Early diagnosis and treatment of the osteonecrosis of the femoral head (ONFH), a refractory disease, is imperative to prevent femoral head collapse; however, the existing solutions remain controversial. This study assessed the safety and efficacy of extracorporeal shock wave therapy (ESWT) combined with multiple drilling and intramedullary drug injection, a novel cocktail therapy, as a randomized controlled trial (RCT) model to postulate an alternative therapy for patients with early-stage ONFH.Femoral head necrosis patients aged 20 to 60 years with stage ARCO I-II were recruited. One hundred twenty eligible participants were randomized into four groups in a 1:1:1:1 ratio: extracorporeal shock wave therapy combined with multiple drilling and intramedullary drug injection (group EMI), extracorporeal shock wave therapy (group E), multiple drilling combined with intramedullary drug injection (group MI), and multiple drilling . The primary outcomes included effective rate, subchondral collapse rate of the femoral head, lesion size, and grade of bone marrow edema. Secondary outcomes included the Harris Hip Score and the visual analog scale. All outcomes were measured at the screening visit (baseline) and at the planned time intervals during treatment and follow-up, and the efficacy was statistically analyzed according to the intention-to-treat sub-populations and per-protocol sub-populations.To examine the clinical efficacy of ESWT combined with multiple drilling and intramedullary drug injection to provide a safe and more effective method for treating early-stage ONFH.ChiCTR1900020888; Pre-results. Early diagnosis presents several challenges. Early identification and treatment of patients with acute pain in the hip and groin area could be helpful in improving the diagnosis.[Core decompression (CD), often used in conjunction with concurrent nonvascularized bone grafting, is a common method for treating the pre-collapse.Since 2001, clinicians have been interested in exploring the use of extracorporeal shock wave therapy (ESWT), a biophysical therapy for treating early-stage femoral head necrosis.This study studied another treatment that has not been reported internationally: intramedullary drug injection. This method was the first reported in 2000 by Professor Zhongchao Jiang in China, which has been used in clinical practice for a long time with good clinical efficacy (48.8–80%), but most of them are retrospective studies with insufficient evidence.Despite extensive research on the treatments for the early-stage femoral head necrosis, such as CD with/without graft, bone-marrow implantation, bone grafting with/without vascularization, growth-factor-based treatment, mesenchymal stem cells, and proximal femoral osteotomies, there is still little evidence of treatment in stage I patients, and the optimal joint protection method is still unknown.1.The new cocktail of extracorporeal shock wave therapy + multiple drilling + intramedullary drug injection could achieve better clinical efficacy,2.The clinical effect of ESWT alone would be no worse than that of multiple drilling, and3.the clinical efficacy of intramedullary drug injection would be explored.Thus, here an RCT protocol was formulated to verify this hypothesis.22.1To examine the clinical efficacy of ESWT combined with multiple drilling and intramedullary drug injection to provide a safe and more effective method for treating early-stage ONFH.2.2This study is a prospective, single-center RCT, which was designed in accordance with the principles of the Declaration of Helsinki. All participants would be provided in-depth information on the protocol, risks, and right to withdrawal from the research. A signed informed consent would be procured from all participants. The protocol adheres to the SPIRIT 2013 Statement.[2.3Posters for recruitment would be posted in the outpatient and orthopedic inpatient departments of the Hospital of Chendu University of Traditional Chinese Medicine. Other methods of recruitment would include local print media, social media, flyers, and word of mouth. Formal recruitment for the study began in June 2020 and would continue until June 2021. Patients who would consent to participate would be examined and diagnosed by the physician .2.41.Patients between the ages of 20 and 60 years.2.Patients with a clinical diagnosis of ONFH,3.Patients who would voluntarily participate in the trial and agree to sign the relevant written consent.2.51.Patients with a history of hip fracture or surgery2.Patients who consumed hormones or alcohol within the last 1 month.3.Patients undergoing any other treatment.4.Patients who were pregnant or lactating.5.Patients who were allergic to relevant drugs used in the course of this test.6.Patients with other hip diseases or serious medical diseases.2.6The randomization sequence would be generated in advance by the clinical supervisor (WXL) using STATA , and would be sealed in opaque envelopes. After the baseline testing, the researchers  would open the envelopes by serial number in the presence of the clinical supervisor (WXL). It was difficult to implement the blinding since the treatment methods could be known by the subjects and the doctors. The efficacy and safety evaluation would be performed by blinded third-party data analysts.2.72.7.12) would be administered, followed by rest for 1 month, after which, the next course would be initiated.[Here, we would use the Orthopedic ESWT Device . The patient would be placed in the supine position with adduction and internal rotation of the affected limb. The anterior and the lateral part of the hip would be marked at 3 to 5 points using an X-ray or ultrasound depending on the lesion size. Lidocaine gel would be applied to the skin at those points. During treatment, attention would be paid to avoid shock wave damage to the femoral arteries and nerves. The study would include two courses of treatment; each course would include weekly treatments for 4 weeks, and each time 3000 to 5000 pulses  would be designed in advance. The patients would be in the supine position, with the affected hip raised 5 to 10 cm with a 15° internal rotation. The insertion area would be draped aseptically, followed by the administration of local anesthesia. A 2.5 mm Kirschner wire would be inserted percutaneously, 3 to 5 cm distal to the greater trochanter, and would be fluoroscopically guided towards the necrotic area until 0.5 cm beneath the subchondral bone.2.7.3The patient would be placed in the supine position, with the affected hip raised 5 to 10 cm with a 15° internal rotation to expose the greater trochanter of the femur. After skin preparation and draping, anesthesia along with 10 ml of 5% lidocaine would be administered to infiltrate from skin to the periost, layer by layer. The lateral greater trochanter of the affected hip was designated as the insertion point, and an 18-gauge puncture needle was penetrated into the cancellous bone area of the femur neck fundus, followed by extraction of the needle core along with 5 to 10 mL of medullary blood using a 10 mL syringe. Then, the compound osteopeptide injection (5 mL) and Chuanxiong Danshizine injection (5 mL) were slowly injected . After the injection, the needle core was slowly introduced, the puncture needle was pulled out, and a sterile gauze was applied for compression dressing; thus, completing the operational procedure. This treatment would be administered weekly for a period of 4 weeks, followed by a 1-month resting period, and repeated. This study treatment would include two courses.2.82.8.1In this group, the first course of treatment would include ESWT, followed by Multiple drilling and intramedullary drug injection. Then, for the next 3 weeks, they would receive ESWT and intramedullary drug injection on a weekly basis. After a 1-month resting period, the patients would receive weekly ESWT and intramedullary drug injection for 4 consecutive weeks.2.8.2The patients in this group would receive two courses of ESWT.2.8.3The patients in this group would receive multiple drilling and an intramedullary drug injection as the first treatment. Then, they would receive an intramedullary drug injection on a weekly basis for 3 weeks. After a 1-month rest period, the patients would receive weekly intramedullary drug injection for 4 consecutive weeks.2.8.4The affected hip would undergo the multiple drilling processes once to decompress, and the patients would be followed-up.All patients participating in the trial would receive rehabilitation guidance and training.2.9Table 2.9.12.9.1.1The effectiveness would be evaluated at the last follow-up visit. The intervention would be considered effective if the necrotic area of the femoral head does not increase, and the ARCO stage does not progress. Effective rate =  × 100%.2.9.1.2The subchondral femoral head collapse would be evaluated by plain Radiography or CT scans.2.9.1.3The lesion size would be determined by 3-D MRI measurements.2.9.1.4The patients would undergo an MRI scan before inclusion as well as 4 and 12 months after inclusion to evaluate the BME of the femoral head. BME would be classified as follows: grade 0 (no BME), grade 1 (perinecrotic BME), grade 2 , grade 3 (BME extended into the neck of the femur), and grade 4 (BME extended into the intertrochanteric region).2.9.22.9.2.1VAS would be used to evaluate the pain degree of the affected hip.2.9.2.2HHS would be used to evaluate the hip function of the affected side.2.9.3The following baseline demographic parameters would be recorded: age, gender, course of the disease, ARCO stage, history of smoking, alcohol consumption, glucocorticoid use, and concomitant disease.2.10Z-Test with unpooled variance. The significance level of the test would be .05 . Due to the limited sample size, this study would conduct a non-inferiority study of ESWT compared with multiple drilling. Sample sizes of the group E (24) and group M (24) would achieve 81.098% power to detect a non-inferiority margin difference between the group proportions of −0.20. Statistical analysis would be done using the one-sided Z test (unpooled). The significance level of the test would be .025. Additionally, we also designed group MI (multiple drilling + intramedullary drug injection) to observe the efficacy of this method and to explore whether the addition of the intramedullary drug injection could further increase the efficacy compared with the control group (group M); compared with group EMI, based on group MI, whether ESWT could further increase the efficacy. Estimating a dropout rate of 20%, we planned to enroll a total of 120 patients, with 30 patients per group.The primary outcome would be the effective rate . Previous studies have revealed the effective rate of multiple drilling to be 61.5%,2.112.11.1 to enter the data, and data managers would review the electronic data for consistency with the data on the CRF. During this period, if any problem occurs, the inspector shall be notified on time, and the investigator would be questioned. All questions/answers between them would be exchanged in the form of question sheets, which would be preserved for future reference. The paper and the electronic version of the CRF would be kept in the Hospital of Chengdu University of Traditional Chinese Medicine for 10 years, with restricted access.The researchers involved in the trial would be trained in good clinical practice. The