertebrate‐infecting DNA and RNA viruses in children ≤18 years, and subsequent development of IA or T1D. Analysis of bacteriophage has been excluded from this review. The unbiased viral mNGS in early life and childhood has potential to comprehensively identify diabetogenic viruses increasing the IA/T1D risk or viruses affording protection. This may present new opportunities to intervene through antiviral medications or vaccination.22.1,This review is registered on PROSPERO (23 July 2020), registration number CRD42020188737. Two reviewers (Clare L. Faulkner and Yi Xuan Luo) independently conducted a systematic search for observational studies investigating the association between virome composition and/or abundance, and IA or T1D. EMBASE and MEDLINE databases were searched (2000–1 June 2020) using the strategy in Appendix Eligible studies were observational , using mNGS to characterise the virome in any clinical specimen in children aged ≤18 years who developed IA and/or T1D. Age restriction was imposed because IA often develops in childhood, suggesting viruses exert influence early in life. IA was defined as persistence of one or more autoantibodies against T1D‐associated autoantigens  in ≥2 time‐separated consecutive samples. Transplacental autoantibodies were excluded, defined as transient presence of the same autoantibody in a child <18 months and his/her mother. T1D was defined using American Diabetes Association criteria.n = 2), an independent advisor (Ki Wook Kim) was consulted to reach consensus decision. Case reports/series, uncontrolled studies, reviews and animal studies were omitted based on exclusion criteria.Two reviewers (Clare L. Faulkner and Yi Xuan Luo) screened titles and abstracts of identified studies Figure  and then2.2n = 4).Data extracted included publication authors, year and geographical location; study design; study participants; number of cases/controls; age; level of pre‐existing IA/T1D risk (human leukocyte antigen [HLA] genotype and family history); sample type, number and collection protocols; virus detection method and positivity threshold; rates of virus positivity in cases/controls; examined outcome (IA or T1D); measures of effect and funding. Original authors were contacted for insufficient or missing published data (Two reviewers (Clare L. Faulkner and Yi Xuan Luo) independently assessed the quality of included studies using the National Health and Medical Research Council (NHMRC) level of evidence scalep‐values for viruses present in children with IA or T1D versus controls from the extracted data using the Mantel–Haenszel method. Virus positivity was defined as virus material present in ≥1 study sample as detected by mNGS. Analysis was performed both for the number of case/control individuals and the number of case/control samples positive for virus. We used both fixed‐ and random‐effects models; only results from random‐effects models are presented due to heterogeneity of study populations. Statistical heterogeneity was explored using Cochrane's Q Test (χ2) and the I2 statistic, which indicate the proportion of variance of the summary effect attributable to between‐study heterogeneity. A p < 0.10 was considered a statistically significant heterogeneity, while I2 ≤ 25% and ≥75% were deemed low and high heterogeneity, respectively. Subgroup analyses were performed for geographical location, stool versus plasma, consecutive virus shedding and studies using comparable detection thresholds, and pooled ORs were calculated. Sensitivity and influence analyses were conducted by the study size. Data analysis was completed in Review Manager, Version 5.4 (Cochrane Collaboration),p ≤ 0.05.We calculated ORs with 95% CIs and  Q Test χ2 and theOur systematic review is reported using meta‐analysis of observational studies in epidemiology and Preferred Reporting Items for Systematic Reviews and Meta‐Analyses guidelines.3,,The search returned 778 publications (51 duplicates), leaving 727 articles for review. Title and abstract screening identified 12 publications for full‐text review. Four were excluded: three were repeat data sets3.1,Six studies defined IA as positivity for ≥1 T1D‐associated autoantibody; two defined as ≥2 autoantibodies . Stool versus plasma subgroup analysis gave pooled ORs 1.20  and 0.66 , respectively  and 0.87 , respectively.Seven studies investigated vertebrate‐infecting viruses and IA. No significant heterogeneity was observed, unless stated. Due to insufficient sample data, positivity for