Adropin is a peptide hormone which modulates energy homeostasis and metabolism. In animals with diet-induced obesity, adropin attenuates adiposity and improves lipid and glucose homeostasis. Adropin promotes the proliferation of rodent white preadipocytes and suppresses their differentiation into adipocytes. By contrast, the effects of adropin on mature white adipocytes are unknown. Therefore, we aimed to evaluate the effects of adropin on lipolysis, lipogenesis and glucose uptake in white rodent adipocytes. We assessed the effects of adropin on the mRNA expression of adiponectin, resistin and visfatin. White preadipocytes were isolated from male Wistar rats. Differentiated 3T3-L1 cells were used as a surrogate model of white adipocytes. Lipolysis was measured by the evaluation of glycerol and free fatty acid secretion using colorimetric kits. The effects of adropin on lipogenesis and glucose uptake were measured using radioactive-labelled glucose. The expression of adipokine mRNA was studied using real-time PCR. Our results show that adropin slightly promotes lipolysis in rat adipocytes and 3T3-L1 cells. Adropin suppresses lipogenesis in rat adipocytes without influencing glucose uptake. In addition, adropin stimulates adiponectin mRNA expression and suppresses the expression of resistin and visfatin. These results indicate that adropin may be involved in controlling lipid metabolism and adipokine expression in white rodent adipocytes. Enho), which was originally identified in 2008 by Kumar et al.  glucose  incorporation into lipids . In brieRat preadipocytes were seeded and differentiated in 24-well plates. Next, cells were incubated in a glucose-free KRB buffer containing 0.1% BSA for 30 min. After the incubation, cells were washed with PBS and incubated for 30 min in KRB buffer in the presence or absence of adropin (10 or 100 nmol/L). Next, 18.5 kBq of deoxy-D-glucose, 2-[1-14C] glucose (Perkin Elmer) and 0.1 mmol/L 2-deoxyglucose were added, and cells were incubated for 6 min. Then, ice-cold PBS containing 20 μmol/L of cytochalasin B was added. Then, KRB was aspirated, and cells were washed (3 times) with PBS. The cells were lysed using 0.1% SDS. Lysates were transferred into scintillation liquid and β-radiation was measured using a Liquid Scintillation Analyzer Tri-Carb 4810 TR (Perkin Elmer).Gapdh.Total RNA was extracted using Extrazol reagent . cDNA was synthetized using FIREScript RT cDNA Synthesis MIX with Oligo (dT) and Random primers . cDNA was amplified using EvaGreen qPCR Mix (Solis BioDyne) on QuantStudio 12K Flex . The sequences of PCR primers are shown in g at 4 °C for 10 min), and then the supernatants were collected. Furthermore, we determined protein concentration by a BCA Protein Assay Kit (Thermo Fisher Scientific). Every sample containing 30 μg of protein was diluted in a loading buffer and then denatured . SDS-PAGE (5–12% Tris-HCl gel) was applied to separate proteins based on their molecular weight. Afterwards, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked for 1 h at RT in 5% BSA in TBST . We incubated the membranes with a primary antibody  and washed them three times (for 10 min each) with TBST. Next, the membranes were incubated with a secondary antibody for 1.5 h at RT and washed again (as described above). Following the last wash, the signal was detected using chemiluminescence  and visualized by ChemiDoc MP Imaging System . Then, the membranes were stripped and the process was repeated for the total HSL antibody. The dilution was 1:1000 for primary antibodies and 1:5000 for secondary antibodies.The Western blot technique was used to detect phosphorylated and total HSL. Cells were differentiated and then incubated with adropin (100 nmol/L) for the indicated time points (0–60 min). Next, cell plates were placed on ice and washed with ice-cold PBS. RIPA buffer (Sigma-Aldrich), with an addition of protease and phosphatase inhibitor cocktails , was used to lyse cells and to isolate proteins. Tubes with collected cells, after incubation on ice (10 min), were centrifuged (14.000× p < 0.05 (*) was considered to be statistically significant. All experiments were repeated at least two times.Data were compared using one-way ANOVA followed by the Bonferroni post hoc. First, we evaluated the effects of adropin on lipolysis in differentiated rat primary preadipocytes. The successful differentiation of both white fat precursor cells is shown in Hsl mRNA expression assessed in rat adipocytes treated with adropin (10 and 100 nmol/L) for 3 h. However, adropin (100 nmol/L) promoted HSL phosphorylation in these cells  in rat adipocytes. As shown in se cells b,c.As shown in Next, we evaluated the effects of adropin on the mRNA expression of selected adipokines, such as adiponectin, resistin and visfatin. As demonstrated in Pparγ, Scd1 and Fas in white adipose tissue [In this study, we report that adropin modulates lipid metabolism and the mRNA expression of adiponectin, resistin and visfatin in mature white fat rodent cells. Firstly, we found that acting on differentiated rat adipocytes and 3T3-L1 cells, adropin slightly promotes the secretion of glycerol and FFA, the hallmarks of lipolysis . In our Hsl mRNA levels but enhanced the phosphorylation of HSL at serine 563. It is worth noting that the phosphorylation of HSL at serine 563 is promoted by lipolysis-inducing factors [The regulation of lipolysis in white adipocytes is complex, and it is modulated by numerous metabolic, hormonal and environmental signals . Neverth factors ,26,27,28In addition to lipolysis, intracellular fat content in adipocytes is modulated by other processes, such as lipid synthesis termed as lipogenesis . TherefoIn adipose tissue, lipogenesis depends upon the availability of carbohydrates in circulation, as well as their uptake by adipocytes . TherefoNext, we evaluated the influence of adropin on the mRNA expression of selected adipokines involved in controlling energy homeostasis and metabolism, such as adiponectin, resistin and visfatin ,33. We fIt is worth to note that adiponectin is able to improve insulin sensitivity , which rBy contrast, it was found that resistin impairs glucose tolerance and insulin action in rodents . FurtherOur study has several limitations. First of all, we did not elucidate the mechanism conferring the activation of HSL or lipolysis by adropin and its putative receptor GPR19. However, it was found that in tilapia hepatocytes, adropin promotes LPL expression via a cAMP/PKA-dependent mechanism , which wIn summary, we found that by acting on white rodent adipocytes, adropin slightly stimulates lipolysis while suppressing de novo lipid synthesis. However, these effects were moderate. Furthermore, adropin stimulates the expression of adiponectin; however, it downregulates the expression of resistin and visfatin . Overall


Better markers of early response to neoadjuvant chemotherapy (NACT) in patients with breast cancer are required to enable the timely identification of non-responders and reduce unnecessary treatment side-effects. Early functional imaging may better predict response to treatment than conventional measures of tumour size. The purpose of this study was to test the hypothesis that the change in tumour blood flow after one cycle of NACT would predict pathological response.In this prospective cohort study, dynamic contrast-enhanced MRI was performed in 35 females with breast cancer before and after one cycle of epirubicin and cyclophosphamide-based NACT (EC90). Estimates of tumour blood flow and tumour volume were compared with pathological response obtained at surgery following completion of NACT.Tumour blood flow at baseline  reduced slightly after one cycle of NACT (0.28 ± 0.18 ml/min/ml). Following treatment 15 patients were identified as pathological responders and 20 as non-responders. There were no relationships found between tumour blood flow and pathological response. Conversely, tumour volume was found to be a good predictor of pathological response  at both baseline (area under the receiver operating characteristic curve 0.80) and after one cycle of NACT (area under the receiver operating characteristic curve 0.81).The change in breast tumour blood flow following one cycle of EC90 did not predict pathological response. Tumour volume may be a better early marker of response with such agents. Typically, a change in tumour size is demonstrated after two or more cycles of NACT2 but several studies have examined functional imaging techniques to assess response after only one cycle.3–5 Amongst the functional imaging techniques 18F-FDG PET has been used to assess changes in tumour glucose metabolism following NACT6 and a number of PET studies have looked specifically at breast tumour blood flow with either [15-O] H2O PET7 or dynamic 18F-FDG PET.8 However, repeated PET imaging is impractical for routine clinical use9 and so MRI techniques that measure parameters related to flow, via dynamic contrast-enhanced (DCE) MRI, have also been studied.10 A difference between these MRI approaches and PET imaging of blood flow is the lack of absolute quantification by MRI. Moreover, there has been a lack of consistency in study methodology and the reporting of pathological response in MRI studies over the years.10–12Neoadjuvant chemotherapy (NACT) offers an increased rate of breast conserving surgery, can downsize advanced breast tumours to allow surgery and with the addition of human epidermal growth factor receptor 2 (HER2)-targeted therapy (to NACT or adjuvant chemotherapy) can improve survival in HER2-positive breast cancer.13 to measure tumour blood flow at baseline and after one cycle of NACT in order to test the hypothesis that the change in blood flow after one cycle of NACT would predict pathological response as defined according to international recommendations.14 Analysis of the HTR data to estimate blood flow also allowed us to assess related haemodynamic parameters such as blood volume, capillary permeability surface–area product (PS) and interstitial volume and, by using the HSR data, we were able to assess tumour volume.In this study, we used a recently developed interleaved high spatial resolution (HSR)/high temporal resolution (HTR) MRI technique2) and cyclophosphamide (600 mg/m2)] followed by three 3-weekly cycles of docetaxel (100 mg/m2). In patients with HER2 positive tumours, docetaxel was accompanied by trastuzumab, and in some (more recent) cases pertuzumab. Blood samples, taken as standard of care during clinic visits, were used to estimate haematocrit at each MRI visit.In this prospective study consecutive patients, with newly diagnosed primary invasive carcinoma of the breast due to undergo NACT with curative intent, were approached and asked if they were willing to take part. All those agreeing to join the local Research Ethics Committee approved study provided written informed consent after the nature of the procedures had been fully explained. Each underwent a block sequential regimen of NACT: three 3-weekly cycles of EC90  when they were not (assessed using the Shapiro–Wilk test). To determine whether differences existed between the measured parameters, estimates from pR and pNR were compared using the non-parametric Mann–Whitney Between August 2015 and April 2018, 40 female patients  gave written informed consent to enter the study. MRI data were obtained in all 40 patients at baseline (7 [4 - 12] days before the first administration of NACT). However, one patient was unable to tolerate the complete imaging protocol and technical problems in one other (back coil was switched on and off intermittently) precluded