original data would be recorded correctly on the case report form (CRF) on a timely basis, which would be reviewed and signed by the inspector and handed over to the data managers. Investigators would also log into the Clinical Trial Management Public Platform  and analyzed using the Kruskal–Wallis H test; Nemeyi tests would be applied for multiple comparisons. For the ordinal data, we would use the Kruskal–Wallis H test to assess the difference between the four groups; and Nemeyi tests would be used for multiple comparisons. Categorical variables would be expressed as frequencies and percentages and analyzed with the chi-square test or the Fisher's exact test. We would use the Cox's proportional hazard model to examine the survival status of the subchondral femoral head collapse between the four groups. Repeated measurement ANOVA would be used to analyze the repeated measurement data, such as VAS scores and HHS. Statistical analyses would be done according to the intention-to-treat principle on SPSS 22.0 Windows  with two-tailed tests wherever appropriate, and P < .05 would be regarded as statistically significant.Continuous data with a normal distribution pattern would be presented as mean ± standard deviation (SD) and analyzed using the one-way ANOVA, and LSD-2.122.12.1The Operational Guidelines for the Establishment and Functioning of Data and Safety Monitoring Board by the WHO would be used as the reference to establish the Data and Safety Monitoring Board. The committee would be an independent advisory group and would ensure the quality of research as well as protect patient rights and health. Every three months, the Data and Safety Monitoring Board would conduct a surveillance inspection and appraisal. Additionally, the ethics committee would conduct a follow-up review every 12 months.The project leader could suspend the trial and report to the results to the ethics committee and project sponsor if serious safety problems are found during the trial or the treatment effect of the test group is found to be too poor, even invalid with no clinical value, or other reasons.2.12.2All adverse events would be routinely reported and documented during the treatment and follow-up. An adverse event is defined as any undesirable experience experienced by the study participant, which may/may not be directly related to the intervention. All adverse reactions will be handled appropriately.2.13Neither the patients nor the public would have any involvement in the design/conduct of this study, and in the outcome measures.2.14The Medical Ethics Committee of Hospital of Chengdu University of Traditional Chinese Medicine provided ethics approval for this clinical trial (ID: 2019KL-042). If the protocol needs to be amended, approval from the ethics committee is required before implementation. The results of this study would be published in peer-reviewed journals and presented at international conferences. All forms of reports will be kept confidential for participants.3Currently, there is limited evidence regarding the pathological mechanism of ONFH. It is known that venous stasis or damage or interruption of the arterial blood supply causes the apoptosis of bone cells and bone formation-related cells, resulting in sparse trabecular bone and micro-fractures. This induces the initiation of the self-healing process, while simultaneously causing immune damage and the formation of osteosclerosis band. The osteosclerosis band hinders the growth of new capillaries, which accelerates the progression of necrosis in the bone tissue and causes the femoral head collapse and secondary osteoarthritis in the late stage.According to the pathological mechanism of ONFH, combined with previous research and clinical practice, we hypothesized that the new cocktail therapy of ESWT + multiple drilling + intramedullary drug injection could achieve better clinical efficacy in the treatment of early-stage ONFH. First, multiple drilling (2.5 mm) could not only realize “decompression,” but could also break through the osteosclerosis band barrier, providing a channel for the drainage of necrotic liquefied tissue in the femoral head as well as the growth of new capillaries.[If this hypothesis is verified, our findings will provide a minimally invasive, safe, accessible, and effective treatment for patients with early-stage ONFH and provide evidence-based medical recommendations for the clinical promotion of this treatment.Conceptualization: QianChun Li, Rigao Chen, Yang Yu, Xinling Wang, Zhongchao Jiang, Xiaohong Fan.Data curation: QianChun Li, Rigao Chen, Yang Yu, Xueya Feng, Leiming Jiang, Botao Chen, Peng Xin, Tong Li, Yin Shi, Qiang Jian, Zhongchao Jiang, Xiaohong Fan.Formal analysis: QianChun Li, Rigao Chen, Yang Yu, Xueya Feng, Zhongchao Jiang, Xiaohong Fan.Funding acquisition: Xiaohong Fan.Investigation: QianChun Li, Rigao Chen, Yang Yu, Xinling Wang, Botao Chen, Peng Xin, Tong Li, Yin Shi, Qiang Jian, Zhongchao Jiang, Xiaohong Fan.Methodology: QianChun Li, Rigao Chen, Yang Yu, Xinling Wang, Zhongchao Jiang, Xiaohong Fan.Project administration: Rigao Chen, Xinling Wang, Zhongchao Jiang, Xiaohong Fan.Resources: Zhongchao Jiang, Xiaohong Fan.Software: QianChun Li, Xueya Feng.Supervision: Rigao Chen, Xinling Wang, Xiaohong Fan.Validation: QianChun Li, Rigao Chen, Yang Yu, Zhongchao Jiang, Xiaohong Fan.Visualization: QianChun Li.Writing – original draft: QianChun Li, Xueya Feng, Botao Chen, Tong Li, Yin Shi, Qiang Jian.Writing – review & editing: Rigao Chen, Yang Yu, Xinling Wang, Leiming Jiang, Peng Xin, Zhongchao Jiang, Xiaohong Fan.


The discrimination improvement of an array of four highly sensitive 30 MHz gas quartz crystal microbalance (QCM) sensors was performed and compared to a similar system based on a 12-MHz QCM. The sensing polymeric films were ethyl cellulose (EC), poly-methyl methacrylate (PMMA), Apiezon L (ApL), and Apiezon T (ApT) and they were coated over the AT-cut QCM devices by the drop casting technique. All the sensors had almost the same film thickness (0.2 μm). The fabricated QCM sensor arrays were exposed to three different concentrations, corresponding to 5, 10, and 15 μL, of ethanol, ethyl acetate, and heptane vapors. The steady state sensor responses were measured in a static system at a temperature of 20 °C and relative humidity of 22%. Our results showed that the 30-MHz sensors have a higher sensitivity than 12-MHz ones (around 5.73 times), independently of the sensing film and measured sample. On the other hand, principal component analysis and discriminant analysis were performed using the raw data of the responses. An improvement of the classification percentage between 12 MHz and 30 MHz sensors was found. However, it was not sufficient, especially for low concentrations. Furthermore, using partition coefficient and discriminant analysis (DA), an improvement of 100% classification of the three samples was achieved for the case of the 30-MHz sensor array. Volatile Organic Compounds (VOCs) are one of the leading causes of indoor air pollution problems. These pollutants are emitted from different sources such as the volatilization of detergents, spray painting industry, inks, chemical agents in hospital disinfection, and even cooking. They can negatively affect human health, through several symptoms or diseases, including headaches, nervousness, or even cancer ,2,3. MorA variety of sensing methods such as electrochemical resistors, optical sensors, field-effect transistors (FETs), surface acoustic wave (SAW) sensors, surface plasmon resonance (SPR), and quartz crystal microbalance (QCM) have been used for VOCs analysis ,11,12,13−2 Hz−1 [−2 Hz−1, which would allow us to measure lower concentrations with better resolution in the sensor responses [Several factors influence the detection sensitivity of QCM gas sensors such as the kind of sensitive polymer, the film thickness, the functional groups affinities of the polymeric material to VOCs, environmental conditions , cluster analysis (CA), analysis of variance (ANOVA), principal component analysis (PCA), and discriminant analysis (DA) ,33,34,35In this work, a comparison of the responses for two QCM gas sensor arrays with four different polymeric sensing films is described. The arrays are composed of 12 MHz and 30 MHz fundamental resonance frequency QCM sensors, respectively, and they were used to measure and classify some selected VOCs with specific characteristics. The studied VOCs were ethanol, ethyl acetate, and heptane, which are important air pollutants , and theWe showed that the response of the 30-MHz QCM sensors array (30QCM-SA) is around 5.73 times larger than that of the 12-MHz QCM sensors array (12QCM-SA). Using PCA and DA, we were able to determine the responses’ correlation of the four sensors considering the concentration of the target compounds at controlled temperature and relative humidity. Finally, in order to eliminate concentration, sensing film thickness, and resonances frequency effects, we used the partition coefficient for the recognition and classification of these VOCs. The results showed that the sensitivity and the classification percentage were improved by using the 30QCM-SA.The sensor sensitivity depends significantly on the QCM operation frequency. It can be verified by the Sauerbrey equation (Equation (1)), where the frequency change ( surface .(1)Δf= The sensing film average thickness .The partition coefficient logarithm ng films . K is deIn the case of QCM sensors, Substituting Equation (6) in Equation (4), we obtain Equation (7) to determine K ,39.(7)K® grease L , and Apiezon® grease T . The VOC samples measured were ethanol , ethyl acetate , and heptane . EC, PMMA, ApT, hp, and EtOAc were purchased from Sigma-Aldrich, while ApL, EtOH, and chloroform were purchased from Supelco Analytical Products, J.T. Baker, and Meyer Corporation, respectively. All reagents were of an analytical grade and used without any purification.The polymeric materials used as sensing films were ethyl cellulose , poly(methyl methacrylate) , ApiezonThe gas sensors were fabricated using HC49U encapsulation, AT-cut, silver electrode QCM with the fundamental resonance frequency of 12 MHz and 30 MHz. For 12 MHz, the crystal wafer diameter was 9.7 mm and the electrode radius was 2.4 mm, while, for 30 MHz, the wafer diameter was 6.5 mm with an electrode radius of 1.25 mm. These electrode dimensions do not present a large deviation from the Sauerbrey equation, according to Huang et al. ,40. The The experimental setup used in this study is shown in The QCM gas sensors were placed inside a Teflon chamber (1 L volume) together with a relative humidity sensor  and a temperature sensor  for monitoring the environmental conditions. The QCM sensors were connected to an oscillator circuit located outside the chamber. The QCM frequencies were measured by a home-made frequency meter with a 1-Hz resolution and a sampling rate of 1 data per second . The meaThe VOCs’ liquid samples were injected into the chamber via a septum valve by employing a microliter syringe  and waited until the steady-state has been reached. The purge of the chamber and the recovery process of the polymeric sensing films were carried out with an extraction pump .The QCM sensor responses were measured for three different concentrations corresponding to injections of 5, 10, and 15 μL of ethanol , ethyl acetate , and heptane . The concentrations