We present the case of a giant cell tumour of soft tissue (GCT-ST) presenting as a slow-growing paraspinal mass. Imaging investigations revealed a well-circumscribed subcutaneous lesion containing fluid–fluid levels and an internal solid nodule. The imaging findings resulted in only a tentative differential which included haematoma or complex epidermoid cyst but failed to provide a definitive diagnosis. The patient underwent an image-guided biopsy from which a histopathological diagnosis of a GCT-ST was made. GCT-ST is a primary soft tissue neoplasm that is clinically and histologically similar to giant cell tumour of bone. Given its rare occurrence, there is very little published literature on the characteristic imaging findings of GCT-ST to help with its diagnosis which is usually only made histologically. The aim of this case report is to highlight our specific imaging findings and add to the limited pre-existing imaging data on GCT-ST. Giant cell tumour of soft tissues (GCT-ST), also known as “giant cell tumour of low malignant potential“ is a rare presentation of the relatively common pathological entity of giant cell tumours. It is clinically and histologically similar to giant cell tumours of bone, classically presenting as painless masses within the extremities and the trunk. Given its rare occurrence, very little can be found in the literature regarding the imaging characteristics of GCT-ST other than that presented in a few case reports. We report a case of a paraspinal GCT-ST and show how the imaging findings correlate with the histological features of GCT of bone.T1W and T2W signal change within the fat along the lateral margin (curved arrow). The imaging findings were not characteristic of any specific pathology in this location and a differential diagnosis of possible haematoma or complex epidermoid cyst was given. The patient was referred to the local sarcoma service and had a subsequent image-guided biopsy from which a histological diagnosis of GCT-ST was made (A fit and well 30-year-old female presented to her primary care practitioner with a 12 month history of a slow-growing left paraspinal lump in the absence of any trauma. She had an initial ultrasound scan  which shwas made . Followiwas made .1 However, subsequent detailed pathological analysis of these tumours found that they lack cytologic atypia, even in the presence of increased mitotic activity and vascular invasion and thus they were reclassified by Folpe et al as “giant cell tumours of low malignant potential“.2 This is a primary soft tissue neoplasm that is clinically and histologically similar to giant cell tumour of bone because it demonstrates multinodular aggregates of round to spindle-shaped neoplastic cells, admixed with numerous, uniformly scattered osteoclast-like multinucleated giant cells (3 It occurs predominantly in the fifth decade of life and has no sex predilection. These tumours usually develop in the superficial soft tissues of the extremities and less frequently within the trunk and head & neck.4 GCT-ST is expected to run a benign course as the malignant potential of these tumours, as determined by their histological characteristics is considered to be low and metastases are very rare.5 Complete surgical excision with clear resection margins is expected to be curative.GCT-ST was first described by Salm and Sissons in 1972 where it was initially considered under the umbrella term of “malignant giant cell tumour of soft parts“.nt cells .3 It occ4,6,7 and thus characteristic radiological findings for this tumour have not been documented. In the present case the ultrasound image (8 The ill-defined low signal change within the fat lateral to the lesion corresponded to an area of histiocytic infiltration and fibrosis within the adipose tissue consistent with reactive changes, presumably to possible previous extravasation of fluid into the adjacent fat. With the benefit of hindsight it can be appreciated that the imaging findings do resemble the features seen with GCT of bone and secondary ABC development. However, given the atypical location and rarity of this tumour the correct diagnosis was not considered, even as part of the differential in this case. This case report highlights the imaging findings of benign GCT-ST, which are similar to another published case report7 also presenting ultrasound and MRI evaluation. In that case, the mass was also subcutaneous with two well-defined simple cystic areas demonstrated on ultrasound and MRI, but showed no evidence of ABC development. Ultrasound features were very similar to the current case but MRI features differed with better demonstration of fluid–fluid levels and multiloculation seen in the current case. Two other case reports showed quite different ultrasound and MRI features and are summarised in Table 19,10. In conclusion, we present imaging findings for a rare presentation of benign GCT-ST which should be considered in the differential diagnosis of subcutaneous complex cystic masses particularly if fluid–fluid levels are seen suggesting secondary ABC development.GCT-ST is relatively rare with only a few reported cases,nd image  shows a nd image . The MRInd image . The cysGCT-ST are rare benign tumours most commonly found in the superficial soft tissues of the extremities.GCT-ST should be considered in the differential diagnosis of a subcutaneous complex cystic mass on ultrasound or MRI.The presence of fluid–fluid levels within such a mass is another helpful diagnostic clue as it is suggestive of secondary ABC formation.