the measurement of tumour blood flow. Following a single cycle of EC90, 37 patients were scanned (three patients declined further MRI scans). Technical problems (as previously) affected one further scan, so blood flow estimates were obtained in 36 patients. Paired baseline and cycle one data were available for 35 patients and the results presented below reflect these 35 patients only .The immunohistochemical subtype (IHS) of the 35 tumours were: 2 HER2 enriched (oestrogen receptor and progesterone receptor negative and HER2 positive), 9 triple negative and 24 luminal B (oestrogen receptor and/or progesterone receptor positive and HER2 positive or HER2 negative with high levels of Ki-67). Following the completion of NACT and surgery, pathological analysis revealed 6 patients (17%) had a pathological complete response (RCB-0), 9 patients had minimal residual disease (RCB-I), 12 patients moderate (RCB-II) and 8 patients extensive residual disease (RCB-III). Thus, there were deemed to be 15 pR (43%) and 20 pNR (57%).3 and patients going on to a pR had tumours that were significantly smaller than those that went on to pNR (p = 0.003). There was a small but significant decrease in tumour volume following one cycle of NACT  and tumour volume at baseline and after cycle one were good predictors of pR (area under the ROC curve 0.80 (95% CI 0.66 to 0.95) and 0.81 (95% CI 0.66 to 0.95), respectively). Using a volume threshold at cycle one to predict response failure, 12 patients with tumours of 9 cm3 or larger went on to pNR  and all 15 patients who went on to a pR had tumours smaller than 9 cm3 (100% specificity). This test had a positive predictive value of 100%.Pathological complete response to NACT varied as a function of IHS – neither of the patients with HER2 enriched tumours, 3/9 (33%) patients with triple negative tumours and 3/24 (12.5%) patients with luminal B tumours had a complete pathological response. Less variation was seen in pR where 1/2 (50%) patients with HER2 enriched tumours showed pR compared to 3/9 (33%) with triple negative tumours and 11/24 (46%) with luminal B tumours. Luminal B tumours that were HER2 positive responded better to NACT: 9/12 (75%) HER2 positive tumours had a pR compared to only 2/12 (17%) HER2 negative tumours. Median enhancing tumour volume at baseline was 4.8 [2.3 to 22.5] cmT1 at baseline (1287 ± 87 ms) showed no change following one cycle of NACT (1285 ± 92 ms) and showed no significant variation as a function of IHS (Tumour blood flow at baseline (0.32 ± 0.17 ml/min/ml) and after cycle one (0.28 ± 0.18 ml/min/ml) showed little variation as a function of IHS  or pathon of IHS .p = 0.007) after one cycle of NACT and this was mirrored in an increase of blood T1 from a baseline value of 1660 ± 64 ms to 1766 ± 109 ms (p = 0.00002) after one cycle of NACT.15Patient haematocrit decreased from a baseline value of 0.41 ± 0.03 to 0.39 ± 0.03  of NACT.7 We did not see this effect in our study following just one cycle of NACT. There was, on average, a decrease in blood flow of 12% but this was subject to considerable variation, with both increases and decreases seen in both pR and pNR (8 who saw a large variability in blood flow response in HER2 negative tumours after one cycle of EC chemotherapy (HER2 positive patients in that study experienced a large decrease in blood flow but they were selectively treated with docetaxel and trastuzumab). The primary hypothesis of this study - that change in blood flow after one cycle would predict pathological response - was not supported by our resultsAt least one previous [15-O] H and pNR . Our res10 was predictive of response. This mirrors results seen in the ACRIN 6657 trial that highlighted the importance of enhancing tumour volume, measured at cycle one, to recurrence-free survival.19 At cycle one of our study, 12 of the 35 tumours were 9 cm3 or larger . These patients could potentially have been spared further NACT since all failed to respond (pNR). Of the remaining 23 patients with tumours smaller than 9 cm3, 15 went on to a pR leaving only 8 non-responding patients. Further work is required to identify a specific imaging phenotype to identify those tumours at the early stages of NACT.Conversely, tumour volume, which was identified as a more reliable marker of response than tumour diameter in a previous review,20 Previous quantitative MRI studies have typically reported parameters such as Ktrans (volume transfer constant) without measuring patient-specific arterial input functions21–23 or by measuring the input function inadequately24 . It is well recognised that measurement of the arterial input function is very challenging.11 As in the current study, some reports have highlighted the importance of tumour size after a single cycle of NACT as a predictor of response while tracer kinetic parameters have been less useful21,25; others found neither size nor tracer kinetic parameters useful at this timepoint.22 Although the current study lacks direct validation of the blood flow estimates, the values reported very closely reflect previously published estimates obtained using the ‘gold-standard’ of [O-15] H2O PET in a similar group of patients.26 Moreover, the lack of variation in tumour blood flow as a function of IHS seen in the PET study26 is similarly seen in our data . Despite the well-established variation in response to NACT of the different IHS of breast cancer, there appears to be very little difference in their haemodynamic characteristics at baseline and no clear patterns of change following one cycle of NACT. The decrease of blood flow seen in responders later in the course of NACT by others7 is not yet apparent after one cycle. Measurements of blood flow may prove to be more revealing when assessing NACT regimens with expected antiangiogenic effects.8This is the first published study to report absolute estimates of breast tumour blood flow and related parameters measured using MRI in response to treatment.T1 measurements plus a few additional DCE acquisitions) and this could be reduced if a faster B1-insensitive T1 measurement sequence were employed. The interleaving of HTR data with the clinical HSR data did not have a significant impact on clinical reporting.13 Perhaps more significant limitations are the small number of patients studied and the post-processing overhead both for tumour volume estimation and tracer kinetic analysis, which currently rely on in-house software. Further work is required to develop user-friendly tailored software for these applications.The additional time required to acquire data for blood flow estimation amounts to less than 10 min  show little variation with tumour IHS at baseline or in response to one cycle of NACT and, contradicting our primary hypothesis, blood flow change following one cycle of NACT did not predict pathological response. In contrast and reflecting the findings of a recent multicentre trial, enhancing tumour volume measured after one cycle of NACT appears to show promise for prediction of pathological response.


Mediastinal mature teratomas are often benign, asymptomatic, and incidentally detected during routine chest roentgenography. Enzymes secreted by intestinal or pancreatic tissue in teratomas may lead to mediastinitis or the rupture of adjacent tissues. Herein, we present a case of a patient who experienced sudden onset of chest pain followed by the perforation of a mediastinal teratoma.A 10-year-old boy presented with chest pain 2 days before admittance to the hospital. Chest radiography showed an anomalous mass shadow, and computed tomography showed an anterior mediastinal mass. Radiography revealed an increase in the mass shadow size and dullness of the left costal phrenic angle. Magnetic resonance imaging revealed pleural effusion and intratumoral haemorrhage, indicating perforation of the tumour. Emergency excision and thymectomy via sternotomy were performed. Pathology confirmed that the mediastinal tumour presented no immature or malignant elements.In the present case, the onset of chest pain occurred 2 days before admission, and the initial computed tomography did not reveal tumour perforation. Subsequent chest radiography and magnetic resonance imaging indicated that the tumour had perforated. Surgical tumour excision was planned at the time of admission; however, once perforation was confirmed, emergency surgery was performed. The pleural effusion had high cancer antigen 19-9 levels, and this was expected as the pleural effusion contained pancreatic digestive enzymes.The perforation of a mediastinal mature teratoma cannot be predicted based on the symptoms, tumour size, or onset of pain alone. Once perforation is confirmed, surgical excision should be performed immediately. These t2A 10-year-old boy with no medical history who was experiencing chest pain for 2 days was admitted to our hospital. Chest radiography showed a mass shadow in contact with the aortic shadow, and computed tomography (CT) showed an anterior mediastinal tumour . Blood tOnce the sternotomy was performed, the left thoracic cavity where the tumour was protruding was intentionally opened. There was a moderate volume of viscous pleural effusion. The tumour was involved with the left phrenic nerve and showed adhesions to the thymus. Tumour excision and thymectomy were successfully performed, and the left phrenic nerve was detached from the tumour using Metzenbaum scissors. The thoracic cavity and mediastinum were irrigated with 5,000 mL of saline solution after the excision.The patient’s recovery was uneventful. His chest tube was removed on the fifth postoperative day, and he was discharged the following day. Phrenic nerve palsy was not observed. Pathology confirmed that the mediastinal tumour was a mature teratoma with no immature or malignant elements .Fig. 3Pa3Reportedly, 50–62 % of mature teratomas are asymptomatic ,5. In coCystic teratomas may perforate adjacent organs but tumour diameter and wall thickness are not associated with the risk of perforation . The effTherefore, it is important to perform surgical excision immediately after confirming tumour perforation.4We identified two important clinical issues in the present case. First, the perforation of a mediastinal mature teratoma cannot be predicted solely based on the symptoms, tumour size, and timing of onset. Second, it is important to perform immediate surgical excision once the tumour has been perforated. The present case demonstrates the importance of early diagnosis of a ruptured mediastinal teratoma using radiography and MRI. Though a ruptured mediastinal teratoma is not particularly rare, it is rare to locate images of a teratoma before and after the rupture. However, our case report successfully captured these images.The authors report no declarations of interest.I have no sources of funding should be declared.Our institutional review board gave echical exemption for this study.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Tsuyoshi Uchida: Writing – original draft, Data curation, Formal analysis.Hirochika Matsubara: Writing – review & editing.Tamami Hada: Writing – review & editing, Data curation, Formal analysis.Daisuke Sato: Writing – review & editing, Data curation, Formal analysis, Conceptualization.Norio Hasuda: Writing – review & editing.Hiroyuki Nakajima: Writing – review & editing, Supervision.Not applicable.Tsuyoshi Uchida.Not commissioned, externally peer-reviewed.The datasets generated and analysed during the current study are not publicly available as this is a case report, but they are available from the corresponding author on reasonable request.