were calculated using Equation (5). All measurements were repeated five times at Typical tapping topography mode AFM images , showing the bare QCM electrode, its profile as well as EC, PMMA, ApL, and ApT sensing films deposited on the QCM electrode, which are shown in The electrical impedance curve of the coated QCM were measured to observe the frequency shift caused by the deposited polymeric sensing film. The measurement of impedance curves is necessary since they allow the observation of the stability and the effect of the sensing film deposition onto the QCM electrodes. i et al. . GeneralEquations (2) and (3) were used to estimate the sensing film thickness . The fact that the responses are similar can cause EtOAc not to be classified correctly and that the array cannot distinguish between different VOCs. On the other hand, for 30-MHz sensors, the responses are different for those sensors (149 Hz for ApL and 72 Hz for ApT) and, since their values are higher, we can be confident that this better reflects the characteristics of the sensor array. EtOH and hp response curves behavior was the same for the four sensing films (data not shown).The difference between all the sensitivity patterns is related to the sensing film affinity to the VOCs. The mutual solubility between the two materials is better since their solubility parameters t of ApL ,46,47.PCA and DA analysis were performed using the responses of both arrays to the three samples under study, and at the three concentration values mentioned above. The results are shown in The factors of concentration, sensing film thickness, and resonance frequency QCM can cause problems with the classification and recognition of the VOCs. Therefore, to eliminate such factors, a feature that can be used is the partition coefficient ith VOCs . MoreoveTo assess the capability of QCM sensors’ array for discrimination between EtOH, EtOAc, and hp, PCA and DA were employed. A study of the discrimination improvement of an array of high-frequency gas sensors based on 30-MHz QCM was performed using EC, PMMA, ApL, and ApT deposited over the QCM electrodes by the drop-casting method. The sensor responses were measured and they showed different sensitivities for ethanol, ethyl acetate, and heptane. Moreover, the obtained results were compared with a conventional 12-MHz QCM sensors array. A sensitivity increment was achieved using 30-MHz QCM devices. Theoretically, the increment must be 6.25. However, there are different aspects, such as sensing film structure, affinity, molecular weight, or environmental parameters (temperature and humidity) that change the sensitivity and selectivity behavior. We obtained an average sensitivity increment of 5.73.PCA and DA results using raw data showed that, in general, MP values are higher for the 30QCM-SA, which means that it is more effective to discriminate the samples. Furthermore, the results using partition coefficient data revealed an evident increment in the array performance for 30 MHz QCM, obtaining a classification of 100% for the three measured samples.


Primary renal extra-skeletal Ewing sarcoma is a rare neoplasm, often metastatic at diagnosis, and with a poor outcome. A multimodal approach is often the treatment of choice in this aggressive neoplasm. We present a case of primary renal extra-skeletal sarcoma in a 45-year-old woman who underwent tumor resection without clear margins. After no response to the first cycle of chemotherapy, we documented an early onset of local recurrence. The patient refused any other treatment and died four months after surgery. The ES family is a group of poorly differentiated tumors made up of small round blue cells and it recognizes the rearrangement of the ESWR1 gene as a pathognomonic sign [ES is the second most common pediatric bone tumor, with a peak of incidence between the first and second decade and 80% of cases arise from the skeleton . In adulHerein, we report a case of an adult woman affected by an EES treated with surgery and chemotherapy. Secondary aim was to perform a review of the literature about EES and its therapeutic management.2A 45-year-old female with persistent chest pain underwent chest-abdomen CT scan for suspicion of pulmonary thromboembolism. The imaging revealed a heterogeneous retroperitoneal mass, sized as 17 cm in diameter, extending from the superior left renal pole and infiltrating posterior abdominal wall and partially the diaphragm . The tumThe patient underwent a laparotomic left radical nephrectomy with adrenalectomy and partial resection of psoas muscle and diaphragm, which were macroscopically involved by tumor, in order to achieve a radical surgery. Hilar and para-aortic lymphadenectomy were also carried out. Operative time was 200 min. Estimated blood loss was 550 cc. There were not any perioperative complications. No blood transfusion was needed. The patient was dismissed on post-operatory day 13.Macroscopic examination showed a 200 × 150 × 90 mm grey-yellow, friable mass with capsular interruption and containing necrotic and hemorrhagic areas. Surgical margins were unclear at level of psoas and diaphragm resection. Histologically, the tumor was made up of nodules separated by sclero-hyaline septa and it was composed of small to medium-sized cells with oval nuclei, small nucleoli, granular chromatin, and scanty cytoplasm. The mitotic activity was high. On immunohistochemistry, the neoplastic cell resulted to be diffusely positive for CD99, focally positive for CD56, and focally and weakly positive for synaptophysin. The neoplastic cells were negative for cytokeratin (CKAE1/AE3), epithelial membrane antigen (EMA), S-100, leucocyte common antigen (LCA), CD2, CD20, CD79 alpha, CD117, LAT, CD10, MPO, TdT, CD68PGM1, NPM1, CD38, PAX5, CD31, CD34, and desmin. The proliferative index (KI67/MIB-1) was about 70%. The genetic analysis revealed the presence of EWSR1 rearrangement. The immuno-morphological analysis highlighted a malignant aggressive neoplasm with EWSR1 rearrangement, in keeping with EES. Lymph nodes were free of tumor. Four weeks after surgery, the patient underwent ESFT 2001 chemotherapy protocol, including 5 cycles of injectable vincristine (1.6 mg), ifosfamide (2.2 g), mesna (440 mg), and etoposide (110 mg). The protocol, the most suitable in this case, comprehensive of adriamycin in addition to vincristine et ifosfamide, was not administered due to the poor heart ejection fraction.After the first cycle of chemotherapy, the patient required emergency care for dyspnea. An X-ray exam showed left pleural effusion and a chest drainage was placed. A CT scan disclosed a huge recurrence in the surgical site and a peritoneal carcinomatosis as shown in Informed consent: Written informed consent was obtained from the patient for publication of relevant medical information and all of accompanying images within the manuscript.3ES was first described by Stout in 1918 and later on in 1921 by James Ewing who characterized this tumor describing it in the diaphysis of long bones. ES in children and adolescents is defined as a bone tumor, which may occur at any site within the skeleton, but preferentially affects the trunk and the diaphysis of long bones . Howevert  chromosomal translocation, which fuses EWSR1 to the FLI1 ETS family gene to generate EWSR1-FLI1 fusion transcripts which induce mitotic defects leading to genomic instability and subsequent malignant transformation [About 85–90% of EES cases present a somatic reciprocal ormation ,8,9.A first challenge for this kind of lesion is the diagnosis. Clinical symptoms are not characteristic when present.As in other pathologic conditions, radiological findings are usually not typical and present a wide spectrum of imaging features and metastatic patterns. The imaging features of primary EES on contrast-enhanced CT or magnetic resonance imaging (MRI) are bulky heterogeneous masses with frequent local invasion or with compression of adjacent organs. Retroperitoneal masses are usually large at diagnosis with 50% of lesions bigger than 20 cm . Most caIn recent literature, more and more cases of extra-skeletal sites in adults are reported. Probably, this increase is related to the availability of molecular diagnostic techniques and not to a true increase in its incidence .ES of the kidney is a rare tumor in adults and it was described for the first time in 1975 by Seemayer and colleagues. Some authors suggested that the origin of ES in the kidney could be neural cells invaginated into the kidney during their development or embryonic neural crest cells migrating into the kidney and undergoing tumorigenesis . The occAt diagnosis, the most common symptom is pain (in 63%), followed by hematuria in 41% and a palpable mass in 25% . In our Regarding renal ES , 57% of In our case, the huge mass showed imaging features of malignant tumor, and for this reason, we performed a surgical treatment without biopsy. Due to bulky size and wide blood supply of the tumor, a preoperative renal angio-embolization was carried out in order to reduce the risk of intraoperative bleeding and to facilitate surgical procedure, thus decreasing operative time and perioperative morbidity. Other important benefits of preoperative embolization included the potential role of an early ligation of the renal vein before the renal artery has been fully controlled, according to the indications given by Robson. In literature, the real usefulness of preoperative renal embolization is still debated. However, a recent prospective, randomized study showed preoperative renal embolization to be a safe and well-tolerated procedure that allowed to reduce median blood loss in patients with huge kidney cancer who underwent embolization before nephrectomy compared to patients who did not undergo preoperative embolization .Prognosis is poor in metastatic disease ,27,28,29Multimodal approach including surgery associated to the adjuvant chemotherapy is the standard therapeutic approach, with radiotherapy (RT) playing an optional role in localized and nonsurgical tumors. However, 32.5% of patients received only surgical treatment, while 13% received only chemotherapy. In the 87% of the patients who received surgical treatment, a nephrectomy was performed. About 5.5% of surgical patients received neoadjuvant chemotherapy, 47% adjuvant chemotherapy, and 15% adjuvant chemotherapy and radiotherapy. The mean overall survival (OS) in the group of patients treated by multimodal approach was 20.8 months, whereas that in the patients who received only surgery was 10.3 months ,30,31,32The key point in the management of these tumors is to obtain a complete surgical debridement with clear negative margins, but this is affected by the disease stage at diagnosis ,36. In o4Reporting this case, we would point out that in presence of a renal mass, especially if huge, it has to be borne in mind that EES can occur primarily in the kidney. The local invasion should be well-evaluated before the surgery because, in case of EES, only a complete surgical ablation of the tumor could improve cancer-specific survival.In adult, advanced ES is a dramatic condition with inauspicious outcome. Often, successful rate of surgical treatment may be affected by complexity to obtain negative surgical margins due to disease extension. Due to this, surgery in advanced disease may be considered as an important step of multimodal treatment.


