MET amplification in circulating free DNA (cfDNA) from 174 patients with cancer and 49 healthy controls and demonstrated the accuracy of the analysis to detect its alteration in patients. Importantly, a significant correlation between cfDNA concentration and MET copy number (CN) in cancer patients  was determined. Furthermore, we evaluated two approaches to detect the presence of MET on circulating tumor cells (CTCs), using the CellSearch® and Parsortix systems and monitored patients under anti-EGFR treatment (n = 30) combining both cfDNA and CTCs analyses. This follow-up provides evidence for the potential of MET CN assessment when patients develop resistance to anti-EGFR therapy and a significant association between the presence of CTCs MET+ and the Overall Survival (OS) in head and neck cancer patients . In conclusion, we develop specific and noninvasive assays to monitor MET status in cfDNA/CTCs and demonstrate the utility of plasma MET CN determination as a biomarker for monitoring the appearance of resistance to anti-EGFR therapy.MET alterations may provide a potential biomarker to evaluate patients who will benefit from treatment with MET inhibitors. Therefore, the purpose of the present study is to investigate the utility of a liquid biopsy-based strategy to assess MET alterations in cancer patients. We analyzed  Receptor tyrosine kinases (RTKs) are crucial regulators of key cellular processes such as cell growth, differentiation, neovascularization, and tissue repair. Hepatocyte growth factor receptor (MET or c-MET) is an RTK produced predominantly in cells of epithelial origin . Its onlDeregulation of the MET pathway has been associated with cancer growth and metastasis in several types of tumors  ,3, takinMET amplification has been also described in diverse tumor types, such as non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, colorectal cancer, medulloblastoma, and glioblastoma, and has been associated with bad prognosis and poor survival [MET protein overexpression and/or amplification are frequently found in different cancers. In fact, survival ,10,11,12survival . On the survival ,14. In asurvival ,7.MET amplification and/or MET overexpression may be a potential biomarker for the evaluation of patients who will benefit from treatment with MET inhibitors [MET amplification rates can be detected, depending on the detection techniques [All these data indicate that hibitors . Howeverchniques , for exaMET genomic changes have mostly been associated with metastatic patients and normally appear with the progression of disease [MET copy number (CN) status in cfDNA and the MET expression in CTCs from a cohort of cancer patients with different tumor types and evaluated its clinical potential to detect the appearance of resistances and to guide the treatment with anti-MET drugs in a noninvasive manner.In the same line, MET protein levels can be dependent on the antibodies employed, which can recognize different MET epitopes and domains, showing different membrane and/or cytoplasmic staining intensities by immunohistochemistry (IHC) . Moreove disease . Therefo disease . TherefoMET gene CN changes and expression levels. We also included the MET-negative prostate cancer cell line LNCaP and the cancer cell lines A549  and PC3 (prostate), purchased from the ATCC, in the study. The cell lines were routinely cultured in ATCC’s recommended growth medium at 37 °C, 5% CO2, and 95% humidity.The MET Genetic Alteration Cell Panel was purchased from the American Type Culture Collection . The panel was composed of five human tumor cell lines  showing various degrees of MET CN data of the eight cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE) database (http://www.broadinstitute.org/ccle/home), determined using the Affymetrix Genome-Wide Human SNP Array 6.0 platform. Next, genomic DNA (gDNA) was extracted from the same cancer cell lines with a QIAamp DNA Blood Mini Kit , according to the manufacturer’s suggested protocol. DNA quantity was assessed using a NanoDrop spectrophotometer .MET amplification, including 112 patients diagnosed with diverse metastatic cancers  who had progressed to at least one line of therapy and also 34 patients with non-metastatic tumors. They were recruited between August 2017 and October 2019 at the Medical Oncology Service of Complexo Hospitalario Universitario de Santiago de Compostela and Hospital HM La Rosaleda of Santiago de Compostela. We also included 28 stage IV NSCLC EGFR mutated patients treated with tyrosine kinase inhibitors at the Medical Oncology Service of Hospital Universitario Son Espases  (MET CN. These patients had a progression to first-line tyrosine kinase inhibitors (TKIs) and some of them even to osimertinib in second-line (irreversible EGFR TKI). In addition, we recruited 49 healthy volunteers as controls for testing the specificity of the MET CN analyses. Among the patients recruited, those diagnosed with lung or head and neck cancers starting with anti-EGFR treatment (n = 30), were monitored by means of cfDNA and CTCs analysis before the initiation of therapy (baseline), at 12 and 24 weeks after the onset of therapy, and at disease progression in order to evaluate the dynamics of MET amplification and overexpression within tumor evolution.The study design for testing the utility of blood samples to assess the MET status in cancer patients is shown in , Spain) . Frozen All individuals gave written informed consent approved by Santiago de Compostela and Lugo, and Islas Baleares Ethics Committees  prior to enrolling in the study. The protocol approved by the Ethics Committee was conducted according to the Declaration of Helsinki.g, and 10 min at 6000 g; both at room temperature), and then stored at -80 °C for further processing. cfDNA was extracted from 3 mL of plasma using a QIAmp Circulating Nucleic Acid Kit , according to the manufacturer’s instructions. cfDNA yields were assessed using a Qubit 1X dsDNA Hs Assay Kit, according to the manufacturer’s instructions .Ten milliliters of peripheral whole blood from controls and patients were obtained by direct venipuncture directly with Streck Cell-Free DNA blood collection tubes . Plasma was separated within 96 h after blood collection as a result of two sequential centrifugation steps (10 min at 1600 n = 8) and cfDNA isolated from plasma  was amplified using ddPCR to analyze MET CN. The ddPCR experiments were performed according to the manufacturer’s protocol . Each assay contained 20 μL reaction mixture of 10 μL ddPCR Supermix for Probes , 1 μL PrimePCR ddPCR CN assay for MET with a FAM fluorophore , 1 μL PrimePCR ddPCR CN assay for the reference gene with a HEX fluorophore , and 8 μL DNA template and nuclease-free water. In the case of gDNA from cancer cell lines, we added the HaeIII enzyme  to the mix, according to the manufacturer’s instructions. Droplets were generated using a QX200 AutoDG Droplet Digital PCR System  with Droplet Generation Oil for Probes  and transferred to a 96-well plate. PCR amplification was performed with the following conditions: 95 °C for 10 min, followed by 40 cycles of 94 °C for 30 s, 60 °C for 60 s, and 10 min incubation at 98 °C. The plates were read on a Bio-Rad QX200 droplet reader  and we calculated MET CN using the QuantaSoft Pro software .Genomic DNA extracted from the cancer cell lines (n = 12). The tissue sections were hybridized with MET (SpectrumRed)/CEN7 (SpectrumGreen) Dual Color FISH Probe  following the manufacturer’s protocol. In each case, 60 non-overlapping nuclei were examined by a fluorescence microscope . FISH positivity was estimated using the standard Colorado criteria /CEP7q (green) per cell plus high polysomy; 5 copies > mean MET per cell) [Fluorescent In Situ Hybridization (FISH) analysis was conducted on formalin-fixed paraffin-embedded (FFPE) tissues in a subgroup of patients who had a blood sample taken within 2 months after tissue biopsy (er cell) .® and Parsortix Systems, running identical blood samples with both technologies. All spiked samples were enriched within 96 h of collection.The assays to evaluate MET expression on CTCs were tested using cancer cell lines spiked in whole blood from the healthy volunteers recruited. A total of 7.5 mL of blood from healthy donors was collected in CellSave tubes . Cells were spiked at a final concentration of 30 cells per mL of blood. Concurrently, we employed samples from three healthy donors per each cell line. These samples were analyzed using the CellSearch® system . CellSearch® Circulating Tumor Cell Kit  was used for these specific experiments, including ferrofluids coated with epithelial cell-specific anti-Epithelial cell adhesion molecule (EpCAM) antibodies to immunomagnetically enrich epithelial cells; a mixture of antibodies directed to cytokeratins (CKs) 8, 18, and 19 conjugated to phycoerythrin (PE); anti-CD45 mAb conjugated to allophycocyanin (APC); and nuclear dye 4′,6-diamidino-2-phenylindole (DAPI) to fluorescently label the cells. The C32 cells and human melanoma samples were analyzed using a CellSearch® Circulating Melanoma Cell Kit , which included ferrofluid coated with antibodies targeting the CD146 antigen to capture circulating melanoma cells (CMCs) and fluorescent reagents against anti-high molecular weight melanoma-associated antigen (HMW-MAA-PE (MEL-PE)), anti-CD34-APC for endothelial cells, anti-CD45-APC for leukocytes, and DAPI for staining cell nuclei. The open 4th antibody position of the CellSearch® system was used to evaluate MET expression. We employed the anti-MET fluorescein (FITC)-conjugated monoclonal antibody  at a final concentration of 20 μg/mL. CTCs were identified as EpCAM+, CK+, CD45−, and DAPI+, and the level of MET expression was recorded for each CTC, on a 0–3 scale, by comparison to the MET expression levels of the different cell lines reference.A total of 7.5 mL of peripheral whole blood samples were collected in CellSave tubes  for CTCs enumeration using the FDA-cleared CellSearch+, CD45−, and DAPI+, and the level of MET expression on CTCs was determined, on a scale of 0–3 scale, depending on the MET antibody intensity, according to the results obtained in cell-line controls.A total of 7.5 mL of peripheral whole blood was collected in CellSave tubes  and loaded into the Parsortix microfluidic device . CTCs were then enriched from blood samples in disposable Parsortix cassettes , according to the manufacturer’s guidelines. CTCs were trapped in the Parsortix cassette due to their large size and lower compressibility than the remaining blood cells. After separation, we fixed the sample and carried out immunofluorescence staining. CTCs were subjected to on-cassette-staining with selected antibodies, according to the manufacturer’s guidelines, followed by fluorescence microscopy detection . The selected antibodies included Alexa Fluor (AF)546-conjugated pan-Cytokeratin , AF647-conjugated CD45 , anti-MET FITC-conjugated , and DAPI to fluorescently label the cells. CTCs were identified as CKMET CN between cancer patients and healthy controls. Two-tailed P values of 0.05 or less were considered significant. The cut-off value for the MET CN test to define the presence of MET amplification was determined using the mean plus 3 standard deviations of healthy controls. The Kappa test was used to determine the concordance between MET amplification based on FISH and ddPCR analyses. Paired Student t tests were used to compare the CTCs enrichment potential between the Parsortix and the CellSearch® systems.Statistical analyses were performed using R version 3.4.0. Pearson’s correlation and linear regression were used to evaluate the relationship between ddPCR and SNP6.0 or FISH results. The Mann–Whitney–Wilcoxon U-Test was used to compare the MET amplification and different cancer tumor types . First, we obtained