Evaluation of fluid responsiveness during veno-arterial extracorporeal membrane oxygenation (VA-ECMO) support is crucial. The aim of this study was to investigate whether changes in left ventricular outflow tract velocity–time integral (ΔVTI), induced by a Trendelenburg maneuver, could predict fluid responsiveness during VA-ECMO.This prospective study was conducted in patients with VA-ECMO support. The protocol included four sequential steps: (1) baseline-1, a supine position with a 15° upward bed angulation; (2) Trendelenburg maneuver, 15° downward bed angulation; (3) baseline-2, the same position as baseline-1, and (4) fluid challenge, administration of 500 mL gelatin over 15 min without postural change. Hemodynamic parameters were recorded at each step. Fluid responsiveness was defined as ΔVTI of 15% or more, after volume expansion.R2 of the linear regression was 0.76, between ΔVTIs induced by Trendelenburg maneuver and the fluid challenge. The area under the receiver operating characteristic curve of ΔVTI induced by Trendelenburg maneuver to predict fluid responsiveness was 0.93 [95% confidence interval (CI) 0.81–0.98], with a sensitivity of 82% (95% CI 60–95%), and specificity of 88% (95% CI 64–99%), at a best threshold of 10% (95% CI 6–12%).From June 2018 to December 2019, 22 patients with VA-ECMO were included, and a total of 39 measurements were performed. Of these, 22 measurements (56%) met fluid responsiveness. The Changes in VTI induced by the Trendelenburg maneuver could effectively predict fluid responsiveness in VA-ECMO patients.Trial registration ClinicalTrials.gov, NCT 03553459 (the TEMPLE study). Registered on May 30, 2018 Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is a rescue therapy for patients with refractory cardiogenic shock , 2. DuriSeveral methods are currently available to evaluate fluid responsiveness in critically ill patients –9. MechaThe Trendelenburg maneuver is a “self” volumetric loading maneuver , and hasThis study was designed to investigate whether a change in velocity–time integral (ΔVTI) measured by TTE induced by the Trendelenburg maneuver, could predict fluid responsiveness in patients on VA-ECMO.This study was approved by the Institutional Review Board , and conducted in a 40-bed cardiac surgical intensive care unit (ICU). During the study period (June 2018–December 2019), 22 postoperative patients were prospectively enrolled after informed consent was received from the patient’s next of kin. We included ventilated patients with relatively low VA-ECMO pump flow (2–3 L/min) for whom the decision to perform volume expansion was made by the attending physician . ExBaseline-1: a supine position with a 15° upward bed angulation,Trendelenburg maneuver: 15° downward bed angulation ,Baseline-2: recover to the same position as baseline-1, andFluid challenge: administration of 500 mL gelatin over 15 min without postural change.Throughout the study, patients were sedated with a combination of remifentanil and midazolam, with the aim of achieving a Richmond Agitation–Sedation Scale  of − 5. After 1 min stabilization for each step, VTI, heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), central venous pressure (CVP) and pulse pressure (PP) were recorded. In our center, the tip of the drainage cannula was placed in the right atrium to guarantee adequate blood collection . The pren (the number of replicate TTE); (3) precision = 2 × CE; (4) least significant change (LSC) = CE × 1.96 × √2, indicting the minimal observed change value can be considered as real. These assessments were performed in eight patients at baseline position by obtaining twice by the same operator  and a second operator . The CV was 4% for intra-observer variability, and 5% for inter-observer variability. The intra-observer LSC was 8% for VTI measurements.Transthoracic echocardiography was performed on a CX50 instrument  by the same experienced operator (LL Dong) who was blinded to patient clinical data. VTI was measured at the level of the LVOT, using the 5-chamber apical view. Five consecutive measurements were recorded to calculate a mean VTI value. For TTE measurement, some indices were defined to estimate the reproducibility , 23: (1)Upon patient inclusion, demographic information, surgical procedures, Acute Physiologic and Chronic Health Evaluation (APACHE) II scores, left ventricular ejection fraction (LVEF), laboratory examinations and blood gas analyses were collected. Supportive therapies included VA-ECMO and mechanical ventilation (MV) indices, vasoactive drug doses  , and the0 = 0.5, AUC1 = 0.80, power = 0.9, alpha = 0.05, allocation ratio = 1, lower false positive rate (FPR) = 0, upper FPR = 1.00, type of data = continuous, and alternative hypothesis = two-sided test, therefore the least number of measurements required was 34.Sample size estimation was performed using PASS software. We used the following settings: AUCContinuous variables were expressed as medians (with interquartile ranges [IQR]) and compared between groups using the Wilcoxon or Friedman rank sum tests. For pairwise multiple comparisons, we used the Nemenyi post hoc test. Categorical variables were expressed as numbers (and percentages) and compared using the