In the field of oil and gas exploration, drilling fluid is regarded as the essential “blood” for drilling, which mainly helps to control the formation pressure and remove cuttings from the well. During the drilling fluid cycle, the drilling fluid penetrates into the pores of the formation rock, thus blocking the rock pores and resulting in a decline in oil and gas recovery efficiency. Therefore, it is very important to understand the microscopic mechanism of formation damage caused by drilling fluid. However, as an important component of formation damage, the microscopic mechanism of fluid damage has not yet been clearly revealed. In this study, a new microetching model (MEM), along with displacement equipment, was designed. The pore network of rock samples was extracted from thin-section images and etched to a thin aluminum sheet by laser. Oil-based drilling fluid was used to displace the stratum water in the MEM. The displacement process was recorded by a camera and analyzed. A core flooding experiment, permeability measurement, and SEM observations were performed. The results show that, for low-porosity and low-permeability sandstone, the main forms of formation damage by drilling fluid include solid damage and liquid damage. Solid damage is mainly caused by the blockage of small pores and narrow throats with solid particles of the size 0.1~30.0 μm in drilling fluid, while liquid damage is mainly caused by the water lock and hydrocarbon lock effects formed by the oil–water two-phase interface, gas–water two-phase interface, or the oil–gas–water three-phase interface. In drilling for oil and gas exploration, the drilling fluid in the wellbore mainly plays the role of controlling the formation pressure and removing cuttings from the well ,2,3,4 Fa. DuringAt present, the experimental methods for studying the damage to rocks caused by drilling fluid at the microscopic scale include thin-section observations ,14, scanIn recent years, with the continuous progress of photolithography, the accuracy of etching is improving, and the field of its application is expanding ,24,25. SThe aim of this study is to design a new MEM and fluid displacement technique that involves replicating the pore structure of rock on aluminum sheets using infrared lasers and encapsulating the models with thicker glass and rubber to create MEM that can withstand higher pressures, so as to continue the drilling fluid displacement of stratum fluids in rock pore structures. This new technique can reveal the microscopic mechanism of formation damage caused by liquid components and provide a theoretical basis for the efficient exploration and development of oil and gas.The first step of MEM fabrication is to extract the pore structure from the rock a–e. A stThe second step of MEM fabrication is to etch the pore structure f–i. PrevThe third step of MEM fabrication is encapsulation j. To facAfter obtaining the MEM, the drilling fluid displacement of stratum fluids was carried out through the microetching displacement device, which simulates the damage of the reservoir by drilling fluid under drilling conditions. In this study, the major part of the MEM displacement device a,b was aThe stratum water used for the microetching model displacement experiments was blended according to the ion concentrations of the actual stratum water, and methyl blue was added to facilitate differentiation from the pure water (transparent) in the annulus between the sleeve cavity and the encapsulated MEM. The drilling fluid used in this research was oil-based drilling fluid. The composition of the oil-based drilling fluid is listed in The main process of the microscopic displacement experiment is as follows: (1) Experimental device assembly: The displacement experimental device was assembled in accordance with TM-300 automated permeameter using the unsteady-state method following the standard SY/T 6385-2016. The fluid used for permeability measurement was nitrogen at a temperature of 20 °C, a confining pressure of 5 MPa, and an initial displacement pressure of 3 MPa. When the gas flow rate reaches a stable state, the flow rate and pressure value are read, the permeability can be obtained by Equation (1) [K is the permeability (mD); A is the cross-sectional area of the rock sample (cm2); L is the length of the rock sample (cm); p1 is the inlet pressure (MPa); p2 is the outlet pressure (MPa); ap is the atmospheric pressure (MPa); Q is the flow rate (cm3/s); μ is the test gas viscosity (MPa·s); nitrogen has a fluid viscosity of approximately 0.0173 MPa·s at a temperature of 20 °C and one atmosphere.dR is the permeability decline ratio (%); K1 is the permeability of the rock sample before the damage (mD); K2 is the permeability of the rock sample after damage (mD).To further verify the degree of the damage to cores caused by drilling fluid, four core plugs were also selected to carry out flooding experiments on the core plugs by drilling fluid, and the permeability of the core plugs before and after the damage was measured for comparison. The permeability measurement was performed with a Core Lab CMStion (1) . The perThe core plugs before and after damage were cracked open to obtain their natural sections, and SEM observations were used to reveal the blockage of rock pores by solid particles from the drilling fluid. The SEM observation was performed by an FEI Quanta 650 FEG scanning electron microscope.The rock samples used in this study were collected from the Enping Formation in the Baiyun Sag of Pearl River Mouth Basin . During In order to observe the distribution state and contact relationship of drilling fluid, stratum water, and gas in the MEM more clearly, and to quantitatively analyze the volume change in different fluids during the displacement, image processing was performed on the photos taken by optical microscope . The proThe image of the microscopic model saturated with water after image processing a clearlyThe image of the microscopic model of drilling fluid displacing stratum fluid after image processing a clearlyMore images during the microscopic model saturated with stratum water and drilling fluid displacement experiments are selected for image processing, and the volume percentages of different fluids  are calculated and plotted as a line graph of their change with displacement time a. In theThe 40–70 min point in the microscopic displacement experiment marks the process of the drilling fluid displacement of stratum fluids. When drilling fluid starts to displace stratum fluid, the initial intrusion rate of drilling fluid is very fast, close to the intrusion rate of stratum water in the saturated stratum water stage of the microscopic model (the slope of the blue line from 0 to 30 min is close to the slope of the red line from 40 to 50 min), but after encountering small pores and narrow throats (at 50 min), there is no obvious fluid flow during the following 50 to 70 min, and the microscopic model reaches a fully damaged state. The 50 min time point in the microscopic displacement experiment corresponds to 4 particles in the drilling fluid, 2 grains in the rock, 4 particles in the drilling fluid.The photos of the four core plugs damaged by drilling fluid through core flooding experiments are shown in 4) in drilling fluid ranging in diameter from 0.1 to 30.0 μm move with the drilling fluid to the small pores and narrow throats, blocking the pores and throats and causing solid damage in the reservoir; (2) the white oil and water in the drilling fluid come into contact with the stratum water and natural gas (or crude oil) in the formation, forming an oil–water two-phase interface, a gas–water two-phase interface or an oil–gas–water three-phase interface, and these interfaces reach small pores and narrow throats in rock with the movement of the drilling fluid, generating a very large seepage resistance, forming water lock and hydrocarbon lock effects, and causing liquid damage. In the process of oil and gas production, the solid damage and liquid damage caused by drilling fluid intrusion into the reservoir significantly reduce the efficiency of oil and gas recovery.Combining the results of MEM displacement experiments, core flooding experiments, permeability measurements and SEM observations, it is shown that the main forms of damage of the low-porosity and low-permeability sandstone in the research area by drilling fluid include solid damage and liquid damage: (1) solid particles  in drilling fluid move to the small pores and narrow throats in the rock and then block them; the white oil and water in the drilling fluid mix with the stratum fluid, forming an oil–water two-phase interface, gas–water two-phase interface or oil–gas–water three-phase interface, causing water lock and hydrocarbon lock effects and leading to liquid damage. Therefore, from the perspective of low-porosity and low-permeability reservoir protection and efficient oil and gas development, it is necessary to adjust the particle size of solid components in the drilling fluid, in addition to optimizing the material and ratio of liquid components in the drilling fluid.For the low-porosity and low-permeability sandstone reservoir of the Enping Formation in the Baiyun Sag of the Pearl River Mouth Basin, MEM-based displacement experiments, core flooding experiments, permeability measurements, and SEM observations before and after damage by drilling fluid were carried out. The results show that the main forms of low-porosity and low-permeability sandstone damage by drilling fluid include solid damage and liquid damage; solid particles (especially BaSO


This study aimed to establish the best early gastric cancer lymph node metastasis (LNM) prediction model through machine learning (ML) to better guide clinical diagnosis and treatment decisions.We screened gastric cancer patients with T1a and T1b stages from 2010 to 2015 in the Surveillance, Epidemiology and End Results (SEER) database and collected the clinicopathological data of patients with early gastric cancer who were treated with surgery at the Second Affiliated Hospital of Nanchang University from January 2014 to December 2016. At the same time, we applied 7 ML algorithms—the generalized linear model (GLM), RPART, random forest (RF), gradient boosting machine (GBM), support vector machine (SVM), regularized dual averaging (RDA), and the neural network (NNET)—and combined them with patient pathological information to develop the best prediction model for early gastric cancer lymph node metastasis. Among the SEER set, 80% were randomly selected to train the models, while the remaining 20% were used for testing. The data from the Second Affiliated Hospital were considered as the external verification set. Finally, we used the AUROC, F1-score value, sensitivity, and specificity to evaluate the performance of the model.The tumour size, tumour grade, and depth of tumour invasion were independent risk factors for early gastric cancer LNM. Comprehensive comparison of the prediction model performance of the training set and test set showed that the RDA model had the best prediction performance . The AUROC of the external validation set was 0.73.Tumour size, tumour grade, and depth of tumour invasion were independent risk factors for early gastric cancer LNM. ML predicted LNM risk more accurately, and the RDA model had the best predictive performance and could better guide clinical diagnosis and treatment decisions. Gastric cancer ranks as the fifth most common malignant tumour and third in mortality worldwide , 2. EarlEGC treatment is being gradually replaced by more minimally invasive methods, such as endoscopic mucosectomy and endoscopic submucosal dissection , 5. CompPresently, studies have reported the risk factors for lymph node metastasis in EGC and have established predictive models. However, these results regarding certain risk factors for lymph node metastasis were inconsistent , 13. BecTherefore, this study used ML to compare the efficacy of different prediction models for LNM of EGC to identify an accurate prediction method and accurately guide the selection of clinical diagnosis and treatment plans.in situ; (2) Distant metastasis; (3) Incomplete tumour staging; (4) Incomplete information. The tumour site, grade, and histology were coded according to the International Classification of Diseases for Oncology, version 