MET CN data as determined by microarray (Affimetrix SNP6.0 Array) from the publicly available Cancer Cell Line Encyclopedia database. Then, we compared the data with our ddPCR results in the same cancer cell lines. After the comparative analysis, a strong linear association of MET CN measurements based on Pearson’s correlation  was observed, validating the accuracy of our analytic approach , a threshold of 2.8 was established to define the presence of MET amplification   A in metaMET CN values and clinical characteristics, such as the number of metastatic sites or the treatment lines received prior to sample collection. We also found a high variability of the MET CN status between the different tumor types, which was not significantly different among groups. However, it is remarkable that all patients with renal cancer (n = 3) showed MET CN values over the threshold   .MET CN [MET amplification between cfDNA and tissue samples in 12 patients with melanoma (n = 4), lung (n = 3), ovarian (n = 2), head and neck (n = 2), and skin cancers (n = 1) by ddPCR and FISH, respectively, in order to evaluate the sensitivity and specificity of our MET CN assay compared to the FISH ratio MET/CEN-7 > 4 (based on standard Colorado criteria). It is important to remark that, in these patients, the date of tissue and plasma sampling collection was less than 2 months. The linear association between MET CN analyzed by ddPCR and the MET/CEN-7 ratio analyzed by FISH was relatively moderate . Sensitivity and specificity to detect the MET amplification present in tissue by means of plasma ddPCR were 85.71% and 100%, respectively, demonstrating a good performance of the technique.Currently, in routine studies, FISH is the standard method used to evaluate the MET CN ,17. We t® and Parsortix systems, using preserved blood samples from healthy controls spiked with six human tumor cells , representative of the variability of MET CN and MET expression. The average percentage of spiked cells captured by the Parsortix system was 73.04 ± 19.23% (range 54–99.01%), while, for the CellSearch® system, the average recovery was 71.60 ± 22.58% (range 32.65–91.32%).To complement our approach in characterizing MET status by means of liquid biopsy, we developed a strategy to assess MET expression in the CTCs population. First, we evaluated the enrichment potential of the CellSearch® and Parsortix platforms to isolate the cell lines as result of the differential strategy for the CTCs enrichment applied by both platforms . These cell lines are characterized by a low EpCAM expression explaining the low recovery with an EpCAM-dependent strategy . We analyzed the MET expression in CTCs using the optimized conditions described above , ranging from 1 to 77 CTCs in 7.5 mL of blood (mean = 4.83 CTCs). From these 11 patients, 6 showed MET overexpression (score 2 or 3), representing 20% of all patients analyzed. Using the Parsortix system, at least one CTC was detected in 20 patients (66.67%) at baseline, ranging from 1 to 83 CTCs in 7.5 mL of blood (mean = 8.8 CTCs), being MET overexpression found in 8 patients out of 30 (26.67%). These results evidence a higher efficiency of Parsortix system in comparison to CellSearch®, at baseline, in this cohort of patients with lung or head and neck tumors. In fact, 12 patients (7 patients with head and neck tumors and 5 with NSCLC) were positive for CTCs using Parsortix system but negative using CellSearch® while the inverse situation was observed only in 3 patients. Therefore, the overall positive concordance of both strategies was 50%. Regarding MET status, in 4 patients, MET overexpression was found exclusively using Parsortix system, while 3 patients showed overexpression only using CellSearch®. Overall, 11 patients showed CTCs overexpressing MET using CellSearch® or Parsortix system.To determine whether ed above . At baseMET CN and MET overexpression at baseline with the Progression-Free Survival (PFS) and Overall Survival (OS) of these patients, as a “pilot” study. Using the Parsortix system, we found a significant association between CTCs MET-positive presence (CTC-MET-positive ≥1) and a poorer OS in head and neck cancer patients  = 6.66; MET amplification or MET expression in CTCs isolated by CellSearch® and the patients’ survival, probably because of the limited population analyzed.We also explored the association of MET amplification and MET overexpression analyses were further addressed in 30 patients undergoing EFGR therapy. Eight of the 30 patients died and nine showed progressive disease during the follow-up. Five patients died before the second sampling point and 3 before the third collection point. Regarding CTCs monitoring using CellSearch® system 4 of 12 patients were positive for CTCs and MET overexpression after 12 weeks of treatment, 3 of 9 after 24 weeks, and 1 of 8 at progression. In the case of Parsortix samples, 3 of 7 patients had CTCs with MET overexpression after 12 weeks, 0 of 5 after 24 weeks, and 2 of 9 at progression. In addition, 1 of 20 patients showed MET amplification after 12 weeks, 2 of 17 after 24 weeks, and 1 of 9 at progression, evidencing the amplification acquisition as a response of the treatment pressure. However, MET amplification and MET overexpression results only showed clear changes in a few of the patients within the treatment, suggesting its interest for monitoring, but these results should be considered as with caution because the longitudinal cohort was quite sparse.Longitudinal MET CN status in cfDNA and the MET expression in CTCs were also evaluated. She had 31 CTCs in the CellSearch® system, 4 of them showing MET overexpression (12.9%); whereas 13 CTCs were detected with Parsortix system . Despite not showing radiological progression, there was a serious clinical deterioration and the patient died within two months. Besides, one case of the retrospective study evidenced how MET CN detection may be informative in monitoring the appearance of resistance mechanisms in response to anti-EGFR treatment .This study was designed to investigate the utility of liquid biopsy in assessing MET protein expression and ® and the Parsortix platforms routine. Due to the ultra-rare nature of CTCs, with numbers as low as 1 CTC per 106-107 leukocytes, most assays for CTCs analyses use a combination of enrichment and detection/characterization techniques [® system the only FDA approved device for CTCs enumeration, although there are others based on this strategy . The main limitation of this approach is the lack of a “universal marker” that can be used independently of both the tumor type and the stage of disease progression [®, and ISET®) and microfluidic-based enrichment . The Parsortix system uses a combination of microfluidic and size/deformability-based approaches to separate CTCs from blood samples. Its main advantage over CellSearch® is the ability to enumerate CTCs independently of their epithelial or mesenchymal status. However, cell size-based systems have difficulty to completely separate cancer cells and leukocyte by their size since patient’s CTCs exhibit a high degree of pleomorphism, where the size of the captured tumor cells can vary from 4-30 µm [For CTCs analysis, we implemented the MET expression detection using the CellSearchchniques . It is igression . To solv 4-30 µm . Therefo 4-30 µm .® system, indicating that an antigen CTCs independent isolation approach can overcome the EpCAM expression heterogeneity that characterizes these tumors. Therefore, some non-epithelial or mesenchymal CTCs from cancer patients would be detected on the Parsortix platform but could escape the antigen-dependent enrichment method of the CellSearch® EpCAM assay, and vice versa with epithelial CTCs, such as reflected our spiking experiments with different cell lines expressing different levels of EpCAM. Therefore, although the good results obtained using Parsortix system, a larger cohort of patients would be necessary to clearly determine which technology should be used to characterize CTCs from patients with both tumors.In our cohort of patients with head and neck and NSCLC, we found that the Parsortix approach identified a higher proportion of CTC-positive patients than the CellSearch® system [® system and by immunocytochemistry on ISET-enriched CTCs from advanced-stage NSCLC patients. They found that MET expression was lower in CTCs analyzed by CellSearch® than using ISET. In fact, only 9 of 256 (3.52%) patients showed CTCs with high MET expression (scores of 2+ or 3+) in CellSearch®, whereas MET-positive CTCs from advanced-stage NSCLC patients were successfully detected using the ISET platform, being 54 of the 75 (72%) patients’ blood samples defined as MET positive. Our data showed that the prevalence of MET expression in CTCs isolated by CellSearch® is greater than previously reported data (20% MET-positive scores of 2+ or 3+), and even higher to the expected MET expression in patients with metastatic cancer. Importantly, using Parsortix system, we found 26.6% of patients with MET-positive scores of 2+ or 3+, being the concordance to detect the presence of CTCs overexpressing MET between both technologies, of 50%. These results showed that MET is expressed on CTCs with a considerable patient dependent and technology variability. Obviously, these results reflect the distinct CTCs enrichment methodologies of the two platforms, and the importance of tumor lineage for the MET-CTC-assay, highly diverse in our cohort of patient.Independently to the CTCs enumeration rate showed by both technologies, our study provides a proof-of-concept for the analysis of MET expression in CTCs to determine MET status in patients that might be eligible in clinical trial testing MET inhibitors, and could be useful to monitor the appearance of therapy resistance. Previously, Zhang et al. developed a noninvasive assay for MET+ CTCs capture and characterization from multiple patients with metastatic carcinomas using the CellSearch® system . Due to ® system , using aMET amplification in plasma samples from cancer patients. Zhang et al. had already shown this capacity, detecting MET CN in genomic DNA from cancer cell lines and tumor samples [MET amplification in plasma, most of them with metastatic disease. Although NGS panels can provide more complete information regarding the status of other driver genes, our goal was the validation of a more sensitive and easier to implement test to monitor MET CN in plasma using ddPCR. For this aim, a cohort of healthy controls and cancer patients with different tumor types and metastatic stages were analyzed to detect MET CN by ddPCR. We found 16.4% of the metastatic patients and 3% of the non-metastatic setting as positive for MET amplification, being higher rates than the previously described using an NGS approach. Besides, we evaluated the concordance of the MET CN detection between plasma and tumor samples obtained at the same time as the disease evolution. Importantly, MET amplification positivity detected by ddPCR was comparable to that detected by FISH. Only one melanoma patient with MET amplification determined by FISH (MET/CEN-7 ratio = 6.1) did not show MET amplification in plasma using ddPCR (CN > 2.8). These results reinforced the value of the MET analysis in plasma samples as a more accessible monitoring tool to guide anti-MET therapies.In addition to the CTCs analysis, we demonstrated the ddPCR approach to be a useful tool for detecting  samples . Recentl samples . In thisMET amplification is a relevant resistance mechanism to first-generation EGFR-TKIs, being detectable in approximately 5%-22% of NSCLC patients with acquired resistance to these drugs [MET amplification [MET amplification, or even MET hyperactivation [MET amplification detected by ddPCR as an acquired resistance mechanism. In this context, our results demonstrated the utility of plasma MET determination as a valuable biomarker for monitoring the appearance of resistance to anti-EGFR therapy. In fact, we reported a case of MET amplification detected in plasma from a NSCLC patient harboring EGFR L858R mutation after disease progression to erlotinib.Actually, se drugs . Osimertfication . There htivation . Targetitivation ,29,30. TMET CN obtained from plasma and tissue. Despite this limitation, we showed that plasma may be a good option for monitoring the molecular evolution of the disease, which is even more relevant in patients for which a tissue-rebiopsy is not feasible. In addition, we implemented the detection of MET expression in CTCs from cancer patients using two complementary technologies and obtaining promising results. Nevertheless, taking into account the limited follow-up of the patients and the heterogeneity of MET overexpression found within the different tumor types and the technologies, the real clinical value of the assessment of MET expression in CTCs must be evaluated also in a larger cohort of patients.Regardless of the value of our findings, some limitations to our study should be kept in mind. The small number of tissue samples (obtained close to plasma collection) that were available did not allow us to carry out a robust comparative study between MET CN on cfDNA using blood from cancer patients. Both molecular alterations were detected in patients with different tumors and represent a good opportunity to characterize these tumors in a noninvasive and dynamic way, as well as guiding the addition of MET inhibitors.In conclusion, we developed specific and noninvasive assays to monitor MET expression on CTCs and