Fisher's exact test. Linear regression analysis was used to demonstrate relationships between percent change of VTI (ΔVTI) induced by the Trendelenburg position, and fluid challenge. Fluid responsiveness was defined as a VTI increase of 15% or more after volume expansion .. [-precision  curves (PRC) were generated and area under precision–recall curve (AUPRC) were calculated [Receiver operating characteristic (ROC) curves were generated to evaluate percent changes in VTI and arterial pressure parameters , induced by the Trendelenburg maneuver to predicts fluid responsiveness. The area under ROC curves (AUROC) were compared using the DeLong test . Sensiti. . To visulculated .p < 0.05 indicated statistical significance. Statistical analyses were performed using R, version 3.6.2 .All statistical tests were two-tailed, and a value of The study flowchart is shown in Additional file Thirty-nine measurements were performed at a median of 4 days (IQR 3–5) after VA-ECMO support initiation Table . Of thesp = 0.019), ΔPP , and ΔVTI  than the Trendelenburg maneuver. The ΔVTI between the Trendelenburg position and fluid challenge was highly related, with an R2 of 0.7614 and a slope of 0.58 . Hemodynamic parameters from each step are shown (Table Data on fluid responsiveness predictions are shown Fig. . The AURTo the best of our knowledge, this is the first study to explore the predictive value of ΔVTI, induced by the Trendelenburg position for fluid responsiveness in patients with VA-ECMO support. Our work demonstrated that ΔVTI monitoring during the Trendelenburg position was a reliable parameter in predicting fluid responsiveness in this population.VA-ECMO could provide extracorporeal life support, as well as reducing the workload of the injured heart, thereby creating favorable conditions for myocardial recovery , 2. DuriFluid responsiveness, as a cardiac response parameter to additional preload, is typically used for septic shock patients ; howeverThe Trendelenburg maneuver is a method that facilitates “autotransfusion”. Geerts et al. concluded that the final changes in cardiac output induced by the Trendelenburg position were similar to PLR . In thisArterial pulsatility, coming from left ventricular ejection, is often considered as a marker of cardiac recovery during VA-ECMO , 35. PP,Our study had several limitations. First, it was conducted in a single center, which may have limited generalizability across different clinical settings. Second, a larger angle of Trendelenburg positioning may have introduced more “autotransfusion” to the central circulation , and wasOur study suggested that an increase in VTI of at least 10%, induced by the Trendelenburg maneuver, was reliable in predicting fluid responsiveness in patients with VA-ECMO.Additional file 1:Figure S1. Study flowchart. Figure S2. Receiver operating characteristics curves and grey zone analyses. Figure S3. The precision–recall curves for changes in VTI, PP, SBP and DBP induced by Trendelenburg maneuver.


Infigratinib is a pan‐FGFR (fibroblast growth factor receptor) inhibitor that has shown encouraging activity in FGFR‐dependent hepatocellular carcinoma (HCC) models. However, long‐term treatment results in the emergence of resistant colonies. We sought to understand the mechanisms behind infigratinib‐induced tumour cell differentiation and resistance and to explore the potential of adding the CDK4/6 inhibitor ribociclib to prolong cell differentiation.Nine high and three low FGFR1‐3‐expressing HCC patient‐derived xenograft (PDX) tumours were subcutaneously implanted into SCID mice and subsequently treated with either infigratinib alone or in combination with ribociclib. Tumour tissues were then subjected to immunohistochemistry to assess cell differentiation, as indicated by the cytoplasmic‐to‐nuclear ratio and markers such as CYP3A4, HNF4α and albumin. Western blot analyses were performed to investigate the signalling pathways involved.Infigratinib induced cell differentiation in FGFR1‐3‐dependent HCC PDX models, as indicated by an increase in the cytoplasmic/nuclear ratio and an increase in CYP3A4, HNF4α and albumin. Resistant colonies emerged in long‐term treatment, characterised by a reversal of differentiated cell morphology, a reduction in the cytoplasmic‐to‐nuclear ratio and a loss of differentiation markers. Western blot analyses identified an increase in the CDK4/Cdc2/Rb pathway. The addition of ribociclib effectively blocked this pathway and reversed resistance to infigratinib, resulting in prolonged cell differentiation and growth inhibition.Our findings demonstrate that the combined inhibition of FGFR/CDK4/6 pathways is highly effective in providing long‐lasting tumour growth inhibition and cell differentiation and reducing drug resistance. Therefore, further clinical investigations in patients with FGFR1‐3‐dependant HCC are warranted. ALBalbuminCDKcyclin‐dependent kinaseCSCcancer stem cellCYP3A4cytochrome P450 3A4FGFfibroblast growth factorFGFRfibroblast growth factor receptorHCChepatocellular carcinomaHNF4αhepatocyte nuclear factor 4 alphaPDXpatient‐derived xenograftTPMtranscripts per kilobase millionInfigratinib is an FGFR inhibitor that is highly effective against FGFR‐dependant