3. Tumour stage was coded according to the AJCC tumour–node–metastasis staging system, 7th edition  database of the National Cancer Institute, which covers basic information for ~28% of US cases. The collection of patient information did not require informed consent because this information was publicly available . From the database, we mainly collected relevant information, including general characteristics, clinical tumour characteristics, pathological characteristics, treatment methods, survival and prognosis. We also collected the clinical data of EGC patients who were treated with surgery from January 2014 to December 2016 at the Second Affiliated Hospital of Nanchang University. The inclusion criteria were as follows: (1) Patients undergoing surgical treatment; (2) A pathological diagnosis of early gastric cancer; (3) Complete survival information. The exclusion criteria were as follows: (1) Multiple tumours  edition . The detGastric cancer patients were diagnosed from 2010 to 2015. The ages were grouped as follows: <50 years, 50–60 years, 60–70 years, 70–80 years, and >80 years. Race was grouped as follows: White, Black, and other . Tumour size was divided as follows: <2 cm, 2–5 cm, >5 cm and NA. Tumour grade was divided as follows: Grade I, Grade II, Grade III, Grade IV, and NA. Tissue classification included signet ring cell carcinoma and non-signet ring cell carcinoma. The depth of tumour invasion included T1a and T1b. The location of the primary tumour was grouped as follows: cardia, fundus, gastric body, antrum, pylorus, lesser curvature, greater curvature, and overlapping/NOS.For descriptive statistics, chi-squared test or Fisher's exact probability method was used to compare categorical variables. Binary logistic regression was used to analyse the risk factors for lymph node metastasis of EGC. The results were represented by odds ratios (ORs) and 95% confidence intervals (CIs) using the caret package  and establishment of different prediction models. Missing values were detected using the mice package and filled with predictive mean matching. The database patients were randomly divided into a training set and a test set at a ratio of 8:2, and hospital patients were used as the external verification set. The training set was used for model development, and the test set was used for evaluation and verification. Considering that the proportion of patients with LNM was too low, we used the ROSE package to balance the training set. Since then, 7 types of ML algorithms have been established by the training set, including the generalized linear model (GLM), RPART, random forest (RF), gradient boosting machine (GBM), support vector machine (SVM), regularized dual averaging (RDA), and the neural network (NNET). During the training process, 10 cross-validations were performed for each model to maintain the stability of the models, and the best hyperparameters were selected using random search. In the test set, the F1-score value, AUROC, sensitivity and specificity of each model were used to comprehensively evaluate the model, compare the performance differences of different prediction models, and conduct difference testing. Finally, the independent external verification set was used to further validate the accuracy and generalization ability of the best prediction model .According to our inclusion and exclusion criteria, the SEER database involved 2,294 patients, including 1,839 cases in the training set and 458 cases in the test set. The LNM rate of the training set was 14.5%. The LNM rate of the test set was 14.4%.A total of 227 cases were identified in the external validation set, and the LNM rate was 51.1%. P < 0.05) . Tumour size, tumour grade and infiltration depth were independent risk factors for LNM in patients with EGC (P < 0.05)  . We condwith EGC . The ext < 0.05) .The parameters of the training set were adjusted to balance the model and avoid overfitting the model. After balancing the parameters of the training set, we found that the GBM model had the best predictive ability, with AUCROC = 0.825 . The AUCEndoscopic mucosal resection and endoscopic submucosal dissection have been widely used to treat EGC and have been established as the standard method to treat early upper gastrointestinal tumours in Japan . AccordiIn recent years, studies have shown that tumour size, tumour grade, depth of invasion, nerve invasion, and ulcers are risk factors for LNM in patients with EGC, and a prediction model has been established , 28. HowIn the present study, we used univariate and binary logistic regression analyses to show that tumour size, depth of invasion, and tumour grade were independent risk factors for LNM in EGC, a finding that was consistent with most research reports . We founOur model contained three important factors: tumour size, tumour grade and depth of tumour invasion. In previous reports, tumour diameter affected lymph node metastasis in early gastric cancer, and the larger was the tumour diameter, the higher was the risk of LNM in patients . This phThe present study used seven predictive models based on machine learning and the SEER database to compare the performance of different predictive models to obtain the model with the best predictive performance plus clinical data as external verification. We comprehensively verified the calculation methods used in most studies in recent years to establish the LNM prediction model for EGC and obtained the best prediction model. To our best knowledge, this report is the first to use ML to explore the establishment of the best LNM prediction model for EGC.However, this study has several limitations. First, because of the scant clinicopathological information in the database, fewer influencing factors were identified in the model. Second, the number of samples finally included in this study was small, leading to certain limitations in machine learning that are more suitable for large sample data. The small sample size is a relatively common problem, and how to solve this problem is the focus of future research, for which we will continue to work. Finally, although we corrected the sample imbalance problem in the SEER dataset as much as possible, this problem may still interfere with the results and affect the generalization ability of the model.In summary, we compared the performance of seven prediction models using ML algorithms, among which the RDA model had the best performance. The model included three important predictors—tumour size, tumour grade, and depth of tumour invasion—and the external validation set also showed that the model had accurate predictive capabilities and some applicability. The study findings can better help doctors make clinical diagnoses and allow patients to benefit from better treatment.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.HT and ZZ conceived and designed this study. HY and HL collected and assembled the data. HT and ZN analysed and interpreted the data. HT, ZN, and HL drafted the manuscript. CH and JL prepared the figures and tables. All the authors read and approved the final manuscript.This report was supported by the National Natural Science Foundation of China (Grant Numbers: 81860433 and 82103645), the Natural Science Youth Foundation of Jiangxi Province (Grant Number: 20192BAB215036), Jiangxi Province Natural Science Key R&D Project-General Project (Grant Number: 20202BBG73024) and Training Plan for Academic and Technical Young Leaders of Major Disciplines in Jiangxi Province (Grant Number: 20204BCJ23021).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Urokinase-type plasminogen activator receptor (uPAR) is an attractive target for the treatment of cancer, because it is expressed at low levels in healthy tissues but at high levels in malignant tumours. uPAR is closely related to the invasion and metastasis of malignant tumours, plays important roles in the degradation of extracellular matrix (ECM), tumour angiogenesis, cell proliferation and apoptosis, and is associated with the multidrug resistance (MDR) of tumour cells, which has important guiding significance for the judgement of tumor malignancy and prognosis. Several uPAR-targeted antitumour therapeutic agents have been developed to suppress tumour growth, metastatic processes and drug resistance. Here, we review the recent advances in the development of uPAR-targeted antitumor therapeutic strategies, including nanoplatforms carrying therapeutic agents, photodynamic therapy (PDT)/photothermal therapy (PTT) platforms, oncolytic virotherapy, gene therapy technologies, monoclonal antibody therapy and tumour immunotherapy, to promote the translation of these therapeutic agents to clinical applications. Urokinase-type plasminogen activator receptor (uPAR), also known as CD87, is encoded by the PLAUR gene and belongs to the lymphatic antigen-6 superfamily , 2. uPARClassically, the function of uPAR is to act as a receptor for the zymogen form of uPA (pro-uPA) and trigger a cascade of proteolytic events that leads to the degradation of ECM , 16. Onc88SRSRY92 sequence, initiating both angiogenic and inflammatory processes -uPA19–31 -uPA21–30) and WX-360-Nle  [2) [2 (pERERY-NH2) [2) [2) [2]  [Pseudomonas exotoxin A (PE38)] )  block the;H29C]) , α325  , p25 (AE9C]) ) ) [2)  and poly9C])   block thA1-48Ig) , and humA1-48Ig)  also inhA1-48Ig) , IPR-803A1-48Ig) , IPR-301A1-48Ig) , IPR-357A1-48Ig) , 7 126]19,31uPA1A1-48Ig) , MS#479 A1-48Ig) , CompounA1-48Ig) , and docA1-48Ig)  inhibit A1-48Ig) , 132, DTA1-48Ig) , DTAT13 A1-48Ig) , 135, eBA1-48Ig) –141, ATFA1-48Ig) , 143, PAd PAI-2] , DTU2GMCd PAI-2] , ATF-PE3 (PE38)]  exert anHowever, although research has been conducted for more than 30 years, none of these treatments have advanced into clinical application. The pleiotropic nature of uPAR interactions and function, uPAR structural flexibility, species specificity of the uPA-uPAR interaction, limitations of tumour models, the characteristic that uPAR expression is increased on tumour cells and tumour-associated stromal cells, and the baseline expression of uPAR in the glomeruli of normal kidneys that may result in potential “on-target off-tumour” toxicity are all the main hurdles to the development of uPAR inhibitors , 147–152In recent years, with the interdisciplinary integration of cell biology and materials science, many innovative tumour-targeted therapeutic technologies targeting uPAR have emerged, providing new development directions for precise and efficient tumour therapy. uPAR-targeted nanoplatforms carrying therapeutic agents have great potential in enhancing active tumour targeting, improving delivery efficiency, reducing drug toxicity, increasing the hydrophilicity of hydrophobic drugs, achieving tumour diagnosis and treatment integration, and in multimodal synergistic antitumor applications. uPAR-targeted PDT/PTT platforms may be regarded as promising cancer therapeutic strategies due to their unique advantages such as minor trauma, improved selectivity and reduced side effects. uPAR-targeting oncolytic measles virus (MV-uPA) is an innovative biological strategy associated with potent antitumour effects. uPAR-targeted clustered regularly interspaced short palindromic (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) gene-editing technology may provide new therapeutic trearments for aggressive cancers. uPAR-targeted monoclonal antibody therapy may provide new breakthroughs for the development of anticancer therapy. uPAR-targeted chimeric antigen receptor (CAR) T-cell immunotherapy and antibody-recruiting molecules (ARMs) have the ability to target uPAR-expressing cancers for immune-mediated cell death. Therefore, this review focuses on some new applications of uPAR in the six fields described above Fig. .Fig. 2uP24-IONP-Dox) that showed a stronger inhibitory effect than non/single-targeted IONPs on a human breast cancer patient-derived xenograft model and markedly inhibited Wnt/β-catenin signalling and the cancer stem-like phenotype by decreasing the levels of the Wnt ligand, CD44 and uPAR  that simultaneously targeted the LHRH receptor (LHRH-R) and uPAR and exhibited a significant MRI contrast in PCa cells. Importantly, the IONPs carrying PTX (LHRH-AE105-IONPs-PTX) showed two times higher cell cytotoxicity than IONPs targeting a single molecule . Park et ability . Belfioriposomes . Wang etiposomes .24 peptide (ATF24-PEG-Lipo-β-E); these liposomes showed better targeting efficiency and higher cytotoxicity than nondecorated liposomes and exerted a synergistic effect on inhibiting the growth of KU-19-19 bladder cancer with Cis [b-PEG-NPs  that simultaneously antagonized miR-21-induced inhibition of apoptosis and miR-10b-induced metastasis to achieve TNBC therapy [Our research group also developed β-elemene-loaded liposomes modified with ATFwith Cis . Devulap therapy . TherefoAmong anticancer treatments, PDT and PTT are widely regarded as promising cancer therapeutic strategies due to their unique advantages such as minor trauma, improved selectivity, remarkable spatial/temporal resolution and reduced side effects . PDT depLi et al. engineered a U11 peptide modified gold nanocluster platform carrying the cathepsin E (CTSE)-sensitive PDT prodrug/imaging agent CRQAGFSL-5-aminolevulinic acid (5-ALA)/-cyanine 5.5 (Cy5.5) (AuS-U11), which showed excellent efficacy with endomicroscopy-guided PTT/PDT through the combination of active tumour targeting and enzyme-triggered release of 5-ALA and Cy5.5 in a PANC1-CSTE orthotopic tumour model  Fig. 4)4). Li et2-Avastin@PEI/AE105), and observed a prominent photodynamic killing effect and anti-angiogenic activity by targeting uPAR and smart light-controlled drug release in a HeLa tumour model [2−xS NPs and the sonosensitizer Rose Bengal (RB)  for OSCC-targeting and synergetic PTT/sonodynamic therapy (SDT) [125I) interstitial brachy-therapy (IBT-125-I). Furthermore, uIGNs prevented pancreatic tumour metastasis, as evidenced by real-time monitoring of metastatic tumours (less than 2 mm) using CT and NIR imaging [In addition, Yu et al. developed uPAR-targeted polyetherimide-AE105 peptide (P-AE105) conjugated gold nanostars (GNS) carrying an iridium (Ir) complex that exerted enhanced anti-TNBC effects through the ROS-induced p53 apoptotic pathway, and showed excellent PT/photoacoustic (PA)/X-ray computed tomography (CT) imaging properties . Hu et aur model . Zuo et py (SDT) . Hu et a imaging . The uPAOncolytic virotherapy is an emerging platform that represents a novel frontier for cancer treatment. Redirecting viral tropism to specific tumour targets is a promising strategy in the field of oncolytic viruses, which may increase safety and inhibit distant metastases of tumours . RecentlMV-h-uPA or MV-m-uPA, an Edmonston vaccine strain of oncolytic MVs constructed by the ATF of human or murine uPA and mutant MV-H glycoprotein, was able to replicate, and induce cytotoxicity in a species-specific manner. In vivo, MV-h-uPA successfully inhibited tumour growth (inhibition rate of 76% at Day 39), prolonged survival  and reduced metastatic progression in an MDA-MB-231 tumour model . In addiTumour stroma-selective targeting by uPAR retargeted MVs is also associated with enhanced antitumour effects. For example, MV-m-uPA inhibits breast cancer cell proliferation by selectively targeting fibroblasts, and delays tumour progression and prolongs survival in mice bearing a human MDA-MB-231 tumour model . MV-CD46The development of efficient and reliable methods to generate precise, targeted changes in the genome of living cells is a long-standing goal for biomedical researchers. In uPAR-targeted gene therapy technologies, adenovirus-mediated antisense uPAR therapy first emerged as an effective tool for cancer treatment. For example, an adenoviral vector containing the uPAR antisense sequence (Ad-uPAR), an adenovirus containing uPAR antisense and p16 sense expression cassettes (Ad-uPAR/p16), an adenovirus expressing antisense uPAR and uPA sequences (Ad-uPAR-uPA), an adenovirus vector containing antisense uPAR and cathepsin B sequences (Ad-uPAR-Cath B), and an adenovirus expressing antisense uPAR and MMP-9 sequences (Ad-uPAR-MMP-9) were all successfully constructed and inhibited tumour growth and metastasis in gliomas and lung cancer models –193.Subsequently, RNA interference (RNAi) technologies, including siRNAs and short hairpin RNAs (shRNAs) targeting uPAR , were developed to prevent tumour progression. Compared with siRNAs/shRNAs targeting uPAR, siRNAs targeting uPAR and uPA or siRNAs targeting uPAR and cathepsin B exerted a better antitumor effect by inhibiting tumour cell proliferation, migration and invasion and angiogenesis and promoting tumour cell apoptosis , 194–198Streptococcus pyogenes has generated a considerable level of excitement. The RNA-guided CRISPR/Cas9 system is a powerful RNA-guided genome editing tool that utilizes a guide RNA (gRNA) to cleave the desired sequence in the genome and remove existing genes or add new genes. Due to the advantages of being fast, precise, and highly efficient, targeting uPAR with CRISPR/Cas9 technology has been successfully applied in a variety of malignant tumours to enhance the treatment effect [V200 cells. Furthermore, uPAR knockout inhibited MDR to 5-FU, Cis, DTX, and Dox [Recently, a new tool based on bacterial Cas9 from t effect . Targetit effect . Wang et and Dox . Biagion and Dox .Kd = 10 × 10–9; IgG Kd = 2 × 10–12) by forming a stable complex with uPAR and disrupting the uPA/uPAR interaction. LeBeau et al. found that 30 mg/kg 2G10 IgG prevents the growth of TNBC, and 177Lu-labelled 2G10 completely eliminates tumours in orthotopic breast cancer models [A variety of monoclonal antibodies targeting uPAR have been developed, and exert antitumor effects by blocking the uPA/uPAR interaction or inhibiting the interactions between uPAR and integrin, EGFR, FPR, and Vn. The 2G10 antibody binds tightly to uPAR (Fab r models . Harel er models . Durisetr models .Kd ≈ 1 nmol/L), and the binding of ATN-658 to uPAR is not affected by the binding of uPA to uPAR. ATN-658 mainly inhibits the activation of downstream signalling pathways by inhibiting the uPAR/integrin interaction. ATN-658 inhibits the growth and liver metastasis of pancreatic cancer in situ and completely inhibits retroperitoneal infiltration; the antitumour effect is more obvious when this antibody is combined with Gem [Kd ≈ 1 nmol/L) and does not block the uPA/uPAR interaction [ATN-658 is a humanized monoclonal antibody that binds to the D2D3 region of uPAR with high affinity , a hinge region that provides flexibility to the scFv, a transmembrane domain, and intracellular signalling/activation domain(s) , 216. CAarcinoma .ARMs are antibody-binding molecules that exert antitumour effects by delivering endogenous antibodies to tumour tissues and destroying tumour cells via the activated immune system . Jakobsc64Cu-DOTA-AE105 are being conducted to diagnose aggressive cancers and determine cancer aggressiveness. These two agents are expected to be administered to patients in the future.uPAR is an attractive target for the treatment of cancer because it appears to be expressed at high levels in tumours but low levels in normal tissue. uPAR also plays a comprehensive role in the development of tumours and is closely related to tumour proliferation and apoptosis, invasion and metastasis, prognosis, and tumour MDR, providing a basis for the development of multiple therapeutics agents targeting this protein. This review has summarized multiple new applications of uPAR as a target in nanoplatforms carrying therapeutic agents, PTT/PDT platforms, oncolytic virotherapy, gene therapy technologies, monoclonal antibody therapy and tumour immunotherapy in recent years. The development of therapeutic strategies that target tumours via uPAR recognition has proven its potential in animal models, but no uPAR-targeted therapeutic agents have been developed or evaluated in cancer clinical trials to date. Recently, ATN-658 has been humanized (huATN-658) and is awaiting clinical translation; and phase I clinical trials with Among uPAR-targeted therapeutic strategies, uPAR-targeted nanoplatforms also have great potential to achieve translation from laboratory findings to the clinic. Based on the high expression of uPAR on the surface of a variety of tumour cells, uPA/ATF/AE105/AE147/PAI-2/U11 modified nanoplatforms provide the possibility of reducing or overcoming the therapeutic limitations of conventional chemotherapy or PTT/PDT through targeted delivery to tumour cells without obvious toxicity to healthy tissue. Moreover, recent studies have a key role for the tumour microenvironment in promoting tumour proliferation, invasion and metastasis . uPAR ex


Background: Protamine can decrease the risk of hemorrhage during carotid recanalization. However, it may cause severe side effects. There is no consensus on the safety and efficacy of protamine during surgery. Thus, we conduct a comprehensive review and meta-analysis to compare the differences between the protamine and the no-protamine group.Method: We systematically obtained literature from Medline, Google Scholar, Cochrane Library, and PubMed electronic databases. All four databases were scanned from 1937 when protamine was first adopted as a heparin antagonist until February 2021. The reference lists of identified studies were manually checked to determine other eligible studies that qualify. The articles were included in this meta-analysis as long as they met the criteria of PICOS; conference or commentary articles, letters, case report or series, and animal observation were excluded from this study. The Newcastle-Ottawa Quality Assessment Scale and Cochrane Collaboration’s tool are used to assess the risk of bias of each included observational study and RCT, respectively. Stata version 12.0 statistical software  was adopted as statistical software. When I2 < 50%, we consider that the data have no obvious heterogeneity, and we conduct a meta-analysis using the fixed-effect model. Otherwise, the random-effect model was performed.Result: A total of 11 studies, consisting of 94,618 participants, are included in this study. Our analysis found that the rate of wound hematoma had a significant difference among protamine and no-protamine patients . Furthermore, the incidence of hematoma requiring re-operation (0.7%) was significantly lower than that of patients without protamine (1.8%). However, there was no significant difference in the incidence of stroke, wound hematoma with hypertension, transient ischemic attacks (TIA), myocardial infarction (MI), and death.Conclusion: Among included participants undergoing recanalization, the use of protamine is effective in reducing hematoma without increasing the risk of having other complications. Besides, more evidence-based performance is needed to supplement this opinion due to inherent limitations. Ischemic stroke accounts for the mortality of approximately more than ten million lives per year all over the world , and it We systematically obtained literature from Medline, Google Scholar, Cochrane Library, and PubMed electronic databases. We utilized controlled vocabulary to build the search terms such as the National Library of Medicine in this study. All four databases were scanned from 1937 when protamine was first adopted as a heparin antagonist until February 2021 for the keywords of protamine, carotid endarterectomy, and carotid artery stenosis in combination with Boolean logic . The speStudies are considered eligible if they fulfilled the predefined inclusion criteria: (1) population: participant with carotid stenosis; (2) intervention: all patients strictly undergoing carotid recanalization; (3) comparison intervention: use of protamine to no-protamine group; (4) outcome measures: one or more of the following outcomes were reported: complications of wound hematoma, hematoma requiring re-operation, wound hematoma with hypertension, transient ischemic attacks (TIA), myocardial infarction (MI), stroke, and death; and (5) official published prospective and retrospective studies in English.The exclusion criteria are listed as follows: (1) conference or commentary articles and letters, (2) atypical patients and outcome data, (3) case report and case series, and (4) animal observation.Data were extracted by using a form prepared in advance and from the eligible researchers. Each relevant study was independently captured by two authors for the following essential details: the first author of the study, publication year, type of study, quality assessment, endpoints, and study characteristics including the number of populations in total, mean age, and gender ratio, among others. All disagreements were