Perfect channel state information (CSI) is required in most of the classical physical-layer security techniques, while it is difficult to obtain the ideal CSI due to the time-varying wireless fading channel. Although imperfect CSI has a great impact on the security of MIMO communications, deep learning is becoming a promising solution to handle the negative effect of imperfect CSI. In this work, we propose two types of deep learning-based secure MIMO detectors for heterogeneous networks, where the macro base station (BS) chooses the null-space eigenvectors to prevent information leakage to the femto BS. Thus, the bit error rate of the associated user is adopted as the metric to evaluate the system performance. With the help of deep convolutional neural networks (CNNs), the macro BS obtains the refined version from the imperfect CSI. Simulation results are provided to validate the proposed algorithms. The impacts of system parameters, such as the correlation factor of imperfect CSI, the normalized doppler frequency, the number of antennas is investigated in different setup scenarios. The results show that considerable performance gains can be obtained from the deep learning-based detectors compared with the classical maximum likelihood algorithm. In recent years, the heterogeneous wireless network, which can support high-density and high-rate traffic, has attracted much research interest from both academic and industry sectors ,2,3. TheOn the other hand, the architecture of heterogeneous wireless networks brings up many challenges to the physical-layer security ,7,8. SevDeep learning technology has already shown astonishing capabilities in dealing with complex network in mobile communications, such as channel estimation, signal detection, modulation recognition and channel equalization. For example, Ref.  used a 2Although there are some works on learning-based channel estimation algorithms, with imperfect CSI, the impacts of CNN-based MIMO detector on physical-layer security performance is still an open question. Motivated by that, in this paper, we will discuss the deep learning-based secure MIMO communication algorithm for heterogeneous networks with imperfect CSI. In the heterogeneous networks, there exist both macro BSs and femto BSs, which can serve a group of users within different areas. In general, the macro BS provides the massive connections and the large-scale cell coverage in the hot spots. The femto BS can help enlarge the wireless coverage and the data rates of the user in network edge. In some practical scenarios, secure messages transmitted from the macro BS to the users may be intercepted by the femto BS. In this case, with the help of zero-forcing algorithm , the macWe employ the deep learning-based technique for secure MIMO communications in heterogeneous networks, which can exploit the benefits of CNN learning model to produce more accurate CSI and meanwhile reduce the bit error rate (BER) of the receiver.We provide the detailed framework of deep learning-based detectors, where imperfect CSI as well as the original messages or ideal CSI are included in the training set, and can be used in different application scenarios.We present simulation results for deep learning-based detectors in heterogeneous networks. With the help of the CNN technique, the proposed detectors show obvious performance gain over the MLD with acceptable computational cost.The main contributions of this paper are as follows:Notations: We use x, respectively, M to F.Considering Gaussian wiretap channel, Fritschek et al.  introducWith perfect CSI, the deep feedforward neural network (DFNN) with three layers was adopted for time-slot wireless powered system in . In the In addition, the channel fading matrix is modeled as time-varying and flat fading using the classical Jakes model . Thus, tn denotes the sample time and Thus, the channel fading matrix can be calculated asn is omitted without loss of generality.Please note that in the following sections, the sample time It is well-known that the deep learning networks can effectively capture the correlation features of the training data set. Spatial correlation or antenna correlation, which may achieve dimensionality reduction, is an important challenge for MIMO systems ,39. AlsoSince the time correlated MIMO channel model is adopted in this paper, we can use DCNN to obtain more accurate CSI from the outdated CSI. Actually, considering the phase rotation introduced by the channel matrix, the outdated CSI is necessary to assist the data recovery at the user.Due to the open nature of the heterogeneous networks, the femto BS may intercept the signal transmitted from the macro BS to the users. To prevent the information leakage to the femto BS, the macro BS can zero-forcing the equivalent channel matrix of the femto BS by the null-space technique. In this case, the macro BS first obtains the CSI P is the transmission power of the macro BS, Please note that Since the equivalent channel fading matrix of the femto BS is zero-forced, we only need to observe the signal-noise-ratio (SNR) of the associated user. We define the average normalized SNR received at the user asClassically, the receiver obtains the wireless CSI through pilot signals transmitted from the BS, and there exists a time difference between the channel estimation and the data packet transmission. Thus, the estimated CSI is an imperfect version of the instant of packet transmission. To reduce the analysis complexity, it is assumed that the estimation of The standard maximum likelihood detector (MLD) with imperfect CSI can be employed to detect the original message asThe imperfect CSI, which is introduced by the noise or delay of channel estimation, will greatly deteriorate the system performance of standard MLD. To overcome the effects of the imperfect CSI in secure MIMO communications, two types of deep CNN (DCNN)-based detectors are proposed in this section, which can be used in different application scenarios. The deep CNN models are first trained with predefined loss functions and then used to generate the refined CSI N one-dimension convolutional layers excluding the input layer. In the input layer, the channel fading matrix n-th convolutional layer, there are The details of the DCNN model is given as channels . Please N=4 layers, and is described by As to the detailed architecture of the DCNN, we must find the trade-off between complexity and performance. It is noted that the fully connected DNN, which may hold better performance, while its computational complexity is proportional to the square of the number of nodes. On the other hand, both the training data set and the training time required by fully connected DNN the is too large to be satisfied. There also exist some powerful CNN models, such as VGG  and ResNIn the following sections, two different methods, denoted as DCNN type-I and type-II, are introduced to train the DCNN model.During the DCNN model training, the loss function is calculated batch by batch and used to optimize the weight and the bias of the DCNN model . Please The training architecture of DCNN type-II is given as in By using of the SoftMax function, the normalized likelihood probability of each candidate message can be given asi-th candidate message is correct, otherwise We use Accordingly, for probability distributions of Then, the cross-entropy y, the beamforming matrix x are needed, which enlarge the application scenarios of the DCNN type-II. On the other hand, without the help of the accurate CSI, DCNN type-II leads to deteriorated performance compared with type-I, which can be validated in the simulation results.Compared with the loss function in , only thx directly in a supervised manner, and the outdated MIMO channel matrix could be further employed as side information by inputting it to the DCNN. We use DCNN type-III to denote the new DCNN model. Although the detailed architectures of DCNN type-II and the suggested DCNN type-III are different, they are functionally equivalent as a black box with DCNN kernels. In other words, the MLD module in DCNN type-II can be seen as part of the functions of DCNN type-III.Please note that the DCNN model could also output the ground-true symbol In this section, simulation results are provided to verify the proposed DCNN models. The impacts of system parameters, such as the correlation factor of imperfect CSI Specifically, QPSK modulation is employed in all simulation setups. Since the constellations of QPSK modulation is 2-D complex signal, we can generalize the setup to an arbitrary modulation order. We set the data packet length as 600 bits, and each batch consists 10 data packets. During the training phase, a training data set with 10000 batches as well as a validation data set with 1000 batches are fed to the DCNN, and a test data set with 1000 batches is used to evaluate the BER performance of the proposed schemes. The popular TensorFlow framework  is adopty, the beamforming matrix x are needed, which enlarge the application scenarios of the DCNN type-II. On the other hand, without the help of the accurate CSI, DCNN type-II leads to deteriorated performance compared with type-I.Moreover, two types of DCNN-based detectors show almost the same performance with slight gap. Similar results can be obtained from The effect of the normalized frequency In this paper, we investigate two types of deep learning-based secure MIMO detectors for heterogeneous networks. In the considered system, the equivalent channel fading matrix of the femto BS is zero-forced through null-space eigenvectors. The BER of the associated user is adopted as the metric to evaluate the system performance. with the help of deep convolutional neural networks, the macro BS produces more accurate CSI. The impacts of system parameters, such as the correlation factor of imperfect CSI, the normalized doppler frequency, the number of antennas are investigated in different setup scenarios. Considerable performance gains can be obtained from the deep learning-based detectors compared with the classical maximum likelihood algorithm.