hepatocellular carcinoma. However, long‐term treatment with infigratinib resulted in the emergence of drug‐resistant tumour characterised by an increase in CDK4/Cdc2/Rb pathway. The addition of clinically approved ribociclib overcomes infigratinib resistance, delays tumour growth and potentially translating to prolonged patient survival.1In 2018, liver cancer collectively ranked as the sixth most prevalent neoplasm and the fourth leading cause of cancer‐related mortality worldwide.Increasing evidence suggests that cancers arise from a distinct population of tumour‐initiating cells or cancer stem cells (CSCs). First identified in acute myeloid leukaemia (AML),Treating malignant tumours through ‘differentiation therapy’ has been an attractive concept, but clinical development of differentiation‐inducing agents to treat malignant tumours, especially solid tumours, has been limited to date.There have been a number of studies exploring pharmacological agents to drive CSCs into dormant, terminally differentiated cells. In acute promyelocytic leukaemia, all‐trans retinoic acid (ATRA) has been proven to be highly successful in initiating CSC differentiation into more short‐lived granulocytes,Our previous study has established the ability of infigratinib to inhibit tumour growth, normalise tumour vascularisation, reduce intratumoural hypoxia and impair invasion and metastasis in human HCC PDX models.22.1Ribociclib (S7440) and Infigratinib (S2183) were purchased from Selleck Chemicals and were dissolved in 21% Captisol and 30% PEG300 solution (vehicle) for oral administration. Antibodies against FGFR1 (#9740), FGFR3 (#4574), FGFR4 (#8562), AKT (#9272), Rb (#9313), cyclin B1 (#4138), Cdc25C (#4688), Cyclin D1 (#2978), Cyclin E2 (#4132), survivin (#2803), Sox9 (#82630), cleaved PARP (#5625), β catenin (#8480), α‐tubulin (#2144) and phosphorylation‐specific antibodies against AKT Ser473 (#9271), FRS2‐α Tyr439 (#3861), Rb Ser807/811 (#9308), Histone 3 Ser10 (#9701), Cdc2 Tyr15 (#9111) and Erk1/2 Thr202/Tyr204 (#4370) were obtained from Cell Signaling Technology. Antibodies against CYP3A4 (ab124921) and HNF4‐α (ab201460) were from Abcam. The antibodies against FGFR2 (#sc‐122), Erk1/2 (#sc‐94), FRS2‐α (#sc‐17841), GAPDH (sc‐166545) and Cdk2 Thr14/Tyr15 (#sc‐28435‐R) were from Santa Cruz Biotechnology Inc. Anti‐mouse CD31 antibody (#2502) was from BioLegend. Anti‐albumin  antibody was purchased from Sigma‐Aldrich.2.23. For Western blot analyses, mice were treated for 4 days and dosing started when tumour size reached 800 mm3.This study was approved by the SingHealth Institutional Animal Care and Use Committee (IACUC number: 2015/SHS/1020) and the National Cancer Centre Singapore Ethics Board. All animals received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institutes of Health.To determine the optimum dose for the induction of differentiation, mice bearing tumour xenografts were orally treated with vehicle or 10, 15 or 20 mg/kg infigratinib once daily for 4 days. Subsequently, the optimal dose was used to study the antitumour activity of infigratinib in tumour‐bearing mice. To investigate the cell differentiation‐inducing effects of infigratinib, mice were treated daily with either vehicle or 15 mg/kg infigratinib for 12‐21 days. For the drug combination study, mice were randomised into four groups with 10 mice per treatment arm and subsequently orally administered once daily with (a) 200 µL vehicle, (b) 15 mg/kg infigratinib, (c) 75 mg/kg ribociclib or (d) a combination of 15 mg/kg infigratinib and 75 mg/kg ribociclib.3. Infigratinib‐resistant tumours were collected, processed and re‐implanted into treatment‐naïve SCID mice. The infigratinib treatment cycle was then repeated for up to nine passages to maintain resistance. To detect the emergence of infigratinib‐resistant colonies that occurred during the early stages of acquired resistance, tumour tissues were collected when disease progression was observed.To generate infigratinib‐resistant PDX models, HCC01‐0909, HCC21‐0208, HCC26‐0808A and HCC06‐0606 PDX lines were subcutaneously implanted, and mice were repeatedly administered a daily dose of 15 mg/kg infigratinib. Mice were sacrificed once tumours reached approximately 1800 mmTumour volume was measured and calculated over the course of the study, as previously described.2.33VO4. Proteins (80 µg) were resolved by SDS‐PAGE and transferred onto a nitrocellulose membrane. Blots were probed with the indicated primary antibodies, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies, as previously described.Tumours from vehicle‐ and drug‐treated mice were homogenised in buffer containing 50 mmol/L Tris‐HCl pH 7.4, 150 mmol/L NaCl, 0.5% NP‐40, 1 mmol/L EDTA and 25 mmol/L NaF, supplemented with proteinase inhibitors and 10 mmol/L Na2.4Tumours were fixed in 10% formalin overnight and subsequently embedded in paraffin blocks. Tissue sections (5 µm) were immunostained with antibodies against CD31, p‐Histone H3 Ser10 and cleaved PARP to assess microvessel density, cell proliferation and apoptosis respectively. Epitopes were visualised with SignalStain Boost IHC Detection Reagent . To detect markers of hepatocyte differentiation, tissue sections were stained with