discussed until a final decision is reached. The primary study endpoint measurements are relevant to hemorrhagic damage including wound hematoma, hematoma requiring re-operation, and hematoma with hypertension, and secondary endpoints were the composite of ischemic injuries including stroke, TIA, and MI. Herein, in primary endpoints, all hematoma was defined as wound hematoma. In secondary endpoints, stroke was defined as 1 or more of the following: (1) an increase in the National Institute of Health stroke scale (NIHSS) score of >4 points from pre-stroke score; (2) an increase in the MRS score of >2 points from the pre-stroke score; or (3) stroke leading to a modified Rankin scale (MRS) score of 5 or more.I-square (I2), which indicated the percentage of the observed between-study viability. I2 over 25% and less than 75% was considered as moderately heterogeneous or significant heterogeneity. If I2 was under 50%, the endpoint item was considered to be homogeneous, and we ran a meta-analysis by using a fixed-effect model according to the Cochrane Handbook for Systematic Reviews of Interventions. Otherwise, the random-effect model was performed.Stata version 12.0 statistical software  was adopted as statistical software. The risk differences (RDs) or odds ratios (ORs) with the corresponding 95% confidence intervals (95% CIs) were used as measures of the treatment effect of protamine. We accessed the heterogeneity with the Higgins We used the guidance from the Grading of Recommendations Assessment, Development and Evaluation (GRADE) working group to assess the quality of evidence for the primary outcome . The GRAThe screening process is displayed in n = 92,447) and 2 RCTs  with an average age of 76 years , are listed in d 2 RCTs  involvind 2 RCTs  ignored Methodological quality and risk of bias in the included observational studies are assessed by two reviewers independently by using the Newcastle-Ottawa Quality Assessment Scale , which cThere were nearly ten thousand participants, 92% of whom have been undergoing CEA and 8% had been treated with CAS. The detailed results and GRADE assessment of outcomes are shown in p = 0.015, I2 = 77.2%, p = 0.004). A sensitivity analysis was performed to reveal that the heterogeneity was decreased by deleting the study conducted by Dellagrammaticas et al. .We include four independent pieces of research of CEA with 4,706 patients . Among these studies, the incidence of wound hematoma in the protamine group is 3.8% , which is smaller than the no-protamine group . This comparison fully indicates that the group of protamine is associated with a significantly lower incidence of wound hematoma than participants treated with non-protamine . A specific OR of 0.475  and that there is also a significant difference in the subgroup of CEA between the two groups .Analysis of risk of hematoma requiring re-operation between the protamine and no-protamine groups is provided in eight studies. The proportion estimated in protamine and no-protamine groups is 0.7%  versus 1.8% . However, a significant heterogeneity was observed, and a random effects model was used  report the wound hematoma with hypertension. This analysis does not find a significant difference between the two groups . Therefore, we also analyze the subgroup of CEA and reveal that there is no difference among the protamine and no-protamine groups . Moreover, we performed a sensitivity analysis, but no significant difference was revealed in the changes of heterogeneity.Three articles . We think that patients treated with protamine did not have a lower rate of stroke than those treated with no protamine . Perioperative TIA occurred in the protamine  and no-protamine group . No evidence of significant heterogeneity is revealed in these studies . The overall analysis does not prove an apparent difference in TIA rates between protamine and no-protamine groups . Similarly, we find that there is also no difference among protamine and no-protamine patients in the subgroup of CEA  is considered significantly meaningful for artery stenosis. Even though it reduces the risk of stroke, it carries a risk of hematoma . Protamip = 0.005) was obtained, suggesting that the rates of hematoma requiring re-operation was lower than that in patients without protamine. However, we thought the high rates of wound hematoma may be related to a higher risk of hypertension, but there is no difference presented in the two groups . We cannot distinguish whether this result is directly related to protamine use. As far as we know, protamine is a multi-cation strong alkaline polypeptide, which can combine with the glucosaminoglycan of heparin to form a stable complex and inhibit the activity of antithrombin, thus counteracting the anticoagulant effect of heparin and playing the effect of hemostasis. Protamine-induced circulatory changes have been demonstrated by Jastrzebski et al. , the same result as TIA. Even though protamine did not affect the incidence of stroke and TIA by multivariate analysis, the risk of MI has also been studied extensively. In light of the 0.7% rate of MI in our study with protamine showing no significant difference . Furthermore, we should note that the protamine did not change the rate of death among these two groups . Meanwhile, other studies related to this topic did not find an association between protamine and stroke, TIA, MI, and death. We suspect that there may be a certain stimulation factor of protamine that causes a reduction in bleeding.Cerebral recanalization therapy, either intravenous thrombolysis or mechanical thrombectomy, improves the outcomes of patients with artery stenosis , which eOur research has several limitations. The dosage of protamine was not standardized, which may confound the outcomes. Besides the primary inclusion and exclusion criteria, the characteristics of the participants were a little different from each other, potentially causing bias. The number of recent and high-quality studies was too small. Additionally, some heterogeneity was found among included trials due to the different study protocols, patient characteristics, and definitions of clinical endpoints. Moreover, the current study is not registered and there may be a slight deviation, but we strictly followed the procedures of systematic evaluation. Finally, the exact sequence of disease for the included patients cannot be known exactly and protamine might have been administered after a complication occurred rather than before, which might have affected our results.This study has crucial implications. The current meta-analysis demonstrates that surgeons should consider routinely using protamine during carotid recanalization especially for CEA, due to the lower incidence of wound hematoma and hematoma requiring re-operation with its use. These findings, however, have inherent limitations, such as obvious heterogeneity and data from retrospective reviews; therefore, they cannot be regarded robust enough to provide a firm recommendation in clinical practice. With regard to the carotid artery stenting, there were fewer studies examining the effect of protamine; herein, further research is necessary to illustrate whether consistent results exist across all types of carotid revascularization.Protamine, which was primarily isolated from salmon fish sperm, is a small, arginine-rich, positively charged protein with similarities to histones in that it has a role in stabilizing DNA in the sperm head. Despite it being adopted in a variety of vascular and cardiac procedures to reserve systemic heparin anticoagulation, especially for carotid recanalization, this inevitably leads to bleeding and then further causes major or minor strokes, myocardial infarction, or death. Herein, a comprehensive review and meta-analysis are necessary to illustrate the efficiency of protamine for carotid recanalization. This study demonstrated that protamine was effective in reducing hematoma without increasing the risk of having other complications.


This study reports on the E. coli and 344 S. saprophyticus isolates were collected between 2019 and 2020 from 92 medical centers located in 25 countries. Most isolates (68%) tested were cultured from urine specimens collected from patients seen in ambulatory, emergency, family practice, and outpatient medical services. Bacterial identifications were confirmed by MALDI-TOF. Isolates were tested for susceptibility by CLSI methods at a central laboratory (JMI Laboratories). MIC results for oral antibiotics licensed for the treatment of uUTI and drug-resistant subsets were interpreted per CLSI guidelines. A total of 3,562 50/90, 2/2 mg/L) displayed good activity against 3,562 E. coli isolates, with 98.0% of all observed gepotidacin MICs ≤4 mg/L (Table). Susceptibility (S) rates for the other oral agents tested against these isolates were: amoxicillin-clavulanate (79.6% S), ampicillin (45.6% S), ciprofloxacin (72.5%S), fosfomycin (99.0% S), mecillinam (94.1%S), nitrofurantoin (97.3% S), and trimethoprim-sulfamethoxazole (68.2% S). When tested against the drug-resistant subsets, gepotidacin maintained similar MIC50/90 values (2/4 mg/L), except against isolates resistant to fosfomycin (2/8 mg/L). Against S. saprophyticus isolates, gepotidacin  inhibited all isolates at ≤0.25 mg/L. Most oral agents showed S results of >97% against S. saprophyticus isolates, except for penicillin (3.5%S).Gepotidacin  (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Deborah Butler, n/a, GlaxoSmithKline, LLC (Employee) Nicole Scangarella-Oman, MS, GlaxoSmithKline, LLC (Employee) Lindsey Paustian, BS (ASCP), GlaxoSmithKline, LLC (Research Grant or Support) Jennifer M. Streit, BS, GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)


The optimal treatment and management of patients with brain cancer depend on the molecular characteristics of their tumour. Since the tumour changes with time, it is, therefore, essential to characterise the tumour of each patient at the exact time of selecting the most suitable therapeutic strategy. However, obtaining a tumour biopsy for its characterisation is a risky and invasive procedure and, sometimes, not even feasible, leading to a lack of information about the tumour. These challenges can be overcome by using a liquid biopsy of cerebrospinal fluid. Brain cancer cells release DNA into the cerebrospinal fluid, and the analysis of the cell-free circulating tumour DNA can reveal the genetic profile of brain cancer in a relatively noninvasive manner. In this review, we revise the recent results in this field that show how circulating tumour DNA in cerebrospinal fluid can provide diagnostic and prognostic information, identify potential therapeutic targets, monitor the tumour response or resistance to treatment, and help to identify tumour relapse.The correct characterisation of central nervous system (CNS) malignancies is crucial for accurate diagnosis and prognosis and also the identification of actionable genomic alterations that can guide the therapeutic strategy. Surgical biopsies are performed to characterise the tumour; however, these procedures are invasive and are not always feasible for all patients. Moreover, they only provide a static snapshot and can miss tumour heterogeneity. Currently, monitoring of CNS cancer is performed by conventional imaging techniques and, in some cases, cytology analysis of the cerebrospinal fluid (CSF); however, these techniques have limited sensitivity. To overcome these limitations, a liquid biopsy of the CSF can be used to obtain information about the tumour in a less invasive manner. The CSF is a source of cell-free circulating tumour DNA (ctDNA), and the analysis of this biomarker can characterise and monitor brain cancer. Recent studies have shown that ctDNA is more abundant in the CSF than plasma for CNS malignancies and that it can be sequenced to reveal tumour heterogeneity and provide diagnostic and prognostic information. Furthermore, analysis of longitudinal samples can aid patient monitoring by detecting residual disease or even tracking tumour evolution at relapse and, therefore, tailoring the therapeutic strategy. In this review, we provide an overview of the potential clinical applications of the analysis of CSF ctDNA and the challenges that need to be overcome in order to translate research findings into a tool for clinical practice. Central