Antimicrobial stewardship programmes (ASP) are aimed at optimising antimicrobial utilization. However, only few studies have focused on paediatric intensive care units (PICU), where inappropriate antibiotic use occurs frequently. We assessed the effect and safety of a once weekly paediatric infectious disease (PID) ward round with prospective audit and feedback on antibiotic consumption in a multidisciplinary PICU. This study was conducted within 6-months periods before and after the implementation of a weekly PID-ward round. Antimicrobial management and two main recommendations per patient were discussed and documented. The primary outcome was antimicrobial utilization, measured by days of therapy (DoT) and length of therapy (LoT) per 1000 patient days (PD) for all PICU stays. Secondary outcomes included PICU mean length of stay, total mortality, infection-related mortality and cost of therapy. 1964 PD were analyzed during the pre- and 1866 PD during the post-implementation phase. Adherence to the recommendations was 79%. An 18% reduction of DoT/1000 PD was observed in the post-implementation period (p = 0.005). LoT/1000 PD decreased by 11% (p = 0.09). Meropenem and vancomycin usage were reduced by 49% (p = 0.07) and 56% (p = 0.03), respectively. We conclude, that a once weekly PID-ward round with prospective audit and feedback is safe and effective and reduces antibiotic consumption in PICUs. Definite diagnosis of infection is often difficult and PICU clinicians are concerned about delayed treatment of infection. Therefore, empiric antimicrobial therapy is common practice in paediatric intensive care medicine. Antimicrobial utilization among PICU patients is as high as 51–76%5. Data suggest that as much as half of these treatments may be inappropriate and appropriateness of both empiric and definitive antimicrobial therapy remains a challenge7. The main reasons for antibiotic treatment in PICUs are perioperative antibiotic prophylaxis, suspected ventilator-associated pneumonia (VAP) and sepsis7. Moreover, 15–16% of PICU patients acquire HAI; patients after cardiac surgery are particularly at risk9. The most common antimicrobial agents used in PICU patients are 1st and 3rd generation cephalosporins, vancomycin and extended-spectrum penicillins. Notably, vancomycin is prescribed empirically in up to 50%10. Too long and not always well-founded utilization of reserve antibiotics is clearly correlated with selective pressure and the emergence of antibiotic resistances13. PICU clinicians face increasing rates of antibiotic resistances, along with highly sophisticated therapies of critically ill and frequently immunocompromised patients.Children admitted to the Paediatric Intensive Care Unit (PICU) represent a highly vulnerable patient population. Serious infections, both as a primary cause for admission and as a secondary healthcare-associated infection (HAI), are one of the most common problems encountered in a PICU15.Antibiotic stewardship programmes have been developed to improve antibiotic management and utilization. Evidence from adult studies suggests that antibiotic stewardship interventions and collaborations between infectious disease and critical care practitioners can effectively improve antimicrobial utilization and patient outcome in intensive care medicine, including length of stay (LOS) and mortality16. Several antibiotic stewardship strategies have demonstrated promising results: (1) prospective audit and feedback, (2) formulary restriction and preauthorization and (3) structural interventions, e.g. broadening of inflammatory markers testing or screening methods16. Although appropriate use of antibiotics in a PICU is vitally important to ensure optimal clinical outcome neither the best method for, nor the effectiveness of antibiotic stewardship interventions in PICUs have been elucidated. Data on antibiotic stewardship prevalence in paediatric critical care medicine is extremely limited21.In the paediatric setting ABS is practical to implement as well and has no negative effects on patient safetyThe aim of this study was to assess the effect of a once weekly paediatric infectious disease ward round with prospective audit and feedback on antimicrobial use in a multidisciplinary PICU. We compared antibiotic consumption in a pre- and post-implementation period.The study was conducted at the closed 14-bed multidisciplinary PICU of the University Children’s Hospital Tübingen, Germany, which includes general paediatric patients, paediatric cardio-, general- and neurosurgical as well as haematooncological and solid organ transplant patients and offers an active Extracorporeal Life Support System (ECLS) program. The organizational structure of the ward remained unchanged for the study period. During the pre-implementation period in 2017, the PICU consultant in charge mainly directed antibiotic management. Written guidelines for perioperative antimicrobial prophylaxis and PICU empirical antibiotic guidelines were not available. The paediatric infectious disease team (PID-team) comprised a paediatric infectious disease specialist, a microbiologist, an infectious-diseases trained pharmacist and a virologist. The team implemented a weekly PID ward round three months prior to the start of the post-implementation period in 2018.The key feature of the antibiotic stewardship intervention was a once weekly infectious disease ward round using academic detailing and prospective audit with feedback focused on antimicrobial choice and consumption. The main goal of the intervention was an early de-escalation of broad empiric therapy guided by microbiological results, decreased utilization of antipseudomonal agents and increased use of a narrower penicillin-based antimicrobial therapy. The PID-specialist attended the PICU morning round once weekly to get insight into ongoing infectious disease management. History and course of disease, laboratory and microbiological findings, indications and antibiotic choice of all patients on antimicrobials were reviewed by the PID-team. Haemato-oncological patients were excluded. The same day, antimicrobial management of each patient was discussed with the PICU consultant and trainee in charge during the “30-minutes PID-ward round”. We defined seven possible recommendation groups (definitions are given in Online Resource 1). The two main antimicrobial stewardship recommendations for each patient were documented in the chart by the PID-specialist on the same day.We conducted a prospective, pre- and post-implementation cohort study to assess the changes in antibiotic use during the six months of a once weekly PID-ward round on our PICU. The pre- and post-implementation periods were from January 2017 to June 2017 and January 2018 to June 2018, respectively. The primary outcome was antimicrobial utilization, as measured by critical-care unit-related antibiotic days of therapy (DoT) and length of therapy (LoT) per 1000 patient days (PD) for all antibacterial agents. Secondary outcomes included PICU mean length of stay, total mortality, infection-related mortality and cost of therapy. All research of this study was conducted in accordance with the Declaration of Helsinki and carried out in accordance with the guidelines of ICH-GCP. The study was approved by the local ethical review board at the University Hospital Tuebingen (project No 559/2019BO2) with a waiver of informed consent.Demographic data, PICU length of stay, number of ECLS runs, mortality and infection-related mortality and Diagnosis Related Group (DRG)-relative weights were obtained from the hospital information system  by extraction of all PICU patients with one or more PICU stays of at least 24 hours in the study periods. Infection-related mortality was defined as any death that could be attributed to clinically or microbiologically confirmed infection as the immediate or underlying cause. Intensive care case mix index (ICU-CMI) was calculated by taking the sum of all DRG-relative weights divided by the total number of cases in the pre- and post-implementation period.23. DoT was defined as the number of days that a patient receives an antimicrobial agent, regardless of dose. All doses of a specific antibiotic administered on a given day counted as one DoT. The DoT for a given patient on multiple antibiotics was therefore the sum of DoT for each antibiotic the patient received. LoT accounted for the number of days that a patient received any antimicrobial agent, irrespective of the number of different antibiotics. Thus, LoT is always lower than or equal to DoT. For intergroup comparison DoT and LoT were standardized to 1000 patient days, using the PICU length of stay of each patient. To allow for detection of antibiotic consumption patterns and comparison of these data between hospitals, antibiotic density was calculated: DoT and LoT were aggregated and standardized to 1000 patient-days , using the total PDs in the respective period. We classified antibiotics according to the internationally standardized World Health Organization Anatomical Therapeutic Chemical (WHO ATC) classification system. Antibiotic costs were calculated for the median weight of the study population.Critical-care unit-related antibiotic use data were obtained using the antibiotic summary of each patient’s PICU stay from the clinical decision support and patient documentation system . For each PICU stay of a patient of >24 h antibiotic consumption data were extracted into a separate Excel file. Re-admissions to the PICU were counted as separate visits if they were at least 24 h apart. The separate Excel files were aggregated via a custom Python script, computing days of therapy (DoT) and length of therapy (LoT) for each PICU stayAnalysis was performed using Microsoft Excel 2013 and IBM SPSS Statistics Software Version 22 . Descriptive statistics were done for baseline demographic and clinical characteristics of the pre- and post-implementation group. Categorical data was compared using the [chi]2 test. Fisher’s exact test was used in case of small expected observations. The nonparametric Mann–Whitney-U-Test was used for intergroup comparison of continuous variables that were not normally distributed. To assess statistical relevance of the change in antibiotic consumption between pre- and post-implementation period, we compared antibiotic density [DoT/1000PD] of single PICU stays, using Mann-Whitney-U-Test. A p-value < 0.05 was considered statistically significant. A detailed description of the statistical methods is provided in Online Resource 4.Pre-implementation, 165 patients accounted for 183 PICU visits; post-implementation, 182 patients were addressed in 207 PICU visits. During the intervention, antibiotic stewardship recommendations were given for 38% (69 out of 182) of all patients. No statistically significant differences in age, gender distribution, weight or proportion of neonates were noted between the two periods. Distribution of departments of referral as well as the ICU-CMI and number of ECLS runs were similar Table .Table 1 adherence to the recommendations by the PICU team was 79%. In 16 visits (15%) recommendations were not followed, and in 6 visits (6%) no recommendation was given. Almost half of all recommendations given were de-escalation and streamlining as well as stop orders . Total DoT decreased from 2959 days to 2431 in the post-implementation period. Accordingly, there was a significant reduction in total DoT/1000 PD of the PICU stays from 1226 [0–4129] to 1000 [0–3125] (p = 0.005), which represents a median difference of 18% between pre- and the post-implementation periods Fig. . Of noteAntimicrobial consumption was analyzed in detail in the pre-implementation and the post-implementation period  to the given antibiotic stewardship recommendations was 79%. Previous paediatric ASPs have reported comparable adherence of 77–89%30. In 15% ASP recommendations were not followed, mainly because of non-compliance of PICU physicians who felt, that the patient was “too sick” for a narrow-spectrum therapy, or a sudden deterioration of the patient hampered a “step-down therapy”.Sometimes a change in renal function or additional results required other therapeutic adaptions than previously recommended.Judicious and optimal antibiotic treatment in PICU patients is essential in light of the approaching “post-antibiotic era” with even fewer antibiotic options for children compared to adults3. In our study, the majority of recommendations were also related to a reduction of antibiotic utilization with 64% being either de-escalation and/or streamlining, scheduled duration of therapy or stop order.In a point prevalence survey in a similar setting to ours, the main causes of inappropriate use of antibiotics were overly broad spectrum, wrong dosage and unwarranted overlap of spectraet al. reported a total DoT/1000 PD of 1201 before an antibiotic stewardship intervention in the US, which is more or less comparable to ours31. Taking this study as a reference, the antibiotic consumption before and after the implementation was slightly higher in our setting. Antibiotic density measured as DoT/1000 PD decreased by 204 (14%) and was 1303 after the implementation. Others reported similar reductions, largely depending on the setting and baseline antibiotic consumption. A systematic review of the impact of antimicrobial stewardship in critical care in adult populations found reductions of antimicrobial utilization of 11–38% in 24 studies14. Willis et al. and Newland et al. both reported a comparable decrease in overall antibiotic DoT/1000 PD by 11% after the implementation of a Prospective-Audit-With Feedback Antimicrobial Stewardship Program at a Children’s Hospital, not limited to the PICU27. In other PICU settings reductions of 21–64% were described31. In summary, our result seems reasonable when compared to the few studies in similar settings and with