The solidification/stabilization (S/S) method is the usual technique for the remediation of soils polluted by heavy metal in recent years. However, freeze–thaw cycles, an important physical process producing weathering of materials, will affect the long-term stability of engineering characteristics in solidified contaminated soil. In addition, it is still questionable whether using large dosages of binders can enhance the engineering properties of solidified/stabilized contaminated soils. In this study, the three most commonly used binders , alone and mixed in different ratios, were thus added to lead-contaminated soil in various dosages, making a series of cured lead-contaminated soils with different dosages of binders. Afterward, unconfined compression strength tests, direct shear tests, and permeability tests were employed on the resulting samples to find the unconfined compressive strength (UCS), secant modulus ( These reports demonstrated that heavy metal pollution has become the key factor in soil pollution in China.Over the past 30 decades, the rapid development of industry and agriculture have led to soils contaminated by heavy metals worldwide ,3,4,5,6.If heavy metal contaminated soils remain untreated over time, heavy metal contaminants likely will percolate down to groundwater or discharge to rivers . As suchAmong the existing remedial measures for heavy metal contaminated soils, the solidification/stabilization (S/S) technique stands out as being low-risk, relatively cost-effective, simple to operate, and widely applicable to engineering, as well as increasing comprehensive soil strength, and offering good resistance to biodegradation ,21.S/S is a remediation technology that reduces the toxicity of pollutants, by using physical or chemical methods to either immobilize harmful pollutants contained in the soil or to convert such pollutants into chemically inactive forms, preventing their migration and diffusion into the environment ,23. SoliDu et al.  confirmeOn the other hand, researches, done by Aldaood et al. , Du et aUnder the action of freeze–thaw cycles, changes in liquid water cause changes in the three-phase composition ratio of the soil as well as changes in the size and stability of soil aggregates, leading to increases in permeability and decreases in soil stability. In addition, freezing and thawing causes aggregates to release a large amount of inorganic substances, which enhances the organic mineralization of soil, adsorption, and desorption of organics. The freezing and thawing also affect morphological transformation, microbial activity, and the free energy stored in the soil. These changes in the physical and chemical properties of the soil inevitably affect the occurrence, form, and migration of heavy metal pollutants, adsorbed within the soil and the particles of the solidifying agent or colloidal surface ,39,40, lBased on the above evidences, taking the cement/quicklime/fly ash commonly used in soil S/S into consideration , the aimUndisturbed soils used in this work were taken from an industrial site in Chongqing, China, being reddish-brown in color. The collected soil samples were air-dried in the laboratory for about two weeks, and afterward, they were sieved through a 16-mesh polyethylene screen (<1 mm) to remove stones, branches, coarse materials, and other debris. Subsequently, the sieved soil samples were subjected to conventional soil tests in accordance with the Standards for Geotechnical Test Methods (GB/T 50123-1999). The physical property parameters of the soil samples are shown in In this experiment,  process ,46, analThree binders  were selected to remediate lead-contaminated soils in the current research. The cement selected was ordinary Portland cement (OPC325); the main component of quicklime is calcium oxide ; while the fly ash was obtained from the Chongqing Power Plant (secondary). As the total In this research, heavy metal pollutants of predetermined weights , quicklime  and fly ash  alone or mixed, were respectively added into these, and the mixtures of contaminated soils with binders were all mechanically stirred for more than 10 min with a magnetic stirrer. For comparison, specimens without any binder added were also prepared.For the sake of achieving optimal moisture content (OMC), as shown in Mass of prepared mixtures required for each test, calculated based on this OMC, 95% of the maximum dry density and specimen size, was weighed and transferred into a Φ39.1 × 80 mm cylindrical mold. Next, the mold was placed onto a vibrating stand with the frequency and amplitude of 48 Hz and 0.5 mm to obtain specimens of a diameter of 39.1mm, a height of 80mm for the UCS tests and permeability coefficient tests. Similarly, the weighed mixtures were transferred into a sample container with a ring knife and were statically compacted to acquired direct shear tests sample with a diameter of 61.8 mm, and a height of 20 mm. After extracting from the molds by using a hydraulic jack, all specimens were sealed with a plastic film to prevent loss of moisture before being placed into a standard curing room (temperature of 22 °C and relative humidity of 95%) for 56 days.The testing procedure of compound lead-contaminated soils treated with binder mixtures of various dosages  is shown in The cured samples were put into an alternating high- and low-temperature test chamber  at Chongqing University for the freeze–thaw cycle tests. The freeze–thaw cycles were set to 0 days, 3 days, 7 days, and 14 days, respectively. The one-day freeze–thaw cycle is shown in Following the Chinese standard for soil test methods (GB/T 50123-2019), the unconfined compressive strength (UCS) was tested using the YYW-2 strain-controlled unconfined controller . The test was carried out by the displacement control method (1 mm/min) loading, which defined the secant modulus The axial strain rate should be in the range of 1% to 3% strain per minute. The lifting device for testing was raised by turning the handle. When the axial strain was less than 3%, the data were read at every 0.5% of strain (0.4 mm); when the axial strain was equal to or greater than 3%, the data were read at every 1% of strain (0.8 mm). Each test was completed in under 10 min. When the reading of dynamometer peaked, the test ceased after continuing the strain of 3% to 5%; when there was no peak in the reading, the test continued until the strain reached 20%.The shear strength was tested with a ZJ-2 strain-controlled direct shear instrument . The shear rate was 0.8 mm/min. When the sample showed a displacement of 0.2 to 0.4 mm, the measurement of the proving ring and the shear displacement of the box were recorded. When the peak value of the proving ring dial gage was reached, the instrument continued to shear until the displacement reached 4 mm, at which point the damage value was recorded. Where the machine stopped during the shearing process, the damage value was also recorded. If the maximum value of the dial indicator reading was reached during the shearing process, the instrument continued to apply shear stress until the displacement reached 6 mm ,49.The permeability coefficient (constant  when theconstant  was thus3S (3CaO·SiO2), C2S (2CaO·SiO2), and C3A (3CaO·Al2O3), undergo a hydration reaction 2, achieving solidification and remediation of heavy metals; the carbonation reaction, in which Ca(OH)2 is generated after quicklime and CO2 in the air react to form CaCO3, increases the strength of specimens to a certain extent; and the pozzolanic reaction between Ca(OH)2 and soil minerals (such as SiO2 and Al2O3) produces hydrated calcium silicate, calcium hydrated aluminate, and other gel products that improve soil strength.Similarly to cement-cured lead-contaminated soils, the mechanism of quicklime remediation of contaminated soils has four main aspects: The ripening of quicklime, which occurs after quicklime meets water, producing Ca(OH)2 and Al2O3 content. Although the addition of fly ash improved the UCS of specimens, such increases were significantly lower than those seen in samples with cement or quicklime. This is likely to be because the main components of fly ash are CaO, SiO2, and Al2O3, which are effectively hydrated only in an alkaline environment, unlike alkaline activators, such as cement and lime. Additionally, the CaO in fly ash is much lower than that in cement and quicklime , the secant modulus , while the value of


Collagen membranes and matrices being widely used in guided bone regeneration and soft tissue augmentation have characteristic properties based on their composition. The respective proteomic signatures have not been identified. Here, we performed a high-resolution shotgun proteomic analysis on two porcine collagen-based biomaterials designed for guided bone regeneration and soft tissue augmentation. Three lots each of a porcine-derived collagen membrane and a matrix derived from peritoneum and/or skin were digested and separated by nano-reverse-phase high-performance liquid chromatography. The peptides were subjected to mass spectrometric detection and analysis. A total of 37 proteins identified by two peptides were present in all collagen membranes and matrices, with 11 and 16 proteins being exclusively present in the membrane and matrix, respectively. The common extracellular matrix proteins include fibrillar collagens , non-fibrillar collagens , and leucine-rich repeat proteoglycans . The structural proteins vimentin, actin-based microfilaments (ACTB), annexins , tubulins , and histones  were also identified. Examples of membrane-only proteins are COL12A1 and COL14A1, and, of matrix only proteins, elastin (ELN). The proteomic signature thus revealed the similarities between but also some individual proteins of collagen membrane and matrix. Guided bone regeneration (GBR) is a technique in which a membrane covers a bony defect for space provision and excluding soft tissue ingrowth ,2. The oCollagen membranes are derived from xenogeneic sources, including porcine tissues, following a chemical purification process to remove fat and other unwanted tissue components. Most of these tissue sources have a specific type of collagen, but mainly collagen type 1 (COL1), as a structural protein in the extracellular matrix (ECM) of dermal