antibodies against CYP3A4, HNF4α and albumin. Five random images were taken on an Olympus BX60 microscope (Olympus) and subsequently analysed. Image contrast and brightness were uniformly adjusted for clarity.For tumour hypoxia staining, tumour‐bearing mice were intraperitoneally injected with 60 mg/kg pimonidazole hydrochloride 1 hour prior to tumour collection. To identify hypoxic regions, tumour sections were stained with HypoxyProbe Plus Kit HP2 (HypoxyProbe Inc) according to the manufacturer's protocol.2.52) × (π/6)], and the cytoplasmic‐to‐nuclear area ratio was then calculated. An increase in the ratio indicates differentiated cells. Five random fields at a magnification of ×400 from each treatment were captured with an Olympus BX60 microscope (Olympus), with at least 20 cells per field of view analysed.Tissue sections were immunostained with antibodies against β‐catenin to define the cytoplasmic boundary and subsequently counterstained with haematoxylin to visualise nuclei. The areas of the nucleus and cytoplasm were measured using [(Length) × . All authors had access to the study data and had reviewed and approved the final manuscript.The mean tumour volume between treatments was compared using two‐way ANOVA. Student's 33.1To determine the optimum dose for the induction of differentiation markers, mice bearing HCC13‐0109 and HCC01‐0909 PDX models were administered 10, 15 or 20 mg/kg infigratinib for 4 days. Infigratinib reduced the levels of FGFR‐1, ‐2, ‐3 and ‐4 concomitant with a decrease in its mRNA levels Figure . ConsistHistological analyses suggest that infigratinib inhibits tumour cell proliferation, as indicated by a decrease in the number of cells positively stained for p‐Histone H3 Ser10. Compared with the vehicle‐treated group, infigratinib‐treated mice showed a higher density of morphologically distinct intratumoural blood vessels. Vehicle‐treated mice displayed dysregulated and non‐productive blood vessels associated with the presence of large hypoxic regions. In contrast, infigratinib‐treated tumours showed slim blood vessels resembling normal capillaries and a significant reduction in hypoxic regions via vascular normalization Figure . These iConversely, in low‐FGFR‐expressing HCC10‐0505, HCC26‐1004 and HCC2‐1318, infigratinib did not appear to suppress tumour growth, despite the higher dose of infigratinib used (20 mg/kg). Compared with vehicle, p‐Histone H3 Ser10 staining showed similar numbers of staining‐positive cells, indicating the lack of tumour growth inhibition. Furthermore, CD31 staining also exhibited morphologically large and dysregulated blood vessels Figure .https://kmplot.com/).We then analysed the 5‐year overall survival (364 patients) and progression‐free survival  of HCC patients based on the expression of the differentiation markers HNF4α, ALB and CYP3A4. Kaplan‐Meier plots were generated based on liver cancer RNA‐seq data from the publicly available KM Plotter . These results correspond to the smaller tumour weight observed at the end of the treatment cycle Figure . SimilarImmunohistochemical analyses showed an absence of resistant colonies and an increase in CYP3A4 and albumin expression in drug combination‐treated tumours. The majority of tumour cells in the drug combination‐treated tumours had morphologic evidence of the terminal hepatocyte differentiation and exhibited a uniformly higher expression of CYP3A4 and albumin staining throughout the tissue section Figure . This suWestern blot analyses further indicated that treatment with 75 mg/kg ribociclib alone only partially suppress cell cycle proteins such as Cdc25C, cyclin B1 and p‐Cdc2 in HCC21‐0208 tumour. Furthermore, no increase in CYP3A4 and HNF4α expression was detected Figure , consistWhile infigratinib treatment alone was able to normalise blood vessels and suppress tumour proliferation, the combination treatment showed an even higher density of blood vessels, as evidenced by CD31 staining, and less proliferative cells, as indicated by the reduction in cells positively staining for p‐Histone H3 Ser10 Figure . Collect4Hepatocellular carcinoma remains one of the leading causes of cancer‐related mortality worldwide, yet there has been no significant improvement in systemic treatment options in nearly two decades.The FGFRs are members of the tyrosine kinase family, and their dysregulations have been shown to play a key role in liver carcinogenesis.The concept of differentiation therapy pivots towards keeping cancer cells in their dormant state rather than eradicating them completely. Therefore, we investigated the feasibility of long‐term infigratinib treatment. Despite showing an initial response, we observed the emergence of resistant colonies after prolonged treatment with infigratinib, consistent with previous reports.On the basis of this observation, we hypothesised that inhibition of the cell cycle pathway using ribociclib could overcome infigratinib resistance. Clinically, ribociclib is an inhibitor of the cyclin D1/CDK4/6, which is commonly used in the treatment of hormone‐receptor‐positive breast cancer.Mechanistically, ribociclib treatment also appears to slightly upregulate FGFR in some HCC lines, suggesting the presence of a feedback mechanism