similar paediatric antibiotic stewardship interventions.Although, we only reached 38% of all patients with a once weekly PID ward round, we found a significant reduction in median antibiotic utilization (p = 0.005). Antibiotic density was 1507 DoT/1000 PD before the antibiotic stewardship intervention. We cannot state, whether this is a high or low rate of antibiotic consumption, since numbers of studies in comparable settings are scarce. Lee 32. Mean LoT/1000 PD decreased by 83.3 (11%) during the implementation, although this reduction did not reach significance. Since no other PICU studies have evaluated LoT/1000 PD before and during antibiotic stewardship interventions, we cannot compare our data with other PICUs. Other children’s hospitals showed significant decreases of LoT/1000 PD by 7–8% after similar antibiotic stewardship interventions, based on larger cohorts with more than twice the amount of patients and patient days compared to our study33. A prolonged study period with a larger number of cases and patient days may be necessary to further investigate potential reductions of LoT/1000 PD in the PICU setting.Evaluation of the absolute length of antibiotic treatment is important, since the prescription of broad-spectrum antibiotics reduces DoT, whereas the combination of several narrow spectrum antibiotics increases it. LoT is not influenced by this phenomenon and therefore provides additional information about antibiotic utilizationet al. found cefazolin, cefuroxime, vancomycin and gentamicin as the most commonly used antibiotics in his point prevalence survey in a cardiac and medical-surgical paediatric critical care unit3. Cefazolin is used as perioperative prophylaxis in cardiac surgery and the high antibiotic consumption of this agent in our study is explained by a postoperative regimen that favours prolongation of prophylaxis until all medical devices including Central Venous Catheters (CVCs) are removed.Detailed analysis of single agents and antibiotic classes revealed a remarkable decrease in DoT/1000 PD of antimicrobials targeted by the antibiotic stewardship intervention. Cefazolin was the most commonly used single agent before and after the antibiotic stewardship intervention. In line with our findings, Blinova et al. have also shown a reduction of carbapenem consumption in paediatrics through consultation of a paediatric infectious disease specialist. Notably, in the 5 year study period, the proportion of carbapenem utilization decreased from 13% to 4.3% and the incidence of meropenem sensitive Pseudomonas aeruginosa significantly increased34.Meropenem was the second most frequently used antibiotic before the implementation and accounted for 16% of all antibiotics prescribed. DoT/1000 PD of meropenem had been reduced by almost 50% and accounted only for 9% of antibiotics used in the post-implementation period (p = 0.07). Two approaches primarily contributed to the reduction in antibiotic density of meropenem: First, through discussions during the PID-ward round, we tried to switch to penicillin/beta-lactamase inhibitor combinations (e. g. ampicillin/sulbactam or piperacillin/tazobactam) whenever there was no proof of an Extended-Spectrum ß-lactamase (ESBL) or multidrug-resistant pathogen. Second, antibiotic time-out and microbiological diagnostics after 24 h to 48hrs off antibiotics were recommended, if meropenem therapy did not have any effect after 10–14 days of treatment. Thereby, long-term carbapenem courses of 3–4 weeks were avoided. Recently, Hoshina 36. Antibiotic density was significantly reduced by 57% from the pre- to the post-implementation period (p = 0.02). Two other studies in the PICU setting indicate a decrease of vancomycin utilization of 35% and 50% respectively31. The significant reduction in our study is explained by several factors: First, vancomycin utilization was one of the main targets of our antibiotic stewardship intervention. Second, excessive use due to an empiric escalation regimen in the pre-implementation period was high. Recommendations for vancomycin use during the post-implementation period were based on Methicillin-resistant Staphylococcus aureus (MRSA) screening results and probability of catheter-associated blood stream infection due to resistant coagulase-negative staphylococci or the risk of severe Enterococcus faecium infection. Last, we only observe a very low rate of MRSA colonization in our setting.Appropriate prescription of glycopeptide antibiotics was a main target of the antibiotic stewardship intervention, as in previous paediatric studies39.Cotrimoxazole utilization decreased significantly by 70% (p = 0.05). This trend remains rather unclear to us and we can only speculate, that discussion during the PID ward round about indications of antimicrobials may also have influenced the prescription of cotrimoxazole. In contrast to the overall reduction in antibiotic utilization, we observed a significant increase in DoT/1000 PD of ampicillin/sulbactam, from 2% to 7% of all antibiotics prescribed (p < 0.001) in the post-implementation period. This increase was intended by the PID team, since it is well known that penicillin-based therapy has less influence on the intestinal microbiota, causes less selective pressure than broad-spectrum cephalosporins or carbapenems and is therefore classified among the key “ACCESS” antibiotics in the WHO Model List of Essential Medicines for ChildrenDisease severity measured by the ICU-CMI was similar before and during the implementation. To ensure the antibiotic stewardship recommendations did not cause harm, we determined secondary outcome measures including PICU length of stay, ventilator-days, and total hospital mortality. In particular, infection-related mortality remained stable between the study periods. Therefore, we conclude that the antibiotic stewardship intervention with significant reduction of overall DoT/1000 PD is safe.As many other antibiotic stewardship studies, our study is limited to a quasi-experimental pre- and post-implementation design and limited to a single centre. However, we did compare the same seasonal periods and no major changes of the patient or staff structure of the PICU that could have affected antibiotic prescribing practices occurred between the study periods. DoT/1000 PD is a widely accepted measure for antimicrobial use in paediatrics, since it is independent of a patient’s weight, but cannot detect breadth, toxicity and costs of antimicrobial prescription. Furthermore, in this analysis we could not assess the effect of the intervention with respect to patient-related outcomes. As an indirect measure, we used secondary indicators of antibiotic treatment failure - including PICU LOS, ventilator-days and infection related mortality - although they might not be particularly sensitive.In conclusion, this study demonstrates that an ASP with prospective audit and feedback during once weekly ward rounds led by paediatric infectious diseases specialists is associated with good adherence to antibiotic stewardship recommendations. This intervention could significantly reduce antibiotic consumption in a multidisciplinary PICU without negatively affecting patients’ safety. Generalizability, sustainability and clinical outcomes of PICU antimicrobial stewardship need further elucidation.Supplementary Information.Supplementary Information2.


Leptobotiacitrauratea , a loach species, originally described from Dongting Lake, was recently rehabilitated, based on the examination of the holotype and non-topotypical specimens. Several field surveys conducted from 2016 to 2019 in Zhejiang, Jiangxi and Hunan Provinces, P.R. China, yielded many specimens of Leptobotia which were initially identified as L.citrauratea. Molecular and morphological analyses of these specimens demonstrated that two distinct species are involved. One was identified as L.citrauratea, represented by specimens from both the Poyang and Dongting Lake  systems in Jiangxi and Hunan Provinces, and the other species is described as L.brachycephala, represented by specimens from the Ou-Jiang and Qu-Jiang, two coastal rivers of Zhejiang Province, China. Leptobotiabrachycephala resembles L.citrauratea and L.micra in having a row of orange dots or an orange stripe along the dorsal mid-line of the body, extending from the nape to the caudal-fin base – a unique character in Leptobotia. Leptobotiabrachycephala differs from L.citrauratea and L.micra Bohlen & Šlechtová, 2017, in caudal-fin shape and pelvic-fin insertion and proportional measurements including caudal-fin length, head length, predorsal length and anal-fin length. Its species status was further corroborated by position in a molecular phylogenetic analysis, based on the mitochondrial cyt b gene and its minimum uncorrected p-distance (2.9%) from congeneric species. Leptobotia was erected by Bleeker (1870) with the simultaneously-described Leptobotiaelongata  as type species by monotypy. The genus is distinguished from other genera of the family Botiidae by the presence of a simple suborbital spine beneath the eye  gene sequences. Morphological analysis also indicated that two distinct species are involved. One of them was identified as L.citrauratea, represented by specimens sampled from the Poyang and Dongting Lake systems. The other species is an undescribed species represented by specimens from the Ou-Jiang and Qu-Jiang in Zhejiang Province. The present study aims to provide a re-description of L.citrauratea, based on fresh topotypical specimens and the formal description of the undescribed species.Several field surveys conducted by us from 2016 to 2019 in Zhejiang, Jiangxi and Hunan Provinces, yielded many specimens of L.citrauratea, L.elongata and L.brachycephala) were used for morphometric analysis. Voucher specimens are kept in the ichthyological collection of the Museum of Aquatic Organisms at the Institute of Hydrobiology (IHB), Chinese Academy of Sciences, Wuhan City, Hubei Province, China.Specimens were either initially fixed in 10% formalin and then transferred to 70% ethanol for morphological examination or preserved in 95% ethanol for DNA extraction. Seventy-three specimens from the three species  and Dongting Lake; and 22 specimens of L.brachycephala from the Ou-Jiang and Qu-Jiang. Measurements were taken point to point with digital calipers directly linked to a data-recording computer and data recorded to the nearest 0.1 mm. All measurements and counts were made on the left side of each specimen, following the methods of SL) and measurements of parts of the head were expressed as proportions of the lateral head length (HL). Morphometric variation was analysed with Principal Component Analysis (PCA) in Past v.1.89  and H15915 (CTCCGATCTCCGGATTACAAGAC) adopted from 2O) for a total reaction volume of 25 µl. The thermocycling conditions were as follows: initial denaturation for 4 min at 94 °C, denaturation for 50 s at 94 °C, annealing for 50 s at 55 °C and extension for 1 min at 72 °C. After 34–35 cycles, the final extension was done at 72 °C for 10 min and the PCR product was preserved at 4 °C. Sequencing was carried out by the Tianyihuiyuan Biotechnology Company.Genomic DNA was extracted from fin clips stored in ethanol using the TIANamp Genomic DNA Kit  with the recommended protocol. The cyt b sequences were generated from 12 species of Leptobotia. These sequences were used for phylogenetic analysis together with five sequences from two congeneric species  and two sequences serving as outgroup  downloaded from GenBank . Kottelat, 2004:15 ; 2012:16 Leptobotiacitrauratea : Nalbant, 2002: 316 . Bohlen & Šlechtová, 2017: 90 Leptobotiacitrauratea: AMNH 8402, holotype, 50 mm SL; China: Hunan Province: Dungting Lake (photograph examined); collected by Clifford H. Pope, 29 December 1921; IHB 2017100260-65, 201801026314, 201711016295, 201711015676, 201711015673-74, 201711015715-16, 201711016204, 201707028880, 201711015718, 201707028888, topotypes, 17 specimens, 47.0–65.3 mm SL; China: Hunan Province: Nanxian County: Dongting Lake; 29°2'29"N, 112°18'22"E; collected by C.T An, November 2017; IHB 2017056850-60, 2017056862, 12 specimens, 33.3–42.7 mm SL; China: Jiangxi Province: Nanchang City: Gan-Jiang, an effluent of Poyang Lake; 28°32'12"N, 115°49'24"E; collected by D.M. Guo, November 2019.Leptobotiacitrauratea shares with L.micra and L.brachycephala the unique presence of a row of orange spots or an orange stripe along the dorsal mid-line of the body, extending from the nape to the caudal-fin base. It differs from L.micra and L.brachycephala by having a deeply forked (vs. emarginate) caudal fin , pelvic fin inserted slightly posterior or inferior  to the dorsal-fin origin, a longer head  and a shorter predorsal distance (51.4–56.3% SL vs. 58.1–59% SL in L.micra)  and Hemibarbusmaculatus Bleeker, 1871.ina Fig. . The speTaxon classificationAnimaliaCypriniformesBotiidae55DB1420-E1E5-50D3-B8D1-DCCE5CEC1983http://zoobank.org/4A74D0D5-5A3B-4458-8227-B67A591A4143IHB 201909037510, 63.9 mm SL; China: Zhejiang Province: Qingtian County: a stream discharging into the Ou-Jiang; 28°10'20"N, 120°12'51"E; collected by E Zhang and D.M. Guo, 8 October 2018.IHB 2017056858, 2017056869-80, 13 specimens, 54.2–66.8 mm SL; China: Zhejiang Province: Quzhou City: a stream flowing into the Qu-Jiang; 28°57'6"N, 118°51'15"E; collected by D.M. Guo, 15 December 2019.IHB 64VI410-15, 930138-39, 8 specimens, 43.7–60.5 mm SL; China: Zhejiang Province: Longquan City: a stream flowing into the Ou-Jiang; 28°4'12"N, 119°6'54"E; collected in 1964 and 1983.Leptobotiabrachycephala, together with L.citrauratea and L.micra, is distinguished from all other congeneric species by the presence (vs. absence) of a row of orange dots or an orange stripe extending along the dorsal mid-line of the body from the nape to the caudal-fin base  caudal fin with two rounded (vs. broadly pointed) lobes , a slender caudal peduncle (14.6–20.0% SL vs. 12.1–15.8% SL in L.citrauratea and 11.8–13.5% SL in L.micra), a shorter dorsal fin (9.0–11.6% SL vs. 13.7–18% SL in L.citrauratea and 11.0–4.6% SL in L.micra) and a shorter anal fin (8.1–11.4% SL vs. 13.0–17.7% SL in L.citrauratea and 15.3–16.8% SL in L.micra) .ina Fig. . Type spina Fig. . SyntopiThe specific epithet is a Latin version of the Greek words βραχύς (short) and κεφαλά (head), with reference to the short head; to be treated as a noun in apposition.cyt b sequences of species of Leptobotia  lineage and so did those of both L.citrauratea and L.elongata. L.citrauratea was distantly allied to L.elongata, but robustly supported by 100% pp to be sister to L.brachycephala  and Poyang Lake systems ; L.elongata is actually an endemic species of the upper Chang-Jiang Basin.sum Fig. . This isnds Fig. . Additioene Fig. . It is tBotia from the Dongting Lake system: one previously-described species as Botiarubrilabris Dabry de Thiersant, 1872 and two new species, B.purpurea and B.citrauratea. These three species were later referred to Leptobotia where B.purpurea were synonymised with L.taeniops   in this Lake was SL. This specimen, in light of their description, has a button-like fleshy protrusion in the gular area, a character diagnostic for L.rubirilabris within this genus , in having a shorter (vs. longer) suborbital spine just reaching (vs. far beyond) the posterior margin of the eye and no button-like fleshy protrusion in the gular area (vs. present)  , 2012. Tis genus ; thus, int) Fig. .L.brachycephala was confirmed by its morphological and genetic distinction with closely-related congeneric species  and L.microphthalma Fu & Ye, 1983, all of them having a plain brown body and no other colour formation. The fifth group has some rounded light orange spots extending along the mid-line of the dorsum from nape to caudal-fin base and three species are included in this group: L.micra, L.citrauratea and L.brachycephala. The last group has narrow bands on the body or mid-line of the back and three species can be referred to this group: L.guilinensis Chen, 1980, L.orientalis and L.flavolineata.The seventeen species currently included in ern Fig. . The secLeptobotiaelongata: IHB 3609, 58199, 64V2269-70, 4 specimens, 189.6–224.7 mm SL, upper Chang-Jiang Basin at Yichang City, Hubei Province; IHB 2018059214-16, 3 specimens, 170.0–176.5 mm SL, upper Chang-Jiang Basin at Leshan City, Sichuan Province; IHB 790197, 790198-99, 3 specimens, 87.8–167.4 mm SL, upper Chang-Jiang Basin at Luzhou City, Sichuan Province; IHB 790349, 1 specimens, 161.0 mm SL, upper Chang-Jiang Basin at Yibin City, Sichuan Province; IHB 201909035604, 1 specimens, 272.0 mm SL, upper Chang-Jiang Basin at Liangshan Yi Autonomous Prefecture, Sichuan Province; IHB 585410, 501244, 73V1466, 420457, 590452-54, 580451, 8 specimens, 116.8–236.7 mm SL, upper Chang-Jiang Basin at Chongqing City; IHB 201909035958, 201909035934, 2 specimens, 100.7–110.7 mm SL, upper Chang-Jiang Basin at Zhaotong City, Yunnan Province.Leptobotiataeniops: IHB 2017100254-59, 2017110267, 7 specimens, 43.6–112.9 mm SL, middle Chang-Jiang Basin at Yiyang City, Hunan Province; 201807020856, 201807028170, 2 specimens, 61.2–82.4 mm SL, middle Chang-Jiang Basin at Yueyang City, Hunan Province;Leptobotiarubrilabris: IHB 581-032, 2 specimens, 150.2 mm SL, Dongting Lake at Nanxian County, Yiyang City, Hunan Province, South China;Leptobotiatientainensis: IHB 74VI3347-50, 4 specimens, 70.5–91.2 mm SL, Ling-Jiang at Taizhou City, Zhejiang Province;Leptobotiaguilinensis: IHB 2015040803, 1 specimen, 70.3 mm SL, Zhu-Jiang at Guilin City, Guangxi Province;Leptobotiatchangi: IHB 201904029044, 1 specimen, 80.4 mm SL, Qiantang-Jiang at Hangzhou City, Zhejiang Province;Leptobotiapellegrini: IHB 2018099839, 1 specimen, 103.2 mm SL, Zhu-Jiang at Liuzhou City, Guangxi Province;Leptobotiahengyangensis: IHB 2017042831, 2017042828, 2 specimens, 99.7–102.8 mm SL, Xiang-Jiang at Hengyang City, Hunan Province;Leptobotiamicrophthalma: IHB 2016105306, 1 specimen, 78.3 mm SL, upper Chang-Jiang Basin at Leshan City, Sichuan Province.L.bellacauda and L.micra were taken from Data for


Median OS was 56.9 vs 46.8 months with Rd continuous vs MPT (p = 0.15346) and 59.5 months with Rd18. The overall response rate was higher with Rd continuous and Rd18 (78.8% and 79.8%) vs MPT (65.4%). In the 49.0%, 52.9%, and 29.8% of patients with at least very good partial response in the Rd continuous, Rd18, and MPT arms, respectively, the median PFS was 56.0, 30.9, and 40.2 months, respectively. The most common grade 3/4 treatment‐emergent adverse events were neutropenia , anemia , and infections  with Rd continuous, Rd18, and MPT, respectively. These results were consistent with those in the intent‐to‐treat population, confirming the benefit of Rd continuous vs MPT in the Canadian/US subgroup and supporting the role of Rd continuous as a standard of care for transplant‐ineligible patients with NDMM.The phase 3 FIRST trial demonstrated significant improvement in progression‐free survival (PFS) and overall survival (OS) with an immune‐stimulatory agent, lenalidomide, in combination with low‐dose dexamethasone until disease progression (Rd continuous) vs melphalan +prednisone + thalidomide (MPT) in transplant‐ineligible patients with newly diagnosed multiple myeloma (NDMM). Rd continuous similarly extended PFS vs fixed‐duration Rd for 18 cycles (Rd18). Outcomes in the Canadian/US subgroup (104 patients per arm) are reported in this analysis. Rd continuous demonstrated a significant improvement in PFS vs MPT  extended median PFS by 9 months, achieved deeper responses (≈20% increase in ≥VGPR rate), and delayed median TTNT by >14 months vs melphalan +prednisone + thalidomide (MPT). Safety results were generally consistent with the intent‐to‐treat analysis. The results support the role of Rd continuous as a standard of care for transplant‐ineligible patients with NDMM. These agents exhibit multifaceted antimyeloma activity by enhancing immune function, exhibiting direct antitumor  effects, and disrupting aberrant stromal cell support by decreasing cytokine production from interactions between stromal and MM cells.p < 0.00001), with a 21.0‐month median PFS for Rd18. Furthermore, Rd continuous delayed time to next antimyeloma treatment (TTNT) vs Rd18 and MPT . The median overall survival (OS) was also significantly higher with Rd continuous vs MPT , and the median OS with Rd18 was 62.3 months. The overall response rate (ORR) was higher with Rd continuous (81%) vs MPT (67%) and similar to that with Rd18 (79%).Lenalidomide +dexamethasone (Rd) has been a mainstay of treatment in the newly diagnosed MM (NDMM) setting for a number of years. The FIRST study (N = 1623) evaluated lenalidomide +dexamethasone (Rd) in transplant‐ineligible NDMM across 18 countries in Europe, North America, and the Asia‐Pacific region.The inclusion of the Rd18 arm in FIRST enabled evaluation of continuous vs fixed‐duration treatment.The above results support the use of frontline continuous oral therapy for transplant‐ineligible patients with NDMM and established Rd continuous as a standard of care, as reflected by clinical guidelines in Canada, the United States, and Europe.Treatment patterns can vary due to multiple factors, including geography and local guidelines. Thus, this analysis evaluated the outcomes in the subgroup of FIRST patients from Canada and the United States.22.1Details of the FIRST study have been published previously.The primary endpoint was PFS, and the primary comparison was between the Rd continuous and MPT arms (PFS comparison of Rd continuous vs Rd18 arms and MPT vs Rd18 arms was a secondary objective). OS was the key secondary endpoint. ORR, TTNT (which censors deaths), and safety, including second primary malignancies (SPMs), were also secondary endpoints. Time from randomization to second progression or death (PFS2) was an exploratory endpoint.The study was registered at EudraCT (2007‐004823‐39) and ClinicalTrials.gov (NCT00689936). Written informed consent was provided by all patients. Institutional review boards or ethics committees at all sites approved the study before initiation. The study was conducted according to the Declaration of Helsinki and the Harmonization E6 Guidelines for Good Clinical Practice.2.2In the Rd‐containing arms (28‐day cycles), oral lenalidomide (25 mg) was given on days 1 to 21 and oral dexamethasone  was given on days 1, 8, 15, and 22. Treatment was until disease progression (Rd continuous) or for 18 cycles . In the MPT arm (42‐day cycles), oral melphalan  was given on days 1 to 4, oral prednisone (2 mg/kg) was given on days 1 to 4, and oral thalidomide  was given daily. Treatment in the MPT arm was for 12 cycles (72 weeks). Details of starting dose adjustments for renal function and neutrophil counts were provided in a prior publication's supplementary appendix.2.3The data cutoff date  was the same used for the final analysis of OS.33.1This subanalysis was conducted in the 312 patients from sites in Canada (n = 252) and the United States (n = 60), with 104 patients each in the Rd continuous, Rd18, and MPT arms .Rd continuous demonstrated a significant improvement in PFS vs MPT , the median PFS was 56.0, 30.9, and 40.2 months with Rd continuous, Rd18, and MPT, respectively . Furthermore, Rd continuous achieved deeper responses vs MPT, with ≥VGPR rates of 49.0% vs 29.8% and CR rates of 21.2% and 11.5%, respectively. In the Rd18 arm, the ORR was 79.8%, and 52.9% and 22.1% of patients achieved ≥VGPR and CR, respectively.p = 0.02433) as was TTNT . Median PFS2 and TTNT with Rd18 were 39.8 and 29.9 months, respectively.Median PFS2 was significantly longer with Rd continuous vs MPT  with Rd continuous, 16.6 months  with Rd18, and 14.6 months  with MPT. The mean duration of treatment, which takes into account the long‐term treatment of patients with Rd continuous, was 28.3, 13.1, and 11.7 months in the Rd continuous, Rd18, and MPT arms, respectively. The mean number of cycles was 28.9  with Rd continuous, 13.6  with Rd18, and 7.8  with MPT.In the Canadian/US subgroup, neutropenia was the most common grade 3/4 hematologic TEAE, with rates notably higher with MPT (52.0%) than with Rd continuous (28.4%) and Rd18  and delayed median TTNT by >14 months vs MPT. Furthermore, Rd continuous resulted in a longer PFS2 vs MPT, suggesting the benefit of frontline Rd continuous is maintained at relapse. Together, these results further support Rd continuous therapy as the standard of care for transplant‐ineligible patients with NDMM.The demographic characteristics were generally similar between the treatment arms and between this subgroup and the ITT population.Other phase 3 studies have confirmed the activity of Rd continuous in NDMM, including SWOG S0777 (in patients not intended for immediate transplant) and MAIA (in transplant‐ineligible patients).In contrast to the improvement in PFS with Rd continuous in subsequent trials, the median PFS in the VMP arms of the VISTA and ALCYONE trials were similar  despite a >10‐year gap between trials .The safety results in the Canadian/US subgroup analysis were generally consistent with the profile in the ITT population, and no new safety concerns were observed. As noted for the ITT population, hematologic SPMs were less frequent in the Rd arms compared with the MPT arm, and incidence of solid tumor SPMs was similar across all treatment arms.–:CD27+ were prognostic for PFS, which may be reflective of reactivation after treatment with lenalidomide and subsequent transplant.The improved outcomes with Rd continuous may derive from the immunomodulatory effects of lenalidomide. This hypothesis stems from results of studies in transplant‐eligible patients with NDMM, where differences in immune microenvironment after transplant are predictive of outcomes. For example, some  studies have shown that early lymphocyte recovery is predictive of improved survival outcomes following autologous stem cell transplant.The post hoc nature of this analysis is a potential limitation. The smaller number of patients in the subanalysis also reduces the statistical power, which may explain why some outcomes that were significant in the ITT analysis did not reach significance in the subgroup population. The smaller population also limits the ability to conduct segmented comparisons  and further compare outcomes based on baseline demographics, including age, cytogenetic risk, and renal function.Overall, results of the Canadian/US subgroup analysis were consistent with those of the ITT population and support the role of Rd continuous as a standard of care for transplant‐ineligible patients with NDMM. Rd has been further investigated in combinations with bortezomib and daratumumab, resulting in improved outcomes and U.S. Food and Drug Administration approval.AB, MCC, CS: nothing to disclose; NB: consultancy for and honoraria and research funding from Janssen, Bristol Myers Squibb Company, and Amgen; DW: honoraria from and board of directors or advisory committee participant for Amgen, Bristol Myers Squibb Company, Janssen, and Takeda; CC: honoraria from Bristol Myers Squibb Company; KS: honoraria and research funding from and board of directors or advisory committee participant for Bristol Myers Squibb Company; AT: consulting for Roche and Novartis; AD: research funding from Bristol Myers Squibb Company, Takeda, Alnylam, and Pfizer; advisory fees from Akcea, Janssen, Intellia, OncoTracker; KA: advisory board participant for Bristol Myers Squibb Company, Millennium, Takeda, Janssen, Gilead, and Sanofi; DB: employment with Bristol Myers Squibb Company; SR, SS: employment with and equity ownership in Celgene; TF: consulting/advisory fees from Amgen, Bristol Myers Squibb Company, Janssen, Karyopharm, PharmaMar, and Takeda; speakers bureau fees from Amgen, Bristol Myers Squibb Company, Janssen, and Takeda.