tissue, peritoneal tissue, ligament, and bone ,14. For Collagen matrices being used for soft tissue augmentation  can be dCollagens in the extracellular matrix (ECM) are linked to small leucine-rich repeat proteoglycan (SLRP), including decorin (DCN), biglycan (BGN), osteoglycin (OGN), lumican (LUM), and prolargin (PRELP) found in skin . SLRP haHighly sensitive mass spectrometers and improved separation methods have enabled analysis of minute sample amounts of proteins from a wider area of biological materials, such as plasma, serum, urine, bone, and saliva, just to name a few ,27,28,29®, Geistlich Pharma AG, Wolhusen, Switzerland) and three lots of collagen matrix  derived from porcine peritoneum and skin without cross-linking [w/w). After 16 h of incubation at 37 °C, aliquots of 20 µL were prepared and stored in 0.5 mL protein low-bind vials  at −20 °C until injection on the next day. The insoluble part was treated in the same manner as described for the extracted proteins by submerging the material in the respective solution during the sample preparation procedure, with the exception that the protein concentration was not assessed.Three lots of porcine peritoneum-derived collagen membrane , the peptides were separated by nano-reverse-phase  using an UltiMate3000 nano rapid separation liquid chromatography HPLC  separation system. Both, the trap- and separation columns were operated at 50 °C, and the UV peptide detection at 214 nm served as quality control for the HPLC separation. The samples were loaded onto the trap column using a loading solvent of 2% acetonitrile  in an aqueous mix of 0.1% trifluoroacetic acid (TFA)/0.01% heptafluorobutyric acid (HFBA), both purchased from Sigma-Aldrich, Vienna, Austria, at 30 μL/min and precooled to 3 °C . Nano seSus scrofa database  was set to 1%, and a decoy database search was used for estimating the FDR. Analysis was performed with Scaffold software  to visualize and validate complex MS/MS proteomics experiments. The STRING database was used to show protein-protein interactions . Peptides were introduced into the nano electrospray source (ESI) after the UV cell, and the ionization was performed using a stainless-steel needle with a 20 µm inner diameter and a 10 µm tip. The needle voltage was set to 2.8 kV in positive mode, and the top 10 ions were selected for MS/MS analysis (fragmentation). The resolution was set to 70,000 for full MS scans. Ions with a single charge were excluded from the MS/MS analysis, and fragmented ions were excluded for 60 s from further fragmentation. Raw MS/MS files were analyzed using Proteome Discoverer 2.4  and by searching the Swissprot 00008227 , versiong-db.org ).The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository ,40 with In our high-resolution shotgun proteomic analysis of three independent lots each of collagen membrane and matrix, 48 and 53 common proteins, respectively, were identified . The vasThe 37 common proteins consist of 20 members of the ECM . FibrillWe also identified proteins that are not considered to be characteristic of the ECM, such as vimentin (VIM), a structural protein that, along with tubulin-based microtubules  and actin-based microfilaments (ACTB), comprises the cytoskeleton. In addition, annexins , histones , and microfibril associated protein 4 (MFAP4) were identified as being present in three lots of membranes and three lots of matrices. Thus, the rather similar proteomic signature of the membranes and matrices includes the expected members of the ECM, as well as proteins that are linked to cellular components.The shotgun proteomic analysis of independent lots of collagen membrane and matrix also identified 11 proteins that were exclusively present in membranes and 16 proteins in matrices . Among tThe 16 proteins that all three lots of the matrix have in common include elastin (ELN), elongation factor 1-alpha 1 (EEF1A1), TGF-beta-induced protein ig-h3 (TGFBI), periostin (POSTN), and asporin (ASPN). In addition, we also identified ANXA2, LRP2, histone H3, myosin-11 (MYH11), and serpin H1 (SERPINH1).Collagen membranes and matrices, even though being widely used in regenerative dentistry, are poorly characterized at the protein level. To serve as a scientific basis studying the impact of the protein composition on the clinical behavior of collagen membranes and matrices, we performed a proteomic analysis of a porcine peritoneum-derived collagen membrane used for GBR procedures, and a collagen matrix derived from porcine peritoneum and skin usually applied for soft tissue augmentation. Thus, there were two independent clinical indications for biomaterials. Considering that the membrane and the matrix contain peritoneum tissue and that the collagen matrix also contains collagen from skin, we expected rather similar proteomic signatures for the two biomaterials, with the collagen matrix being more heterogenous.The main finding of this analysis was that the overall composition of both biomaterials was rather similar. Among the 37 common proteins occurring in all lots of both biomaterials, 20 were considered collagens and another five proteins were linked to the leucine-rich proteins. Thus, together, 68% of the common proteins are related to the extracellular matrix. Considering that the composition of the biomaterials reflects the composition of the extracellular matrix of the original tissue, we could confirm the presence of COL1 and COL4 in both biomaterials . The proApart from the collagens, other proteins of the extracellular matrix were identified. BGN, DCN, LUM, OGN, and PRELP all belong to the SLRP and are present in skin , exertinCollagen membranes were isolated from xenogenic tissues and subjected to defatting and alkaline treatment. Then, acid-sensitive contaminations were eliminated and the membranes dehydrated with acetone (US Patent number: US5837278A ). It is In general, to produce decellularized biomaterials, removing cells and immunogenic material from the xenogenic tissue is mandatory as the immune system can recognize porcine material as foreign and produce xenoreactive antibodies ,44. MostCollagen membranes and matrices can be distinguished by the presence of skin collagen in the matrix, while the collagen membrane contains exclusively proteins from peritoneal tissue. Our descriptive analysis reveals 11 and 16 proteins that were exclusively identified in collagen membranes and matrices, respectively. What was unexpected, though, was that the membrane, but not the matrix, contains COL12A1 and COL14A1, apart from LRP1, RYR1, and CLTC. Theoretically, all membrane proteins should be identified in the matrix; however, the presence of POST and ASPN, as well as LRP2, HBA, and SERPINH1, may originate from the presence of skin collagen in the matrix. Unfortunately, the qualitative analysis does not provide insights into the relative proportion of the processed porcine peritoneum and the skin used to prepare the matrix.Relative quantification of the proteins comparing the membrane with the matrix, for example, has shown that the matrix has comparatively more COL6 than the membrane. It can only be speculated that it is the COL6 being abundantly expressed by skin fibroblasts  that disBy analyzing the proteome of the collagen membranes ,6, an inEven though not identified by the proteomic analysis, collagen membranes contain growth factors such as TGF-β . However


The subgenotype B5 of EV-A71 is a widely circulating subgenotype that frequently spreads across the globe. Several outbreaks have occurred in nations, such as Malaysia, Thailand, Vietnam, and Japan. Appearing first in Taiwan, China, the subgenotype has been frequently reported in mainland of China even though no outbreaks have been reported so far. The current study reconstructed the migration of the B5 subgenotype of EV-A71 in China via phylogeographical analysis. Furthermore, we investigated its population dynamics in order to draw more credible inferences. Following a dataset cleanup of B5 subgenotype of EV-A71, we detected earlier B5 subgenotypes of EV-A71 sequences that had been circulating in Malaysia and Singapore since the year 2000, which was before the 2003 outbreak that occurred in Sarawak. The Bayesian inference indicated that the most recent common ancestor of B5 subgenotype EV-A71 appeared in September, 1994 (1994.75). With respect to the overall prevalence, geographical reconstruction revealed that the B5 subgenotype EV-A71 originated singly from single-source cluster and subsequently developed several active lineages. Based on a large amount of data that was accumulated, we conclude that the appearance of the B5 subgenotype of EV-A71 in mainland of China was mainly due to multiple migrations from different origins. VP1 sequences , is a fast-spreading pathogen, and causes hand-foot-and-mouth disease (HFMD), which imposes a serious burden on children and infants, worldwide. Reportedly, it may also cause aseptic meningitis, encephalitis, acute flaccid paralysis, and severe respiratory diseases, among others  of C4a evolutionary branch: 1998.4; the TMRCA of C4b subgenotype: 1994.1] before the outbreak that resulted in 353 severe cases and 22 fatalities in the Anhui province of China in 2008 . We purified the PCR products using a QIAquick PCR purification kit  and sequenced them using an ABI 3130 Genetic Analyzer  via the dideoxy chain termination method. We assembled both results from each strand using the Sequencher v5.0.We processed stool samples according to the standard protocol, and inoculated them on rhabdomyosarcoma cell lines (RD)  of the prototype strain BrCr (accession number AB204852) and trimmed to the same length of 891 nt. We made a phylogenetic tree with substitution model of Gama-distributed maximum likelihood composite (ML+Γ4) to clearly classify all sequences into different serotypes, and we save all sequences belonging to the B5 subgenotype as a raw dataset using MEGA 6.06 with bootstrap replication set at 1000 , including seven genomes identified in this study. The SimPlot program (version 3.5.1) was used to carry out similarity plots and bootscanning analyses, with a 200-nucleotide window moving in 20-nucleotide steps. Bootscanning analyses were run using the neighbor-joining method  procedure to construct a Bayes factor (BF) test that identifies