that forces cells to depend on the FGFR pathway to continue to proliferate Figure . ImmunohTaken together, our data suggest that the inhibition of FGFR1‐3 by infigratinib resulted in the induction of cell differentiation and therefore a reduction in cell proliferation. Canonically, blockade of FGFR signalling led to the attenuation of the Ras/Raf/Mek/Erk and PI3K/Akt/mTOR pathways, leading to a decrease in the expression of proteins involved in cancer stemness. As hepatocytes gained resistance to infigratinib, they displayed a dedifferentiated morphology resembling those of cancer stem cells. These cells were characterised by a loss of differentiation markers and an increase in proteins involved in the cell cycle, such CDK4, leading to the inactivation of Rb. However, this was mitigated by the addition of ribociclib, where the combination of both drugs was able to synergistically block compensatory signalling mechanisms, resulting in longer‐lasting differentiation into mature hepatocytes and a significant reduction in drug resistance Figure . These fNone declared.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.Fig S5Click here for additional data file.Fig S6Click here for additional data file.Table S1Click here for additional data file.


R has been applied in a number of contexts, mostly for systems characterized by 2D thermodynamic geometries. Calculations of R in thermodynamic geometries of dimension three or greater have been very few, especially in the fluid regime. In this paper, we calculate R for two examples involving binary fluid mixtures: a binary mixture of a van der Waals (vdW) fluid with only repulsive interactions, and a binary vdW mixture with attractive interactions added. In both of these examples, we evaluate R for full 3D thermodynamic geometries. Our finding is that basic physical patterns found for R in the pure fluid are reproduced to a large extent for the binary fluid.The thermodynamic Ricci curvature scalar  R has yielded a number of interesting results in the study of fluids [R have been made for pure fluids. Much less examined has been R for binary fluids. Pure fluids offer many research topics in a relatively simple thermodynamic geometric scenario. One element of this simplicity is that pure fluids may be represented by a two-dimensional (2D) thermodynamic Riemannian geometry, where just the scalar R gives the full curvature picture.The thermodynamic Ricci curvature scalar f fluids . HoweverR remains the fundamental physical quantity. .The dimension of the thermodynamic phase space grows by one for each added fluid component, and, as the dimension grows, the curvature rapidly becomes more complicated. For example, the binary fluid corresponds to a three-dimensional (3D) phase space, where there are six independent components of the full Riemannian curvature tensor . Which ograngian . This vaR for two thermodynamic examples represented by a 3D thermodynamic geometry: (1) a binary van der Waals (vdW) fluid with just repulsive interactions, and (2) a binary vdW fluid with attractive interactions added. To add conceptual structure to our presentation, we discuss to what extent the 3D results for R follow the same physical principles as the 2D ones. As we show, there is a great deal of correspondence. . (b) As a critical point is approached, does R negative? A negative sign is consistent with effectively attractive interactions. (d) Are the values of R in two coexisting phases equal to each other near a critical point? (e) Are there interesting regimes of positive R? A positive sign is consistent with effectively repulsive interactions. (f) Does the binary repulsive fluid have instances of anomalous negative R, such as is present for the hard-sphere pure fluid [For guidance as to what 3D thermodynamic geometry might tell us, let us pose some questions motivated by findings in the pure fluid ,10,11,12re fluid ?R for the pure fluid and for the binary fluid.We find considerable physical correspondence between T is the temperature, and V is the volume.For a binary fluid, the Helmholtz free energy per volume may be written in terms of its appropriate coordinates ,14,(1)fV, of an infinite closed thermodynamic fluid system The thermodynamic entropy metric originates from the thermodynamic fluctuation theory ,15. Consdynamics  as a meaR. We want R to tell us something about the intrinsic properties of the material comprising the system R in a way involving more than just the properties of the particles; this should be avoided. As emphasized by Callen [V. We recommend that authors always work with open subsystems for calculating R.Let us emphasize a point not always appreciated in the metric geometry of thermodynamic fluctuations. A major project in this geometry is to calculate the thermodynamic curvature y Callen , fluid tg is composed of five nonzero elements, which can be read off from Equation (g with elements In the coordinates Equation :(6)g=1kBT,ρ1,ρ, the metR has units of volume per particle for the binary fluid, the same units as for the pure fluid. These units alone label R as a measure of some sort of size scale within the system. The definition of the Riemannian curvature tensor is ambiguous with respect to a sign, and