nervous system (CNS) malignancies affect both children and adults worldwide and are responsible for substantial morbidity and mortality. An epidemiological study of CNS cancer between 1990 and 2016 revealed that the age-standardised incidence rate has increased by 17.3% globally, with 330,000 incident cases and 227,000 deaths globally in 2016 [CNS cancer consists of primary tumours and intracranial metastases. Most CNS tumours (>90%) occur in the brain, with the remaining located in the meninges, spinal cord and nerves. Depending on the anatomical region and the tumour type, the neurological signs and symptoms will vary and may include headaches, seizures, loss of vision, paralysis, speech disturbance, and motor deficits .CNS tumours are diagnosed using neuroimaging techniques such as magnetic resonance imaging (MRI) or computed tomography; however, to obtain pathological information and molecular diagnosis, tumour biopsies are required. The treatment strategy for primary CNS tumours consists of either obtaining a biopsy or performing a surgical resection, combined, when appropriate, with postoperative radiotherapy and chemotherapy .CNS tumour prognosis is diverse since there are distinct entities with different histopathological characteristics and molecular profiles. Therefore, characterising the tumour specimen is essential for accurate diagnosis and prognosis, as well as to identify potential therapeutic targets. To improve disease control, primary brain tumours or single nodule brain metastases are resected. However, obtaining tumour biopsies is not always possible due to their location, particularly when CNS tumours occur in vital regions such as the basal ganglia or the brain stem. In addition, patients with disseminated disease may not be eligible for such procedures ,5,6.In cases where tumour resection or obtaining a biopsy is possible, the sample obtained may not be representative of tumour heterogeneity ,8,9, andde novo resistant alterations [Tumours evolve over time, particularly under the selective pressure of therapy, which can result in the expansion of pre-existing resistant clones or the acquisition of erations . Thus, terations ,12. The For these reasons, there is an urgent need to develop less invasive methods to identify and validate tumour biomarkers that provide real-time information to aid in diagnosing and monitoring CNS malignancies. Overall, this will help to adjust the therapeutic strategy and guide treatment decisions based on the current tumour profile and its burden.An alternative to a tumour biopsy is a liquid biopsy . Liquid In this review, we will discuss the potential applications of circulating tumour DNA (ctDNA) in CSF as a biomarker for CNS malignancies, the challenges that we need to overcome, and future perspectives for its implementation in the clinical setting.Cells release DNA that then circulate in bodily fluids. The fraction of cell-free DNA (cfDNA) that is shed by cancer cells, presumably undergoing apoptosis or necrosis, is known as ctDNA and carries genomic alterations that can be detected using PCR-based or next-generation sequencing (NGS)-based methods ,15,16,17Increased concentrations of cfDNA have been detected in pathological conditions like trauma, infection and cancer, or even other physiological conditions like exercise .For patients with an extracranial disease, plasma ctDNA has been detected across different cancer types. However, blood may not be a suitable source from patients with CNS malignancies since ctDNA levels were infrequently detected in plasma ,20. ctDNThe proportion of ctDNA in the blood is small and varies depending on tumour characteristics, including type, grade and burden ,19. In cCSF is a clear bodily fluid secreted by the choroid plexus that is present in the subarachnoid space of the brain, the spinal cord and the central canal .CSF is in direct contact with the brain parenchyma, and several studies have shown that CSF is a reliable source of cell-free ctDNA, providing advantages over plasma or serum for the analysis of CNS tumours ,21.Several studies have reported the ability to detect ctDNA in CSF of patients with CNS malignancies. Gene mutations and molecular alterations have been detected in the CSF DNA of CNS cancer patients ,28,29,30CSF samples can be accessed through a lumbar puncture or obtained from the ventricles under certain circumstances. Patients with posterior fossa tumours tend to present with hydrocephalus, a condition in which the CSF accumulates within the cerebral ventricles and/or subarachnoid spaces ,38,39. AAs part of the CNS staging criteria for certain brain tumours, a diagnostic lumbar puncture is routinely performed as standard of care for CSF cytology evaluation of CNS lymphoma, CNS metastasis, and medulloblastoma ,44,45,46CNS tumours are a heterogeneous group of malignancies . In mostThe monitoring of CNS malignancies is currently performed by imaging techniques; however, these are not sensitive for microscopic disease . A complThe distinct clinical applications of the analysis of CSF ctDNA are discussed below for patients with distinct types of CNS cancer.IDH1, IDH2, ATRX, TP53, TERT, H3F3A and HIST1H3B in CSF ctDNA facilitates the molecular diagnosis of diffuse gliomas and provides prognostic information in a relatively noninvasive manner [TERT promoter mutations have been detected in the CSF ctDNA of GBM patients, and shorter OS of patients with high variant allele frequency (VAF) has been observed. The results from this pilot study suggested that VAF levels of the TERT promoter mutation could be a predictor of poor survival [Among diffuse gliomas, glioblastoma (GBM) is the most common malignant brain tumour in adults, with a 2-year survival of 18% and 5-year overall survival (OS) of 4% . Providie manner . In addisurvival . In moresurvival ,23. Moresurvival ,23.H3F3A or HIST1H3B/C; it is usually located in the brain stem, thalamus and spinal cord [Diffuse midline glioma (DMG) is a tumour entity characterised by a K27M mutation in either nal cord . Within nal cord . The ananal cord ,54,55.H3F3A/HIST1H3B mutations was correlated with poor OS while the IDH1 mutation predicted better OS [The molecular characterisation and understanding of DIPG biology have been improved from specimens obtained from rare diagnostic biopsies and postmortem tissue donations ,57,58,59etter OS . MoreoveMGMT promoter methylation status. MGMT promoter methylation causes the loss of MGMT expression, and since it is involved in DNA repair by reversing DNA alkylation, MGMT promoter methylation renders cells more susceptible to temozolomide and is associated with longer survival [MGMT promoter methylation was detected using methylation-specific PCR from genomic DNA extracted from the CSF of glioma patients, with higher sensitivity than from serum [The number of actionable genomic alterations for patients with primary brain tumours is limited. The most relevant biomarker for glioma is survival ,61,62,63om serum .EGFR). EGFR amplification and EGFRvIII mutation have been detected in RNA within extracellular vesicles circulating in CSF [A potential biomarker for GBM is epidermal growth factor receptor . For EGFR mutations, the detection rate was 57.1% (12/21) from CSF ctDNA, which, interestingly, was higher for patients with leptomeningeal  compared with brain parenchymal  metastases [A study of 21 patients with brain metastasis from NSCLC compared the NGS results obtained from different samples to reveal the mutation pattern of driver genes for each patient. Mutations were detected in 95.2%, 66.7% and 39% of patients from CSF ctDNA, plasma ctDNA, and plasma circulating tumour cells, respectively. The most mutated gene was tastases .TP53 and PIK3CA and amplification in ERBB2 and cMYC. Following treatment with T-DM1, extracranial disease control was achieved, and marker levels in plasma decreased. However, the levels increased in CSF ctDNA, consistent with poor treatment benefit to the CNS [The analysis of CSF ctDNA can also contribute to monitoring response to treatment. Metastasis in the CNS developed in a patient with HER2+ breast cancer. Analysis of baseline CSF ctDNA revealed mutations in  the CNS .Altogether, these results indicate that CSF is a more suitable fluid than plasma to reveal the mutational profile of CNS metastases and can aid in diagnosis, tailored treatment selection and monitoring.Malignant B-cells can infiltrate the CNS and are associated with poor prognosis, particularly at relapse . PrimaryctDNA has only been detected in the plasma of a minority of patients with restricted CNS lymphoma ,104. In MYD88 L265P mutation strongly suggests the diagnosis of primary CNS lymphoma, and this mutation has been detected in the CSF ctDNA of patients with CNS lymphoma [Diagnosis and monitoring of CNS lymphoma are challenging, given the difficulties of tumour biopsies and the lack of sensitivity of CSF standard analysis (cytology and flow cytometry) and neuroimaging. The detection of the lymphoma ,108,109,An NGS-based analysis of the CSF cfDNA of 8 patients with CNS lymphoma, at recurrence, detected tumour-derived genetic alterations and showed that the clearance of ctDNA from CSF was associated with sustained tumour responses .The comparison of CSF ctDNA with plasma ctDNA and CSF standard analysis (cytology and flow cytometry) revealed that the analysis of CSF ctDNA better detected CNS disease in patients with B-cell lymphoma . MoreoveCNS tumours are the leading cause of cancer-related mortality in children and adolescents due to the aggressiveness of certain subtypes, including medulloblastoma and high-grade gliomas such as DMG ,111.Medulloblastoma (MB), an embryonal tumour of the CNS, is the most aggressive brain tumour in childhood that can also occur in adults, although this is less common . MB is aPTCH1, TP53), CNVs (MYCN and GLI2 amplification) and arm-level chromosomal aberrations (chromosome 17p loss), providing diagnostic and prognostic information [The study of paediatric patients with MB showed that ctDNA was more abundant in CSF  than plasma (1/13 patients) for patients with negative CSF cytology results. Moreover, exome sequencing of CSF ctDNA recapitulated the tumour mutational burden and the genomic alterations, including MB common mutations , prior knowledge of the tumour genetic profile is required, and new mutations (not present in the primary sample) can appear in the relapse setting. In contrast, whole-exome sequencing (WES), shallow whole-genome sequencing (WGS) or specific gene panels might provide more information to aid with tumour characterisation and monitor residual disease. However, it is important to consider sensitivity, turnaround time and cost-effectiveness.In addition, imaging techniques may sometimes reflect either inflammatory processes from treatment or neoplastic progression . FurtherTo determine the impact of the results and translate them into a tool for clinical practise, standardisation of protocols and larger studies with more patients will be required. The implementation of well-designed and controlled clinical trials will be essential to validate the use of CSF ctDNA as a liquid biopsy for the clinical management of patients.CNS cancer is a dismal disease. It has elevated mortality and disabling effects on patients and is a massive burden on global health care systems. However, early detection and treatment can result in improved outcomes .Several studies of the CSF ctDNA of patients with different types of primary and metastatic CNS tumours have been performed and show promising results, highlighting the potential of CSF ctDNA as a biomarker.The challenges will be to translate these findings into clinically validated assays to improve patient healthcare. Standardisation of protocols and further studies and clinical trials will be necessary to translate the current results into a feasible tool for its implementation in the clinical setting.A liquid biopsy of CSF can characterise the tumour for diagnosis and provide prognostic information, also complementing the information obtained from the tumour sample if a biopsy or resection is feasible . During Altogether, the analysis of CSF ctDNA remains a promising strategy to improve the clinical management of patients with CNS malignancies, and further studies are required to make liquid biopsies a standard clinical tool.