the most parsimonious description of the phylogeographic diffusion process  tree in the Tree Annotator by ignoring the first 1,500 trees, in which nodes' posterior were summarized, and visualization was performed by FigTree. In order to simulate a dynamic process of migration, we converted the MCC tree into a keyhole markup language (KML) file suitable for viewing on Google Earth.VP1 region of EV-A71, of which 5 sequences were identified in the HFMD cases in China. The phylogenetic tree . By contrast, the results of mismatching distribution showed that the simulated curves were in good agreement with measured ones   . Althoug > 0.05) . The firMRCA of the VP1 sequences illustrated that the B5 subgenotype of EV-A71 may have differentiated in 1994.75 [95% Highest Probability Density (HPD): 1988.66–1998.97] and that it has been in circulation for at least 20 years. The main rate of nucleotide substitution is 5.6 × 10−3 [95% HPD: 4.83 × 10−3-6.44 × 10−3]. Except for a few external nodes, the evolution rate has slightly changed during the epidemic period (The Tc period . This maA maximum likelihood tree (1000 bootstrap replicates) was constructed, and we found that seven Yunnan B5 sub-genotype of EV-A71 genomes were separated into two groups, and these two groups of viruses are most similar to B5 sub-genotype of EV-A71 from Thailand and Vietnam, respectively . When weIn geographical reconstruction, the BSSVS has a strong impact on the location root. However, many localities that were weakly supported as root locations showed negligible posterior probability under the BSSVS. Interestingly, when Google Earth was used to reveal the dynamic process, and priors of the minimal rate configuration of epidemiological links were considered, 50 branches connecting 16 locations were identified. Migratory processes have been shown . At a BF cutoff of 3.0 and a verage posterior value cutoff of 0.5, we observed significant connections . Pu'er CVP1 region  Sequences on the VP1 region have rarely been reported in relation to recombination (Mcwilliam Leitch et al., 3CD region, and is considered as an important mechanism underlying the enterovirus evolution (Kyriakopoulou et al., VP1 sequences. In addition, a large number of sequences provide more choices, thereby preventing data bias. Therefore, the VP1 sequences were selected to reconstruct migration characteristics of the B5 subgenotype, especially in China.Commonly, classification of enteroviruses postulates that a series of species, clusters, genotypes, subgenotypes, and clades may be defined via genetic distances or the nucleotide differences in the VP1 regions of sequences from Shanghai, China, in 2011, and Malaysia, in 2003. The sequence showed a strong strict clock signal which was not due to recombination. On the other hand, discovery of the earliest geographical ancestor and determination of the communication relationships between Malaysia and Singapore depends only on posterior probability, and is unsupported by the Bayesian significance tests. The following two aspects are worth consideration. Extensive research on the B5 subgenotype of EV-A71 was performed only following the outbreak in Sarawak, Malaysia, in 2003. By that time, this subgenotype had stably circulated in Malaysia and even migrated to other countries. However, the typing algorithms, based on distance and nucleotide divergence, are often affected by the number of statistical data, samples, or sequences. The two samples from Malaysia and Singapore, 0815-MAA-00 and 5511/SIN/00, respectively, which were involved in the EV-A71 outbreaks with the B4 subgenotypes, enhanced this flaw (Mcminn et al., Posterior probability, an indicator of phylogeography, shows that continuous processes originated in Malaysia, with particular reference to the connections between Malaysia and other countries, such as Japan, Thailand, and Vietnam. We also found population exchanges to be active in those locations, and these may have potentially influenced the viral genotype exchanges. It is consistent with epidemiological evidence indicating that these locations have witnessed many HFMD outbreaks of the B5 subgenotype of EV-A71 in the past years (Mizuta et al., VP1 sequences have fewer dynamic sites or single nucleotide polymorphism sites. This suggests that using genomic sequences may provide more precise processes for tracking the migration events. However, it also indicates the possibility that high frequency recombination may be misleading and may result in conclusions that lead to incredible processes. Secondly, the BF is an algorithm that reveals the significance of connections via the ratio of prior probability to that of the posterior probability. We suggest that this may be likely affected by the number of relatively conserved sequences. When the number of sequences is decreased, some of the low credibility data may disappear. Certainly, we may sometimes lose some roots at the same time. Therefore, in order to explore the migration process accurately, it may be necessary to repeatedly select sequences, not only considering clock signals and population dynamics, but also considering the impact on the posterior probability.In the current study, compared with posterior probability, the BF revealed wide connections, resulting in much interference. This has not been reported in a previous study of the Coxsackievirus A16 (Hassel et al., MN966512-MN966518.The datasets generated for this study can be found in the nucleotide sequences of the entire genome for the seven strains determined in this study have been deposited in GenBank nucleotide sequence database under accession numbers The studies involving human participants were reviewed and approved by Second Ethics Review Committee of the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The patients/participants provided their written informed consent to participate in this study.KH and YZ conceived and designed the experiments. KH, ZH, and YS performed the experiments. KH, YZ, DY, DW, SZ, and WenbX analyzed the data. KH and YZ wrote the main manuscript. KH prepared all the Tables and Figures. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Locust, a grasshopper group rich in nutrients and protein content, can be utilized as a substitute for meat, but it is highly harmful to the environment and food crops worldwide  when compared to animal breeding, which is as crucial as food production  food demand for the growing population, which is estimated to be 9 billion by 2050  report, the insect breeding industries rose from 726 in 2015 to 2,318 (~300%) in 2018. Based on this knowledge, we selected the oriental edible beetle insect, Protaetia brevitarsis seulensis, also known as Kolbe, for genome sequencing (referred to as Kolbe in the rest of the article). Kolbe belongs to the Cetoniinae family, widely used in oriental medicine to treat various diseases. Also, it was approved temporarily as a food material by the Ministry of Food and Drug Safety of Korea (MFDS) in 2014 . The larvae and adults were reared on fermented oak sawdust in a constant rearing room at 25°C ± 1°C, under 50–60% relative humidity (RH) and a 14 h light: 10 h dark photoperiod cycle.Eighteen individual last instar larvae of Kolbe were selected for DNA sample extraction from the whole body for the genomic sequencing. For the genomic DNA isolation, the sample was washed with PBS, sterilized with 70% ethanol, and then anesthetized on ice. The entire body was fixed, and the dissected integument was then cut along the ventral part, and the guts were removed. The carcass was then quickly ground in liquid nitrogen using a mortar and pestle. The ground tissues were used for genomic DNA isolation using a Wizard Genomic DNA Purification Kit  according to the manufacturer's instruction. The quality and quantity of the DNA sample were examined using ultraviolet (UV) absorbance and gel electrophoreses. Additionally, total RNA from four different tissues  and four different developmental stages  were isolated for whole transcriptome sequencing. Briefly, each tissue  was collected from three individual last instar larvae after dissection, as mentioned above. For the collection of hemocytes, three individual last instar larval hemolymph were directly collected into sterile tubes containing anticoagulant buffer  on ice in triplicate, and they were centrifuged for 10 min at 1,000 g at 4°C to remove the supernatant. The tissues were homogenized in a 1.5 ml tube containing TRIzol reagent  using a pestle. In the case of the developmental stage sample, each stage of the three individual samples was washed with 70% ethanol to reduce microbial contamination from its surface. After ethanol volatilization, the individual was then quickly ground into a fine powder in liquid nitrogen, except for the eggs. For the extraction of eggs, 10 eggs were homogenized in a 1.5 ml tube containing TRIzol reagent using a pestle, in triplicate. RNA quantitation was performed by UV absorbance, and gel electrophoreses further confirmed its quality.Q ≤ 20)], and adapters using Trimmomatic-0.32 method , and Illumina (NextSeq500), which is familiar for long and short read sequencing. DNALink, the authorized service provider in South Korea, conducted complete experimental procedures. The Illumina paired-end sequences were initially subjected to the filtering of technical artifacts  and classified into subclasses with the reference Repbase v20.08 database (www.girinst.org/repbase/)  with RMBlastn v2.2.27+.The repeat regions in Kolbe were predicted using RepeatModeler , an ab-initio gene modeler, and a consensus gene modeler. The transcriptomes from the two methods  were mapped to the Kolbe repeat masked draft genome using TopHat, and Cufflink  from fifteen families were used in the ortholog analysis using the OrthoMCL method  of short-read sequences . The 692PRJNA648262. The assembled contigs and its annotation files  are available in figshare: https://figshare.com/s/5e095ac1bf7a63411d23) repository with all the annotations details in Readme file.The complete sequences generated in this study was deposited to the SRA repository under the accession JL, MJ, SS, and YS: genome assembly and annotations. MJ, SS, and YS: manuscript preparation. I-WK, MS, M-AK, SK, JH, EC, and UH: sampling and