any virus was only analysed at the individual level, with no difference between cases and controls, pooled OR 1.03 . For stool versus plasma subgroup analysis, the pooled ORs were 1.14  and 0.80 , respectively . For stool versus plasma subgroup analysis, pooled ORs were 1.15  and 0.80 , respectively. There were minimal differences in effect sizes with sensitivity analysis.Meta‐analyses for specific viruses were conducted where proportions of positive case–control individuals or samples were reported in ≥1 study. Six studies found a significant association between the number of EV‐positive samples and IA . There were no associations between IA and EV‐A‐positive samples or individuals, pooled ORs 1.61  and 1.12 , respectively.Two studies,,p = 0.01; Figure p = 0.0007), but not EV‐A .Three stool studiesp = 0.20). However, for parechovirus‐positive samples and IA , there was significant heterogeneity . In influence analysis, the removal of the largest study outlier  strengthened the magnitude of association, pooled OR 0.44  and low heterogeneity . There was minimal difference in effect size for the number of individuals positive for parechovirus.Four studies reported parechovirus positivity in stool. There was no association between the number of individuals positive for parechovirus and IA, pooled OR 0.83 .Meta‐analyses at the individual and sample level for rotavirus, bocaparvovirus, anelloviruses, sapovirus, norovirus, cardiovirus, circovirus, mamastrovirus, mastadenovirus, kobuvirus, picobirnavirus, erythroparvovirus and roseolovirus showed no associations with IA Table . There w,Meta‐analysis of consecutive shedding of parechovirus, bocaparvovirus, anelloviruses and picobirnavirus found no associations with IA Table . Meta‐anStudy quality subgroup analysis was excluded as NOS scores were ≥8. HLA subgroup analysis was excluded as no studies stratified virus positivity by genotype, and all but twop = 0.02). Notably, human mastadenovirus F, astrovirus, human adenovirus 41, coxsackievirus A2 (CVA2), enteric cytopathic human orphan virus 30, coxsackievirus B3 and human parechovirus were more abundant in cases, while saffold virus, norovirus and rotavirus A were more abundant in controls. Every differentially abundant EV‐A  was more abundant in cases (p < 0.00001). Additionally, one studyp < 0.001).Only one studyp = 0.001).One analysis of very young children <6 months3.4,p = 0.83; and no heterogeneity χ2 = 0.21, p = 0.65, I2 = 0%).Two studiesp = 0.78) and human mastadenovirus F , with no heterogeneity between studies. One studyp = 0.05), but not individuals ; EV‐A was not associated with T1D. Limited studies precluded subgroup and sensitivity analyses.Meta‐analyses for EV, parechovirus, cardiovirus, norovirus, sapovirus, mastadenovirus, bocaparvovirus, mamastrovirus and anelloviruses were not significant for the number of positive individuals Table . Strain‐4This systematic review of 10 observational studies, involving 695 cases and 730 controls, demonstrated associations between virome composition in children ≤18 years and development of IA, but not T1D. This suggests early virome changes may influence initiation of IA, but not progression to T1D. There was a weak association between the number of stool samples positive for EV or EV‐B and IA, with approximately 1.2 times the odds of EV or EV‐B positivity in children who developed IA versus controls; ORs 1.14 (1.00–1.29) and 1.20 (1.01–1.42), respectively. There was 1.5 times the odds of consecutive EV shedding in stool of children with IA versus controls; OR 1.55 (1.09–2.20). Only one study measured consecutive shedding of EV serotypes,,,,,,,,These data suggest that specific gut vertebrate‐infecting viruses present in the gut virome, rather than the presence of any virus, influence IA risk. Research repeatedly reports associations between EV infection and IA initiation, supported by EV RNA in stools,,,We demonstrated an association between IA and the number of virus‐positive samples, but not individuals. Thus, when an individual has more than one positive sample, their IA risk is amplified. This may relate to viral persistence, protracted infection or increased exposure through reinfection. In particular, IA was associated with consecutive or prolonged EV/EV‐B shedding. Consecutive shedding is a strong indicator of persistent infection. Viral persistence is suggested to play a critical role in the development of autoimmunity,,,,The one study that conducted differential abundance analysis