Background: Ventilator-induced lung injury (VILI) via respiratory mechanics is deeply interwoven with hemodynamic, kidney and fluid/electrolyte changes. We aimed to assess the role of positive fluid balance in the framework of ventilation-induced lung injury.Methods:Post-hoc analysis of seventy-eight pigs invasively ventilated for 48 h with mechanical power ranging from 18 to 137 J/min and divided into two groups: high vs. low pleural pressure . Respiratory mechanics, hemodynamics, fluid, sodium and osmotic balances, were assessed at 0, 6, 12, 24, 48 h. Sodium distribution between intracellular, extracellular and non-osmotic sodium storage compartments was estimated assuming osmotic equilibrium. Lung weight, wet-to-dry ratios of lung, kidney, liver, bowel and muscle were measured at the end of the experiment.Results: High pleural pressure group had significant higher cardiac output , use of norepinephrine/epinephrine  and total fluid infusions . This hemodynamic status was associated with significantly increased sodium and fluid retention . Ten percent of the infused sodium was stored in an osmotically inactive compartment. Increasing fluid and sodium retention was positively associated with lung-weight  and with wet-to-dry ratio of the lungs  and kidneys .Conclusion: Increased mechanical power and pleural pressures dictated an increase in hemodynamic support resulting in proportionally increased sodium and fluid retention and pulmonary edema. It is well known that positive pressure mechanical ventilation is associated with hemodynamic impairment and sodium and water retention . In the In a series of animal experiments aimed to elucidate some of the mechanisms of ventilator-induced lung injury (VILI), the animals often required large amounts of fluids and catecholamines to prevent hemodynamic collapse . This apWe hypothesized that an increased fluid balance may worsen the pulmonary function and VILI development, hence, the primary objective of the present study was to investigate whether the applied pleural pressure and positive fluid balance have a synergistic role in dictating the VILI severity, quantified by the increase of lung weight.post-hoc analysis, data of seventy-eight pigs from two previous experimental studies investigating the relationship between mechanical power and VILI  were ventilated for 48 h and were randomized into six groups, 3 of which at low (15 J/min) and 3 at high mechanical power (30 J/min). In each group, the targeted mechanical power was reached with different combinations of PEEP, tidal volume and respiratory rate. In the whole cohort, the tidal volume ranged from 0.5 to 3.8 L, the respiratory rate from 5 to 44 bpm and the PEEP from 5 to 25 cmH2O .2O and a respiratory rate to maintain PaCO2 between 35 and 45 mmHg.In both experiments, anesthesia was induced and maintained with propofol, midazolam and sufentanil. After intubation, volume-control mechanical ventilation was initiated with tidal volume 6 mL/kg, PEEP 5 cmHThe animals had the following devices: endotracheal tube (size 7/7.5 mm); urinary catheter (5 Fr); adult esophageal catheter (8 Fr) Smartcath with esophageal balloon ; central venous catheter (5 Fr) in the jugular vein, ultrasound-guided; swan-Ganz catheter (5 Fr) through an introducer (7 Fr) in the jugular vein, ultrasound guided; arterial PiCCO catheter (5 Fr) in the femoral artery, ultrasound guided. The target temperature was maintained throughout the experiment between 38 and 39°C , by using a thermic blanket. Glycaemia was measured throughout the experiment, if values lower than 60 mg/dL were observed, dextrose 40% was initiated and maintained at the lowest infusion rate, in order to ensure glycemic levels between 60 and 100 mg/dL.At the end of the experiment the animal was euthanized, autopsy was performed and lung, kidney, liver, muscle and bowel samples were sent to the pathology department. Six lung samples were collected for each lung  and one sample for the other organs. Wet-to-dry ratio was calculated as follow: each sample (~2 g of weight) was weighted before and after being heated and dried in an oven at 50 degrees, during 24 h.Both studies were approved by the local ethic committee  .mean)  :mean is the mean airway pressure, ECW is the chest-wall elastance, and ERS is the total elastance of the respiratory system. Pleural pressure was chosen as the study variable because it is the most established determinant of hemodynamic changes, in mechanical ventilation.where PawRespiratory mechanics: plateau pressure, PEEP, tidal volume and esophageal pressure were recorded hourly, as well as their derived variables: total/chest-wall/lung elastances and total/lung mechanical power.Hemodynamic variables: heart rate, pulmonary/systemic blood pressure, cardiac output, pulmonary/systemic vascular resistances were measured every 6 h.Laboratory variables: sodium concentration (in plasma and urine) and osmolarity (in plasma) were measured at 0, 6, 12, 24, and 48 h.Sodium retention  was computed at each timepoint as the infused sodium minus the sodium excreted by urine  was computed as the amount of infused fluids minus the urinary output.Maintenance fluid consisted of 1–2 mL/h of Sterofundin infusion throughout the experiment. Additional fluids were administered in form of Sterofundin or Gelafundin ISO 4% fluid challenge (250 mL in 5–10 min) if: MAP < 60 mmHg; clinical signs of hypoperfusion ; hemodynamic monitoring dynamic indices (pulse pressure variation > 13% or systolic volume variation > 12%). Norepinephrine was initiate if the animal was not fluid responsive.Milliliters of infused solutions times the sodium content of normal saline (0.154 mmol/mL), Sterofundin (0.145 mmol/mL) and Gelafundin ISO 4% (0.151 mmol/mL). All fluids were included in the analysis either if they were infused as fluid challenge, maintenance or for drug dilution.Retained sodium: amount of infused fluid times the sodium concentration of the specific fluid , minus the amount of urine in milliliters times the urinary sodium concentration:+]inf (mmol/L) is the sodium concentration of the infused fluids, Vinf (L) is the amount of fluid infused during an experiment interval, [Na+]u (mmol/L) and Vu (L) are the urine sodium concentration and volume during the same interval. We only included the fraction of dissociated sodium, and excluded the sodium bound to drug molecules.where quations :bas is the baseline intracellular volume.Where ICVaeq) and intracellular (ICVaeq) volumes after the osmotic equilibrium ((3) We computed the new values of extracellular (ECVilibrium :+]beq) and after ([Na+]aeq) the osmotic equilibrium:(4) The following modified mass balance equations were used to calculate the expected sodium concentrations in plasma before ([Na+]bas is sodium plasma concentration at baseline.where [Na+miss):(5) Finally, we used the following equation to assess the amount of sodium that justifies the difference between the measured and the calculated sodium concentration after osmotic equilibrium . Similarly, the fluid balance increased significantly over time, and was significantly larger in the high pleural pressure group . The fluid balance in both groups was similar to the difference in weight-gain measured at the end of the experiment: 2.8 ± 1.8 vs. 5.5 ± 3.0 Kg; (p < 0.01). The positive fluid balance derived by a combination of greater amount of infused fluids and a decrease in urinary output: at the end of the experiment 5.72 ± 2.40 vs. 8.76 ± 3.40 L (p < 0.01) and 2.72 ± 0.89 vs. 2.10 ± 0.93 L (p = 0.01), respectively . Cardiac output was higher in the high pleural pressure group, and unexpectedly, it was significantly associated with greater sodium retention  and the lung wet-to-dry ratio  were significantly different between the low and high-pressure groups; At the end of the experiment, the weight of the lungs  and higher wet-to-dry ratio of lung  and kidney . No association was found between sodium retention and the wet-to-dry ratio of liver, bowel and muscle (see An increase of sodium and fluid retention was positively associated with a higher lung-weight (scle see . As showThe main findings of the present study are: (1) a higher pleural pressure leads to an increase in fluid and sodium retention; (2) sodium and fluid retention were greater when cardiac output was higher and the systemic vascular resistance was lower; (3) about 10% of the retained sodium must be osmotically inactive to account for the measured sodium and osmotic equilibrium, and (4) pulmonary edema and the wet-to-dry ratio were positively associated with the amount of retained sodium and fluid.The effects of mechanical ventilation on the sodium and fluid balance have been known since the first use of positive pressure ventilation . MechaniThe mechanisms of sodium and water retention as a consequence of decreased cardiac output and cardiac failure were described decades ago . Other mOur experiments were designed to study the effects of mechanical power on the lung parenchyma, collectively referred to as VILI. As the experiments were performed in healthy animals, the intensity of ventilation and the thoracic pressure required to produce lung damage were substantially elevated. Hemodynamics were severely compromised immediately after the increase in intrathoracic pressure, and, as a consequence, we observed a significant decrease in sodium excretion and urine volume contraction. This homeostatic response was maintained during the experiment as sodium retention and fluid balance increased linearly until the end of the experiment .As discussed earlier, the decrease in cardiac output is the generally recognized mechanism that explains sodium retention by activating several hormonal pathways. Although several studies have investigated the theory and practice of sodium distribution , the litIn our experiment we were able to separately analyze the distribution of sodium and osmotically active solute particles between the extracellular and intracellular compartments. With this approach, at the end of the experiment, we observed the following: (a) an increase in the extracellular compartment of 111.4% (from 4.75 to 10.04 L) due to the retained volume after the infusions (92.4%) and to the volume shift from the intracellular fluid  , (b) a 3The most striking results, however, are the clinical consequences of sodium and fluid retention in the framework of VILI. Actually, the present work was based on two large experiments aimed at investigating whether the level of mechanical power induced a proportional level of lung injury . SeveralIn conclusion, increasing the intrathoracic pressure led to a significant increase in sodium and fluid retention, which may play a significant role in VILI induced by excessive mechanical power.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES). Oldenburg (Niedersachsen), Germany. Protocol nrs. 16/2223 and 18/2795.SG and LG: conception and design. IP, MB, FR, FC, and FV: data acquisition. SG, MB, SL, PP, MP, FR, IS, PH, TL, and PC: analysis and interpretation. OM, LS, KM, MQ, and LG: drafting the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.LG reports a consultancy for General Electrics and SIDAM. He also receives lectures fees from Estor and Dimar. LS reports financial relationships with Medtronic, Ferrer Deutschland and Merck. Part of the salary support for the author MB was provided by an unrestricted research grant from Sartorius Inc. Göttingen, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.