Concrete has low porosity and compact microstructure, and thus can be vulnerable to high temperature, and the increasing application of various types of supplementary cementitious materials (SCMs) in concrete makes its high-temperature resistant behavior more complex. In this study, we investigate the effects of four formulations with typical SCMs combinations of fly ash (FA), ultra-fine fly ash (UFFA) and metakaolin (MK), and study the effects of SCMs combinations on the thermal performance, microstructure, and the crystalline and amorphous phases evolution of concrete subjected to high temperatures. The experimental results showed that at 400 °C, with the addition of 20% FA (wt %), the thermal conductivity of the sample slightly increased to 1.5 W/(m·K). Replacing FA with UFFA can further increase the thermal conductivity to 1.7 W/(m·K). Thermal conductivity of concrete slightly increased at 400 °C and significantly reduced at 800 °C. Further, combined usage of SCMs delayed and reduced micro-cracks of concrete subjected to high temperatures. This study demonstrates the potential of combining the usage of SCMs to promote the high-temperature performance of concrete and explains the micro-mechanism of concrete containing SCMs at high temperatures. High-performance concrete (HPC) has been extensively used in ultra-high and ultra-large building structures owing to its high strength and excellent durability ,2. HowevUnder high-temperature conditions, cement-based materials are not stable due to their internal physical/chemical changes and formation of cracks. The disappearance of ettringite (AFt) at 100 °C, the decomposition of Calcium Silicate Hydrate (C-S-H) gel and CH at 400–600 °C, and the transformation of C-S-H gel into the nesosilicate form at 750 °C, results in the degradation and damage of concrete . SimilarSupplementary cementitious materials (SCMs) contribute to improve the performance of concrete at high temperatures owing to their pozzolanic effects and super micro-filling capacity . Further2O3·2SiO2·2H2O), as a kind of high activity SCMs, can form anhydrous aluminum silicate (Al2O3·2SiO2) by dehydration at a temperature range of 600 to 900 °C [Fly ash (FA), as the most typical SCM, has been extensively studied for the physical and chemical modification of cement hydration, as well as its hardened properties ,19,20,21o 900 °C ,19,22. To 900 °C . TherefoWe have previously investigated the effects of recipes of SCMs on the workability (slump), mechanical  and physical properties  of concrete after exposure to high temperature . ResiduaIn this study, the effects of SCMs combinations on the thermal performance, microstructure, the crystalline and amorphous phases evolution of concrete subjected to high temperatures are investigated. Four types of HPC formulations were prepared: HPC modified with FA, HPC modified with UFFA, HPC modified with UFFA-MK, and HPC modified with FA-UFFA-MK. The experimental results explain the micro-mechanisms of concrete containing SCMs at high temperatures, and provide theoretical support for the design of concrete with high temperature resistance requirement.Portland cement (P·O 52.5R) was used in this study. 3, respectively. The fine aggregate used was natural river sand with a fineness modulus of 2.6. Broken granite gravel with continuous grading was used. The crushed index, maximum particle size and apparent density of the coarse aggregate are 8.7%, 10 mm and 2.895 g/cmPolycarboxylate-based superplasticizer (SP) was used, with a solid content and water-reducing ratio of 35% and 28%, respectively.In order to investigate the effects of SCMs combinations on the thermal performance, microstructure, the crystalline and amorphous phases evolution of concrete subjected to high temperatures, five different mix proportions were considered in this study. For residual compressive strength test, a specimen with dimensions of 100 × 100 × 100 mm was dried to constant weight after 28 d of curing, and then heated in an electrically controlled furnace , as shown in The thermal conductivity of concrete after exposure to high temperatures was tested by thermal constant analyzer  ,17. For The microstructure of concrete subjected to high temperatures was characterized by Quanta 600 FEG scanning electron microscope . Specimens were broken into small pieces and surfaces were coated by gold to increase its electrical conductivity before testing. X-ray diffraction (XRD) was used to identify the evolution of the crystalline phases of concrete subjected to high temperatures. Nickle-filtered Cu-Ka radiation at 40 kV and 20 mA were used throughout in a PW1390 diffractometer . Scanning speed of 0.020 °/s was used. Differential Thermal Analysis (DTA) thermal analysis  was used to explain the amorphous phases in the hydration products of the concrete at ambient and at high temperatures. Samples were analyzed under an oxygen atmosphere (100 ml/min) at a heating rate of 10 °C /min using alumina crucibles.The thermal conductivity of concrete is an essential parameter for delaying the temperature increase when subjected to high temperatures ,8,17. FiFrom ·K). Replacing FA with UFFA can further increase the thermal conductivity (HPC-2) to 1.7 W/(m·K), which is mainly due to the UFFA’s lower fineness, which can make the HPC denser. For the samples containing 30% UFFA-5% MK (HPC-3) and 20% FA-10% UFFA-5% MK (HPC-4), thermal conductivity increased to 1.9 W/(m·K) and 2.2 W/(m·K) at 400 °C, respectively. It can be concluded that the difference of thermal conductivity is much more evident between concretes than the difference attributed to the temperature itself when the temperature below 400 °C. At 800 °C, thermal conductivity of the concretes modified with UFFA-MK and FA-UFFA-MK fell to 0.3 W/(m·K) and 0.4 W/(m·K), respectively. This may be due to the internal structure of concretes had destructed at 800 °C.It can be observed that the thermal conductivity of the concretes approximately increased by 1%–5% than that of the concrete at RT. Compared with the thermal conductivity of concretes at RT, thermal conductivity of concretes at 400 °C increased by 2.1% (reference concrete), 3.4% (HPC-1), 1.8%(HPC-2), 3.8%(HPC-3), 4.8% (HPC-4). We also found that the thermal conductivity of concretes modified with SCMs was higher than that of the reference concrete. At 400 °C, with the addition of 20% FA (wt %), the thermal conductivity of the sample (HPC-1) slightly increased to 1.5 W/(m2S), portlandite (CH), ettringite (AFt), calcite (CaCO3), brown millerite (Ca4Al2Fe2O10) and lime can be found in the reference specimen, as shown in Compared with the reference concrete, the reflections  can be found in the concrete containing FA-UFFA-MK (HPC-4) after heating, as shown in 2O3 oxide and the decomposition of unburnt coal residues [For the HPC containing fly ash and MK, the peak (# 4) between 160 °C and 180 °C is attributed to the dehydration of the C-A-H and the hydrated calcium silicoaluminates (C-A-S-H) . The pearesidues . Additioresidues .Needle-like crystals of ettringite, CH and C-S-H gel can be found in From This paper presents a study of the effects of SCMs combinations on the thermal performance, microstructure, the crystalline and amorphous phases evolution of concrete subjected to high temperatures. Based on the experimental tests and the microstructure analyses, the following conclusions can be drawn:1Combined usage of SCMs improved the high temperature resistance of concrete after subjected to high temperatures. Formation of the secondary gel and filling capacity by combined usage of SCMs, making the microstructure denser, can exclude propagation of cracks at high temperature.2The thermal conductivity of both the reference concrete and HPC modified with SCMs increased slightly at 400 °C and reduced significantly at 800 °C. Thermal conductivity of concretes modified with SCMs was higher than that of the reference concrete. The thermal conductivity of the HPC modified with MK-UFFA and MK-UFFA-FA at 800 °C dropped to 0.3 W/(m·K) and 0.4 W/(m·K), respectively.3The residual strength of concrete at 400 °C showed a slight increase due to the water result in the decomposition of some hydration products let to rehydrate the anhydrous cement particles. The internal structure cracked and damaged owing to the decomposition of C-S-H gel and CH at 600 °C. For the composite 20% FA-10% UFFA-5% MK mixture after heating to 1000 °C, the unreacted MK particles in the microstructure transformed to mullite as identified in the XRD patterns.4The combined usage of SCMs to concrete contributes to the enhancement of high temperature resistance due to the generation of C-S-H gel and densification of the microstructure. FA not only could be prominently effective at concrete after exposure to high temperatures but works progressively alongside other pozzolanic materials, such as MK. Filling empty pores with different sizes and promoting pozzolanic reactions are two of the reasons that improved the high temperature resistance of concretes.