sequencing. JSH: funding and modeling the study. All authors contributed to the article and approved the submitted version.MJ, SS, and YS were employed by the company Insilicogen Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background and objectives: The aim of this study was to report a case of a patient with Charcot-Marie-Tooth disease type 2 (CMT2) treated with epigallocatechin gallate (EGCG) for 4 months in order to assess its therapeutic potential in CMT2. Materials and Methods: The study included a brother and a sister who have CMT2. The sister received 800 mg of EGCG for 4 months, while her brother received placebo for the same period of time. Both participants were assessed before and after daily administration by means of anthropometry; analysis of inflammatory and oxidation markers of interleukin-6 (IL-6) and paraoxonase 1 (PON1) in the blood sample; and motor tests: 2-min walk test (2MWT), 10-m walk test (10MWT), nine-hole peg test (9HPT) and handgrip strength measurement using a handheld Jamar dynamometer. Results: Regarding muscular and motor functions associated with higher inflammation and oxidation, improvements only observed in the woman in all analysed parameters  after 4 months of treatment with EGCG are noteworthy. Thus, this treatment is proposed as a good candidate to treat the disease. Charcot-Marie-Tooth disease (CMT) is the most common hereditary neurological disorder. It is characterised by deterioration in the functioning of peripheral nerves, with distal predominance, classified in two types according to the nature of the neuronal alteration: CMT type 1 (CMT1), also known as demyelinating CMT, and CMT type 2 (CMT2) where mainly axon damage occurs [There is currently no pharmacological cure and the only treatment available is rehabilitative therapy. However, chronic inflammation ,4 that dRegarding the relation of this catechin with body composition, obesity associated with hypertrophy and hyperplasia of the adipose tissue generates inflammation causing oxidative muscular stress, therefore causing more inflammation which deregulates the myocyte metabolism due to mitochondrial dysfunction. In this sense, EGCG decreases oxidative stress and inflammation related to the metabolic dysfunction of skeletal muscle in obesity, as the expression of antioxidant enzymes is increased. As a result, the production of reactive oxygen species (ROS) is reduced and autophagy related to mitochondria is regulated .Paraoxonase 1 (PON1) is an enzyme that inhibits oxidation of LDL, preventing the production of cytokines, inflammatory mediators and cell adhesion molecules. Thus, inflammatory response and deposition of LDL in the blood vessels is reduced . A decreFinally, IL-6 is a cytokine whose high levels indicate inflammation . In thisBased on the above, the aim of this study was to report a case of a patient with CMT2 treated with EGCG for four months in order to assess its therapeutic potential in CMT2.Treatment procedures for the patients were carried out in accordance with the Helsinki Declaration and were approved on 14 December 2017 by the Ethics Committee of the University of Valencia with code number H1512345043343. Both participants gave written consent to publish their case and data in an international medical journal.InterventionEGCG was administered to the female patient for 4 months, distributed in two doses of 400 mg (capsules at breakfast and dinner) and following an isocaloric Mediterranean diet . The second patient was administered placebo capsules containing microcrystalline cellulose, matching in size and colour. He was told to follow the same instructions: take two capsules a day (breakfast and dinner) and follow the same Mediterranean diet.MeasurementsThe following measurements were taken before and after the 4-month intervention, in the same conditions and at the same time. In the specific case of the test, they were carried out by the same neurologist assigned to each patient before the study.Body composition: Measurements related to weight, size, skin folds and body perimeters and diameters were taken using the Faulkner method, taking into account the protocol currently established by The International Society for the Advancement of Kinanthropometry (ISAK)SECA . The equBlood test and marker analysis: A blood test was carried out at 9 a.m. on an empty stomach, and then the serum was separated from the plasma after centrifuging the samples. PON1 activity was determined using 4-nitrophenyl acetate , with anMotor Assessment: The 2-min walk test (2MWT) was applied, which determines the functional capacity in people with walking difficulties, especially long distances. In this test, the patients were instructed to “walk at a comfortable pace” back and forth along a 60-foot walkway for 2 min . On the The case of a patient was included, alongside her brother, in a randomised double-blind clinical trial for motor disease treatment. The patient and her brother were the only ones from a large sample that had CMT2. In both cases, electrophysiological studies show normal nerve conduction velocities and a decrease in sensitive and motor neurographic responses, in addition to fasciculations, fibrillations and positive waves in electromyography while resting and great motor unit potentials in the one of maximal activation. As far as muscle weakness is concerned, after the exploration prior to the intervention, patients had similar degrees of significant muscular distal amyotrophy to the knee, with a more noticeable weakness especially in plantar and flexor muscles, leading to equinus and varus feet. The genetic cause, in the two study subjects, was mutation in the membrane metalloendopeptidase (MME) gene in homozygosity. As far as the genetic mutation is concerned, the diagnosis report presented by the patients show an alteration in MME gen . However, we do know that they have no familiar history of the disease, being these two siblings the only ones that have developed it among 5 siblings. On the other hand, it is also known the absence of other family members who have developed compatible symptoms with the disease, but there is consanguinity of the parents, which could explain the homozygous mutation with recessive autosomal behaviour.Regarding the severity levels in both patients, the electrophysiological study and mobility tests showed no significant differences between both individuals. The participant treated with EGCG was a 56-year-old woman, weighing 92.4 kg and 156 cm tall, diagnosed in 2008 with CMT2; she was independent in her daily life, with constant weight gain and without any other medical history of interest. Her brother followed a diet of the same characteristics and a placebo treatment as described previously. He was 51 years old, weighing 111.4 kg and 168 cm tall, diagnosed in 2008. In addition, he was also independent in his daily life, with constant weight gain and without any other medical history of interest.2, body fat percentage 28.6% and muscle mass percentage 36.78%. These anthropometric aspects exist alongside muscle weakness, spasms and fatigue. The patient who received placebo had the following anthropometric measurements: waist 120.5 cm, tricipital fold 29 mm, BMI 41.6 kg/cm2, body fat percentage 27.6% and muscle mass percentage 34.25%.Before the intervention, the patient who received EGCG had the following anthropometric measurements: waist 104.5 cm, tricipital fold 35 cm, body mass index (BMI) 37 kg/cm2 (difference of −2.55), body fat percentage 24.76% (difference of −3.82%) and muscle mass percentage 39.58% (difference of 2.8%) (2 (difference of −2.3), body fat percentage 29.65% (difference of 1.99%) and muscle mass percentage 31.46% (difference of −2.79%)  tricipital fold 29 mm (difference of −6 mm), BMI 34.45 kg/cmof 2.8%) . The pat −2.79%) .These changes are expressed in weight loss since the initial weight was 92.4 kg and resulted in 86.2 kg (a difference of 6.2 kg), with a decrease in body fat and an increase in muscle mass . HoweverThese anthropometric characteristics for both patients resulted in improvements in the patient treated with EGCG, yet this did not happen for the patient receiving placebo. In particular, the patient improvement motor skills  and biochemical markers . Changes in the 2MWT especially stand out, with an increase of 50 m (from 60 to 110 m), or the change in IL-6 in serum which decreased from 4.11 pg/mL to 1.07 pg/mL, respectively, and whose value is estimated within normal values .Once our intervention had been conducted, all the analysed variables worsened in the control patient who received placebo. However, an improvement was observed in the patient who received EGCG, showing an increase in lean mass and a decrease in fat mass, alongside improvements in oxidation and inflammation biomarkers (PON1 and IL-6) and greater motor activity in lower limbs and hands. This could be due to an energy improvement at a mitochondrial level, especially skeletal muscle. In this sense, IL-6 was precisely the first myokine described with a function related to obtaining energy from the muscle . Thus, iOur study resulted in observing a large decrease in IL-6 levels by administering high quantities of EGCG for 4 months. Said increase could be on account of an improvement in energy activity in the mitochondria due to the neuroprotective function of EGCG, associated with lower oxidation as indicated in the increase of the PON1 enzyme. Regarding improvements in mitochondrial energy balance, EGCG also improves the activity of the Na(+)/K(+)–ATPase pump that is especially important in muscle and nerve cells in order for correct mitochondrial functioning , which iBoth at a metabolic and functional level, with a decrease in muscle and increase in inflammation and oxidation affecting motor functioning, administering EGCG shows very good results in all these variables. This evidence makes it a possible therapeutic alternative to treat the disease, which could be interesting to try to improve the functional capacity of these patients. Nevertheless, definitive conclusions cannot be drawn regarding the results of a 4-month intervention, in the case of a progressive chronic disease, therefore, we must continue to delve into the understanding of the impact of this type of treatments in CMT2.