found 129 viruses with a ≥2‐fold difference in abundance in the gut of IA cases versus controls.4.1,To minimise bias, we implemented pre‐defined eligibility criteria, screening by independent reviewers, no language restrictions and sources beyond indexed databases. Random‐effects models may have provided more conservative effect estimates by accounting for study population heterogeneity and generating wider confidence intervals.,,Our findings have limitations. Only two studies examined T1D, limiting the analysis of associations between viruses and progression to T1D beyond IA initiation. Of the two studies that analysed T1D, one analysed prospectively collected samples and one analysed samples collected at/after T1D diagnosis. This may limit comparability due to potential differences when examining the virome after, rather than before, diagnosis of the study outcome. IA was predominantly defined as ≥1 autoantibody, despite single autoantibody conferring lower lifetime T1D risk versus multiple antibodies.,Studies applied different positivity thresholds, including 50p100K,,,Despite advances in metagenomics, virome analysis remains challenging, with rapid viral evolution complicating sequencingPositivity for viral nucleic acid is a marker of infection, not proof, as viruses may pass through the gut without productive infection, as with plant viruses and diet‐derived viruses.,,Studies sampled various body sites through stool, plasma and PBMCs. Direct comparison of across sites is difficult, requiring careful consideration of where viruses replicate. For example, EVs and mastadenoviruses preferentially infect and replicate at mucosal surfaces,Finally, this review did not examine bacteriophage to limit scope and adopt the precedent in other studies of focusing on only one class of virus to maximise detection sensitivity.χ2 and I2 limitations in detecting true heterogeneity. Studies were small: six had <50 participants, potentially causing small study effects. However, longitudinal sampling increased statistical power for detecting differences in the virome between cases and controls. Thus, we demonstrated the importance of comparing both number of virus‐positive individuals and samples. One studyOverall quality of included studies was high (NOS scores ≥8). All studies except one matched for ≥3 factors. Meta‐analysis demonstrated little significant heterogeneity; however, results must be interpreted cautiously given 4.2Despite limitations of targeted viral detection in IA/T1D pathogenesis studies, there remains a paucity of large, unbiased virome studies. Our findings must be validated in future studies that (1) include >200 participants and frequent longitudinal sampling preceding IA/T1D onset to improve statistical power and counter small‐study effects; (2) include a wider range of HLA genotypes to consider viral associations with IA/T1D in the context of genetic risk; (3) incorporate multicentre data to reduce geographical bias; (4) employ sensitive enrichment and comprehensive sequencing platforms; (5) integrate differential abundance analysis of viral load; and (6) sample various body sites to characterise viral strains and account for niche variations in viral abundance. Future studies should also include virome analysis during pregnancy to explore the role of antenatal and congenital infections in offspring IA/T1D development.This work was supported by the National Health and Medical Research Council Practitioner fellowship APP1136735 (to Maria E. Craig), the Juvenile Diabetes Research Foundation International Postdoctoral Fellowship 3‐PDF‐2020‐940‐A‐N and the Australian Diabetes Society Lindsey Baudinet Award (to Ki Wook Kim). The authors would like to thank Professors Ondrej Cinek and Kendra Vehik for providing supplementary raw data for inclusion in this systematic review.The author declares that there is no conflict of interests.Maria E. Craig and Ki Wook Kim designed the study and led the study group. Clare L. Faulkner performed the literature search, extracted, analysed and interpreted the data and wrote the manuscript. Yi Xuan Luo independently confirmed the database search and study quality. Ki Wook Kim acted as consultant in instances of uncertainty. Yi Xuan Luo, Sonia Isaacs, William D. Rawlinson, Maria E. Craig and Ki Wook Kim helped interpret the data. All authors were involved in the critical revision of this manuscript. Ki Wook Kim is the guarantor of this study.Supplementary Material 1Click here for additional data file.