The AI-PoP beads were made using Plaster of Paris powder, antibiotic, and water, cured for 24 h, sterilized by ethylene oxide, and stored up to 5 months before testing. For each antibiotic, 20 beads were combined with bovine serum in sterile tubes and incubated at 37°C on a rocker. Serum was replaced at intervals over the 14 days study period, and antibiotic concentrations were determined by high pressure liquid chromatography with mass spectrometry. Separately, in a proof-of-concept study, the growth of E. coli and T. pyogenes in eluent from 10 beads for each antibiotic was quantified by flow cytometry. Antibiotic was detected in AI-PoP bead eluent for 14 days for all but the ampicillin beads, for which antibiotic was detected for 8 days. The concentration of antibiotic in eluent was greater than the minimum inhibitory concentration (MIC) of tested bacteria for the entire study period for penicillin, tetracycline, tulathromycin, and florfenicol. The concentration of ampicillin remained greater than the MIC of E. coli for 4 days and T. pyogenes for 6 days. The colony forming units (CFU)/ml of live E. coli and T. pyogenes was reduced over a 72-h period by 1–3 log10 CFU, with the exception of tetracycline, which reduced CFU/ml of T. pyogenes by <log10 CFU. AI-PoP beads containing penicillin, tetracycline, tulathromycin, and florfenicol elute antibiotic well-above the MIC of selected isolates for the 2 weeks study period. These antibiotics show promise for delivery in joint and wound infections.The objectives of this study were to report a recipe for making antibiotic impregnated Plaster of Paris (AI-PoP) beads using penicillin, ampicillin, tetracycline, tulathromycin, and florfenicol and to determine the  Synovial sepsis, post-operative surgical site infections, and complicated wounds are a devastating cause of increased morbidity and mortality in large animal veterinary patients and can be career or even life-ending –3. Whilein vitro elution of antibiotic from PoP beads has been reported for gentamicin, amikacin, clindamycin, enrofloxacin, vancomycin, tobramycin, and cefazolin  beads constitutes an extra-label drug use under the Animal Medicinal Drug Use Clarification Act of 1994, which also prohibits or limits the use of previously studied drugs in food producing species. Antibiotics elected for use in this study include penicillin, ampicillin, tetracycline, tulathromycin, and florfenicol. These antibiotics were chosen because they are commonly used in food animal practice and extra-label use may be allowed.in vitro elution properties of the AI-PoP beads over 14 days.The objectives of this study were to (1) report a recipe for making AI-PoP beads using penicillin, ampicillin, tetracycline, tulathromycin, and florfenicol and (2) determine the We hypothesized that (1) the elution of all antibiotics from the AI-PoP beads would have a rapid initial phase and complete elution within 2 weeks and (2) the initial concentrations of antibiotic in the eluent would exceed the MIC of bacterial isolates used.2 and a volume of 113 mm3. The AI-PoP beads were allowed to cure in the mold for 24 h at room temperature , sterilized by ethylene oxide, and stored at room temperature . Testing was completed within 5 months of AI-PoP bead manufacturing.Based on methods previously described , 9, AI-PFor each antibiotic, 20 AI-PoP beads and seven milliliters of bovine serum  were aseptically transferred to individual sterile polypropylene test tubes. The eluent from each AI-PoP bead type was tested in quadruplicate; four test tubes of AI-PoP beads were tested for each antibiotic. The number of beads, choice of serum, and volume of serum were chosen based on previous studies , 9 and bConcentrations of five antibiotics were determined using high-pressure liquid chromatography with mass spectrometry detection after protein precipitation of samples with acetonitrile. High-pressure liquid chromatography with mass spectrometry detection was performed using an Agilent 1100 Pump, column compartment, and autosampler  coupled to an ion trap mass spectrometer . Serum samples, serum spikes, serum quality controls, and bovine serum blanks, 50 or 100 μL, depending on calibration curve concentrations of the antibiotic, were mixed with 450 μL of acetonitrile to precipitate serum proteins. The acetonitrile contained an internal standard at a concentration of either 250 or 1,000 ng/mL, depending on calibration curve concentrations of the antibiotic. The samples were vortexed for 5 s and centrifuged for 10 min at 7,500 rpm to sediment the protein pellet. A portion, 20 or 50 μL, of the supernatant was then diluted with water to 1 mL of volume in an autosampler vial. Further dilution of the samples, if needed, was done with blank serum that had been precipitated and diluted identically as the samples. These dilutions were done in autosampler vials fitted with glass inserts and dilutions were 1:5 or 1:10. The vials were then centrifuged at 2,400 rpm prior to analysis. All the quality control samples passed the tolerance of being within ± 15% of the nominal concentration. The limit of quantitation of the analysis was 0.5 μg/mL with a limit of detection of 0.1 μg/mL.The mean observed peak of antibiotic elution was calculated for each antibiotic. The amount of antibiotic eluted was displayed in two different ways: first, as the amount of antibiotic eluted in the first 72 h, expressed as a percent of antibiotic released over the 2-weeks study period, and second, as the total amount of antibiotic released over the 2-weeks study period, expressed as a percent of total incorporated antibiotic. The concentration of antibiotic within the eluent was compared to the MICs of the bacterial isolates used in this study. Some authors consider time dependent antibiotics, such as those used in this study, to be most efficacious when concentration is maintained above the MIC for at least 50% of the dosing interval , thus, wE. coli and a clinical isolate of T. pyogenes. These are two of the most common isolates in cattle from cases of synovial sepsis  broth to a concentration of 107 CFU/mL. This concentration was elected because it was the most concentrated inoculum that could be reasonably achieved using these methods. It has been shown that the risk for surgical site infection is markedly increased when contaminated with more than 105 microorganisms per gram of tissue . Samples were prepared according to manufacturer's instruction, then immediately transported to the flow cytometry laboratory for quantification. The number of events in the bead region was set to 5,000 and the sample was assayed at 488 nm. The data were processed by framing the region around the live and dead populations to yield events data for live and dead bacteria. The bacterial culture density was calculated using outcome events data and dilution factors.The quantification of bacteria subjected to AI-PoP eluent was evaluated against two bacterial isolates, an American Type Culture Collection strain of l sepsis . Togetheanaerobe , 25. In f tissue . Thus, tAll AI-PoP beads eluted detectable concentrations of antibiotic for the 14-days sampling period, with the exception of ampicillin, which eluted detectable concentrations of antibiotic for 8 days. Mean observed peak ampicillin, florfenicol, and tetracycline concentrations occurred at 4 h, mean observed peak tulathromycin concentration occurred at 12 h, and mean observed peak penicillin concentration occurred at 24 h .The amount of antibiotic released in the first 72 h, expressed as a percent of antibiotic released over the 2-weeks study period, was greatest for ampicillin and least for florfenicol . The totE. coli and T. pyogenes for penicillin, tetracycline, tulathromycin, and florfenicol for the entire study period. The concentration of ampicillin remained greater than the MIC of E. coli for 4 days and T. pyogenes for 6 days.The MIC data for each antibiotic and bacterial strain, as provided by the Iowa State University Veterinary Diagnostic Laboratory, is shown in Ampicillin had the shortest elution half-life, and penicillin had the longest . The eluE. coli is reduced by ~1–2 log10 CFU by 72 h for all antibiotics compared to the control sample. The CFU/ml of T. pyogenes is reduced by 3 log10 CFU for all antibiotics except tetracycline by 72 h compared to the control sample. CFU reduction in the tetracycline sample was <log10 CFU.The results of the flow cytometry proof-of-concept study are shown in This study was successful in creating antibiotic impregnated beads using five different antibiotics. The first hypothesis, that the elution of all antibiotics from the AI-PoP beads would have a rapid initial phase and complete elution within 2 weeks, was rejected. Only the elution of ampicillin was complete within 2 weeks; the remaining antibiotics eluted antibiotic for the full 2-weeks study period. The second hypothesis, that the initial concentrations of antibiotic in the eluent would exceed the MIC of bacterial isolates used, was accepted. The concentration of antibiotic in the eluent exceeded the MIC of bacterial isolates for a minimum of 4 days.The amount of antibiotic eluted by the AI-PoP beads was low in this study, ranging from 3.79 to 49.23%. The amount of antibiotic eluted has been shown to be proportional to dissolution of the bead, with the rate of dissolution being affected by increases in porosity . PorositThe elution pattern of penicillin, ampicillin, and tulathromycin in our study is similar to the previously reported elution pattern of most antibiotics , 9, charThis study is consistent with previous reports that found a variable volume of liquid is needed for individual batches in order to obtain an appropriate antibiotic-PoP mixture consistency prior to filling the bead mold , 9. BecaA sampling period of 2 weeks was chosen in this study. Based on the rapid elution of antibiotic from AI-PoP beads observed in a previous study using PoP beads , a similPrevious studies have used eluent to qualitatively describe bacterial inhibition , 8. Flowin vitro work is to relate in vivo. There are many factors of in vivo infection that were not replicated by this study, including microbe-related risk factors such as biofilm, and host-related factors such as an immune response.The choice of antibiotics for systemic administration should be based upon culture and antimicrobial susceptibility data, but because of a variety of factors including cost, time to achieve culture and sensitivity results, negative growth on cultures, and other reasons, the choice is often based upon clinical experience, empirical data, and commonly known bacterial isolates associated with specific conditions. The choice for local delivery of antibiotics is based upon similar criteria, although MIC breakpoints used to identify susceptible and resistant isolates are not predictive when antibiotic drugs are applied topically or locally, since achievable concentrations and effects on clinical outcome are unknown. Additional factors that could potentially influence the performance of local antimicrobials include bacterial burden, wound contamination, and wound exudate. It remains unknown how this The antibiotics chosen for this study were selected because they are labeled for use in food producing species and their extralabel use may be allowed. Food producing species are subject to restrictions on drug use such as provisions within the Animal Medicinal Drug Use Clarification Act of 1994. It is recommended that the Food Animal Residue Avoidance Database be consulted for any extralabel drug use, including those described in this report. It is unknown what the serum or tissue concentrations of these drugs might be after application of AI-PoP bead, so caution should be used when predicting withdrawal times.in vitro inhibition of bacteria by AI-PoP eluent.A limitation of this study is that elution was only investigated for 2 weeks. Elution data showed that a longer period would have been beneficial for penicillin, tetracycline, tulathromycin, and florfenicol. Another limitation is that the mechanical properties of the PoP beads were not characterized, and we were not able to provide further information regarding the rate of dissolution of the beads. A final limitation of this study is the absence of multiple specimens in the proof-of-concept study to determine the in vivo application of penicillin, tetracycline, tulathromycin, and florfenicol AI-PoP beads to corroborate the results of this in vitro study. These antibiotics appear to perform well in eluting antibiotic from AI-PoP beads and show promise for use in food animal species.Future studies should focus on the The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.PM, JS, AK, and DB contributed to the design and completion of this study. VF performed the elution half-life analysis. All authors contributed to the writing, editing, and final approval of 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.