The aim of this study was to identify the main risk factors for health-care-associated infections (HAIs) following cardiac surgery and to establish an effective early warning model for HAIs to enable intervention in an earlier stage.In total, 2227 patients, including 222 patients with postoperative diagnosis of HAIs and 2005 patients with no-HAIs, were continuously enrolled in Beijing Anzhen Hospital, Beijing, China. Propensity score matching was used and 222 matched pairs were created. The risk factors were analyzed with the methods of univariate and multivariate logistic regression. The receiver operating characteristic (ROC) curve was used to test the accuracy of the HAIs early warning model.After propensity score matching, operation time, clamping time, intubation time, urinary catheter time, central venous catheter time, ≥3 blood transfusions, re-endotracheal intubation, length of hospital stay, and length of intensive care unit stay, still showed significant differences between the 2 groups. After logistic model analysis, the independent risk factors for HAIs were medium to high complexity, intubation time, urinary catheter time, and central venous catheter time. The ROC showed the area under curve was 0.985 . When the probability was 0.529, the model had the highest prediction rate, the corresponding sensitivity was 0.946, and the specificity was 0.968.According to the results, the early warning model containing medium to high complexity, intubation time, urinary catheter time, and central venous catheter time enables more accurate predictions and can be used to guide early intervention after pediatric cardiac surgery. All patients were continuously enrolled from January 1, 2016 to November 30, 2017. The exclusion criteria included: clearly diagnosed genopathy, infective endocarditis, perioperative infections of other positions, patients>14 years old, cases of data loss, clinical death within 24 hours after operation. According to the definition of HAIs and criteria above, 2227 children, including 222 HAIs patients and 2005 no-HAIs patients, were enrolled. Capital Medical University Affiliated Anzhen Hospital's Ethics Committee approved the study protocol  and consent was obtained from the patients or their relatives.2.3It is well established that there are many operative methods to address a variety of CHDs. In these circumstances, to standardize the operative methods performed in our patients, we implemented the Risk Adjustment in Congenital Heart Surgery (RACHS-1) score.2.4Data from all patients were collected on a specific form concerning the patient's personal and medical history, symptoms and signs, type of heart disease, and laboratory and imaging findings. The preoperative specific factors evaluated included age, gender, body length, weight, admission hemoglobin level, admission serum total protein level, admission serum albumin level, admission serum prealbumin level, ejection fraction value, the type of heart disease. The intraoperative factors included the category of RACHS-1, the length of the operation, the length of the clamped aorta. The postoperative factors included the use and duration of mechanical ventilation, the use and duration of a urinary catheter, the use and duration of a central venous catheter, the use and duration of antibiotics, the number of blood transfusions, the performance of other invasive operations, the total duration of the hospital stay, and the total duration of the intensive care unit (ICU) stay. Other invasive operations included thoracic drainage tube replacement, re-endotracheal intubation, peritoneal dialysis tube, reoperation, delayed sternal closure, extracorporeal membrane oxygenation, and permanent pacemaker implantation. The study protocol is shown in Figure 2.5Data are presented as the means ± standard deviations for continuous data with a normal distribution, as the medians  for continuous data with a non-normal distribution, or as numbers and percentages for categorical variables.t tests or the Wilcoxon signed-rank test for continuous variables and the Pearson χ2 test for categoric variables. Previous reports suggested standard criteria for the propensity score-matching method, and we followed those criteria in this analysis.Propensity scores were calculated with the following preoperative variables: age, sex, weight, body length, admission hemoglobin level, admission serum total protein level, admission serum albumin level, and admission serum prealbumin level. Matching was performed using a Greedy 5-to-1 Digit-Matching algorithm. After propensity score matching, 222 matched pairs were created .Univariate and multivariate logistic regression were used to identify independent risk factors Table . Receive33.1The preoperative data of the propensity-matched patients are listed in Table 3.2Hospital stay and ICU stay were excluded from the logistic analysis, because these 2 variables were not the risk factors for HAIs but caused by HAIs. According to RACHS-1 score, a RACHS-1 score of 4 or more is called high complexity, a RACHS-1 of 3 is called medium complexity. Medium to high complexity was put together as an independent risk factor, compared with operations with a RACHS-1 of 1 or 2 in our research. Multivariate analysis for the variables after univariate analysis demonstrated that the independent risk factors for HAIs after cardiac surgery were medium to high complexity (P<.001), intubation time (P<.001), urinary catheter time (P<.001), and central venous catheter time (P<.001)  Fig. . When th4Our study confirms that major infection after congenital heart surgery is a complication with significant sequelae. Using a large patient population from a single center, we identified risk factors for healthcare-associated infections and created a clinical tool that can be used preoperatively to estimate a patient's infection risk. We validated the model internally, showing that it has good discrimination.4.1,3 A Japanese hospital studied 526 young patients (under 18 years old) after cardiac surgery from January 2013 to December 2015; 81 patients were diagnosed with postoperative HAIs. The infective rate was 15.4%, including 30 blood-borne infections (5.7%), 30 operative site infections (5.7%), 13 urinary tract infections (2.5%), and 8 pulmonary infections (1.5%). Another retrospective study of 634 CHD patients from the USA reported that 38 patients were diagnosed with HAIs 90 days after surgery, including 19 blood-borne infections (3.0%), 9 operative site infections (1.4%), 6 infective endocarditis (0.95%), and 4 ventilator-related pulmonary infections (0.6%); the total infection rate was 5.99%. Many studies have confirmed–15 that young age, underweight, long operation time, long clamping time, long mechanical ventilation time, and long ICU stay are risk factors for HAIs, but the results were inconsistent to varying degrees in quantitative risk indicator studies. A study in neonates from the USA reported that the risk factors for HAIs also included central venous catheter indwelling time >14 days and >5 postoperative blood transfusions. In addition, the American Society of Anesthesiologists (ASA) score, ICU stay >48 hours, the difficulty of the operation (RACHS-1 score), and cardiopulmonary bypass (CPB) time ≥200 minutes are closely related to the development of postoperative HAIs. Another study from Georgia, USA reported that young patients <1 year old, emergency surgery, reoperation for any reason, reintubation, and a history of hospitalization in other hospitals within the past 3 months have also been shown to be risk factors for HAIs.It has been reported that the incidence rate of HAIs is 2.7% to 8% worldwide. reported that young patients <1 year old, CPB time >105 minutes, aortic cross-clamp time >85 minutes, >3 postoperative blood transfusions, hospitalization time before operation >48 hours could be independent risk factors for SSIs and could guide clinical practice. Other studies reported in foreign countries, including Canada and the United States, indicated that the ASA score, mechanical ventilation time, length of ICU stay, length of postoperative, white blood count before operation and the first day after operation, and duration of vasoactive drug use should also receive attention when attempting to prevent SSIs after an operation. The influence of the use of perioperative antibiotics on SSIs has always been controversial; some of the related studies confirmed that antimicrobial prophylaxis over 48 hours after the operation did not reduce the incidence of SSIs but rather increased the risk of infection, including the rates of drug-resistant Bacillus infections and Clostridium difficile infections.,19 In addition, it has been broadly accepted that the incidences of VAP and CLABSIs are closely related to long durations of invasive catheterization and multiple catheter implantation.Some device-related infections have been paid particular attention by researchers investigating HAIs after operations for CHD; these infections include VAP, SSIs, and CLABSIs. Costello et al4.2P<.001), body length  and weight  kg versus 9.60  kg, P<.001, the data above were not demonstrated in the article). Therefore, we used the method of propensity score matching to effectively remove confounding factors, a number of typical preoperative items including age, sex, weight, body length, admission hemoglobin level, admission serum total protein level, admission serum albumin level, and admission serum prealbumin level were calculated in the method. Admittedly, some preoperative items such as age, weight, and preoperative nutritional status could surely affect the risk of postoperative infection, but we mainly discussed the medical procedures related factors that could generate HAIs in this research.Based on the studies described above, certain preoperative conditions could be high-risk factors for postoperative infections, including low birth weight, poor nutritional status, and preoperative pulmonary infections. Moreover, age, body length, and sex may also be influential factors affecting the incidence of postoperative infections. Under the circumstances, propensity scores were used because we mainly wanted to explore how the intraoperative and postoperative operations influenced HAIs. In fact, we first used all the 2227 patients (including 222 HAIs patients and 2005 non-HAIs patients) enrolled to analyze the differences between 2 groups. We found that some preoperative items could have significant differences including age  years versus 1.00  years, 4.3 we regarded operations with RACHS-1 score≥3 as medium to high complexity, RACHS-1 score<3 as low complexity. We could clearly find out that Medium to high complexity may be the most crucial risk factor for postoperative HAIs, compared with Intubation time, Urinary catheter time, and Central venous catheter time from Tables Operation time and Clamping time, showed no significant difference between 2 groups. It means that extracorporeal circulation technique, myocardial, and respiratory protection technique have made most pediatric cardiac ratios implement safely, while it is the scale and range of operation and patients’ systematic state, especially whether cyanosis preoperatively, that most impact the HAIs.RACHS-1 score is a very important variable for our research. It allows a variety of pediatric operation procedures to be measured in a uniform way. As many articles did,4.4 the model created and validated in this study could have an important clinical impact because it provides a more accurate estimate of an individual patient's risk for major infectious complications. Identification of these high-risk patients is useful in the early stage of the postoperative period by helping parents and providers know what obstacles may lie ahead. In addition, these identified high-risk patients may be targeted for future clinical trials and interventions to reduce this complication of cardiac surgery.Finally, we created a predictive model for HAIs based on the above results. The monitoring indexes included medium to high complexity, intubation time, urinary catheter time, and central venous catheter time. It is also very important to reasonably determine the weighting of these early-warning indicators and warning thresholds. Our research finally determined the weights of the indicators according to their beta coefficients as follows: Medium to high complexity, 5.079; intubation time, 1.049; urinary catheter time, 2.169; central venous catheter time, 0.545. The ROC curve provides the Youden's index  value for each point on the coordinate; the best threshold is usually determined by the optimal truncation point corresponding to the maximum value of the YDI. In this study, when the optimal truncation point was 0.529, the YDI was the maximum value of 0.914. Compared with other warning models,4.5Apart from the complexity of the operations, some factors related to postoperative invasive procedures including intubation time, urinary catheter time, and central venous catheter time are also closely related to postoperative HAIs. This result reminds us of some important concepts in clinical practice: First, remove all unnecessary invasive devices as soon as possible; Second, sufficiently manage all invasive operations, including maintaining aseptic conditions during device implantation, performing adequate postoperative clinical care, and avoiding repeated punctures; Third, it should be a routine for most institutes to monitor the tip culture as an indicator for HAIs. Admittedly, some other invasive operations mentioned in this article, including re-endotracheal intubation, peritoneal dialysis tube, reoperation, extracorporeal membrane oxygenation, and so on, were proved to be independent risk factors for hospital infection. But after propensity matching, the sample size of these operations was rather small in no-HAIs group, we could not make valid comparison between the 2 groups. Another distinctly important factor for hospital infection must be the application of antibiotics. In our research, all of the surgeries were performed with prophylactic antibiotics under anesthesia, primary antibiotic would be continued for 3 days after surgery, and may undergo a combination therapy of 2 antibiotics during stay in ICU, depending on infection control. All antibiotic changes and upgrades are conducted in accordance with the guidelines.5Medium to high complexity, intubation time, urinary catheter time, and central venous catheter time are independent risk factors for HAIs after cardiac surgery. The early warning model containing these 4 factors for HAIs enables more accurate predictions and can be used to guide early intervention after pediatric cardiac surgery.5.1We adjusted for as many potentially confounding variables as possible by performing propensity score matching and multivariable analysis. But there may be selection bias in the patients so that some sample size was too small, such as re-endotracheal intubation and the use of extracorporeal membrane oxygenation. These invasive operations have been proved to be specific risk factors for HAIs, but could not be verified in our research.XXXX.