we use the sign convention of Weinberg [R.It is straightforward to show that Weinberg , where tFluctuations must satisfy thermodynamic stability, requiring maximum entropy of Equation  is positEquation  satisfy R, and x is the concentrationa and b are functions of x reflecting the purely attractive and repulsive parts of the interparticle interactions, respectively:We take the Helmholtz free energy per mole for binary vdW from Konynenburg and Scott :(14)Am=eThe full (extensive) Helmholtz free energy isFor the pure fluid, vdW simplifies by using scaled units . For bina coefficients, First consider the case with only repulsive interactions, with zero Equation , we haveR for repulsive vdW has no dependence on T, nor does it depend on the constants in Equation . R . R detR×(ρR×(ρ(28)RR in an interacting binary fluid system. Our main finding is that the physical interpretation of R for the pure fluid extends very naturally to the binary fluid. The emerging physical picture for the thermodynamic R is thus quite robust. We calculated R for two scenarios involving the van der Waals model for the binary fluid. The first had exclusively repulsive interactions, and the second added attractive interactions, and critical phenomena.We have made a start in calculating the full three-dimensional thermodynamic Ricci curvature scalar R was found to be negative, as shown in R along coexistence curves was beyond the scope of our paper. (e) Our regimes of positive R were all more or less expected, with values all under the low Let us place our results in the context of the expectations in d sphere ; see Fig


Introduction: Limited data currently exist regarding the demographics of principal investigators (PIs) in obstetrics and gynecology (OBGYN), who have received an R01 grant from the National Institutes of Health (NIH).Objective: This study investigated funding differences among gender and advanced degree for PIs in the disciplines of OBGYN.Methods: Retrospective data were collected from the NIH Research Portfolio Online Reporting Tools Expenditures and Results (RePORTER) tool to identify OBGYN PIs who received an R01 grant between 2008 and 2017. Results: Between 2008 and 2017, the NIH awarded 263 R01 grants totaling $113,326,883 in funding to investigators in OBGYN. Male PIs and PIs holding a non-medical degree were awarded the majority of R01 grants . Zero osteopathic (Doctor of Osteopathic Medicine [DO]) physicians were awarded an R01 grant in OBGYN during this time period. Females were awarded larger dollar amounts than males . Allopathic (Doctor of Medicine [MD]) physicians were awarded larger grants than scientists holding a non-medical degree . Both male and female MD physicians were awarded more dollars per grant as compared with PIs holding a non-medical degree .Conclusions: Between 2008 and 2017, a degree disparity was found to exist for investigators who received an NIH R01 grant in OBGYN. Females and investigators holding an MD degree were awarded larger total grants than their male and non-physician counterparts. Further research needs to be undertaken to understand the degree disparity and recent funding trends by the NIH.  Research has been one of the driving forces of medical advancement and improving patient-centered healthcare outcomes over the last several decades. However, these advancements would not be possible without physician scientists and grant funding. The National Institutes of Health (NIH) has been the one of the largest funding sources for research over the last decade and the Research Project (R)-series category is a major funding source for new principal investigators (PIs) . In 2018, departments in obstetrics and gynecology (OBGYN) represented only 1% of all total NIH funding despite being a major provider of primary care services to the population )    (Table The number of physician scientists has decreased over the last decade despite an increase in the overall physician workforce . Based uWhen comparing the gender of those who received a R01 grant in OBGYN, males and females are almost equally represented. The near gender equality in those receiving an NIH R01 grant could be related to an increase in both female faculty members and residents in OBGYN over the last several years . Recent No osteopathic physician received an NIH R01 grant in OBGYN over the time period studied. Similar to these results, no osteopathic physician has been awarded an NIH R01 grant in family medicine, general surgery, and emergency medicine over a similar time period -6. It isTypically, those who hold a dual degree in medicine and another academic field undergo significant research training prior to graduation. In general surgery, males with a dual degree have been awarded larger R01 grant funds as compared to those with only a medical degree . HoweverOver the decade studied, a degree disparity was found to exist in those receiving an NIH R01 grant in OBGYN. Females and those holding an MD degree were awarded larger total grants than their counterparts, but no difference was seen for those MDs holding a dual degree. Further research needs to be undertaken to understand the degree disparity and recent funding trends by the NIH in the specialty of OBGYN.


Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells, the identity of which relies on activation of Notch at cytokinesis. Although Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, the basal pool of Notch is reported to be the main contributor for Notch activation in the pIIa cell. Intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We have previously reported that AP-1 and Stratum regulate the trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that their concomitant loss results in a penetrant Notch gain-of-function phenotype, indicating that they control parallel pathways. Although unequal partitioning of cell fate determinants and cell polarity were unaffected, we observed increased amounts of signalling-competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface, at the expense of the basal pool of Notch. We propose that AP-1 and Stratum operate in parallel pathways to localize Notch and control where receptor activation takes place. Summary: The Notch pathway activation relies on the correct localization of the Notch signalling actors. We report that AP-1 and Stratum ensure the basolateral targeting of Notch during asymmetric cell division. Drosophila has been instrumental in identifying the site of Notch activation at the cell surface. SOPs are polarized epithelial cells that divide asymmetrically within the plane of the epithelium to generate two daughter cells, the fate of which depends on the differential activation of Notch signalling  and the recycling endosomes (RE) towards the plasma membrane (Notch gain-of-function phenotype (GOF). Unequally partitioned Numb controls the endosomal sorting of Notch/Spdo after asymmetric division and prevents their recycling to the plasma membrane , the pIIb cell divides twice to generate the internal cells, among which is one Elav-positive neuron. The pIIa cell divides once to generate the external cells, among which is one socket cell identified by Suppressor of Hairless [Su(H)]. Su(H) can therefore be used as a read-out of cell fate transformations to monitor the effect of individual or simultaneous impairment of Strat and AP-1 -positive socket cells were observed in 6.7% and 7.5% of SOs mutant for strat or depleted of AP-1, respectively . Despite these cuticle defects, the localization of the junctional markers DE-Cadherin, Par3 and Coracle is unaffected by the loss of AP-1 and Strat, suggesting that cell polarity is not altered . However, time-lapse imaging revealed that Par3, instead of being enriched at the posterior pole of SOP prior to mitosis . This defect in planar cell polarity (PCP) regulation is likely caused by the loss of AP-1, as reported in the Drosophila wing . As Par3 is required for the asymmetric localization of Numb and Neur during mitosis . NiGFP is transiently detected at the apical pIIa-pIIb interface at ∼6-9 min after anaphase onset, with a signal intensity peaking 15-20 min before progressively disappearing at ∼30 min . Basal to the midbody, NiGFP localizes in lateral clusters, which appear ∼6-9 min following the anaphase onset and persist longer than the apical pool of NiGFP .We next investigated the effect of AP-1 and Strat on the apico-basal distribution of Notch receptors. We monitored the dynamics of Notch bearing a GFP tag in its intracellular domain  as well as at the lateral pIIa-pIIb interface , and our unpublished observations indicate that Par3 activity is required for the assembly of functional Notch signalling clusters during cytokinesis . On average, 12 NiGFP-Par3 lateral clusters are detected in the wild type at t21 to t35 min . Upon loss of Strat and AP-1, the number of NiGFP-Par3 lateral clusters and total intensity of NiGFP is decreased by 3.1-fold . In parallel, we observed a 2.4-fold increase (mean intensity) in the amount of NiGFP at the apical pIIa-pIIb interface at t21 min after the onset of anaphase. This increase begins at ∼6-9 min after the onset of anaphase, and NiGFP signal persists for longer periods of time compared with the control . In addition to accumulating at the apical interface, NiGFP also localizes in intracellular compartments present in the apical plane . As the lateral clusters are strongly reduced upon loss of Strat and AP-1, our results lead us to hypothesize that the Notch gain-of-function phenotype could, at least in part, come from the apically enriched pool of NiGFP.We found that NiGFP clusters colocalizes with Par3 at the apical . We noticed that 120 s after photobleaching, the fluorescence recovery reaches its maximum in control as well as upon loss of Strat and AP-1. However, the mobile fraction is around 67%±30% upon loss of Strat and AP-1 compared with 31%±15% in a control situation  with a t1/2 of around 40 s±7 s and a t1/2 of 37 s±11 s, respectively . The high recovery rate of the apical pool of NiGFP raises the question of its destiny and is in agreement with Notch activation that can take place at the apical interface upon loss of Strat and AP-1. To determine whether the Notch GOF observed in absence of Strat and AP-1 is ligand dependent, we inhibited Delta endocytosis using a stabilized version of Bearded that is acting as a strong antagonist of Neur (BrdR) . In absence of Strat and AP-1, the overexpression of BrdR also induced a Notch loss-of-function phenotype: opposite to that observed upon loss of Strat and AP-1 only . These data demonstrate that the Notch gain-of-function phenotype observed in absence of Strat and AP-1 is ligand dependent. Together, our results suggest that, in the absence of Strat and AP-1, Delta-mediated Notch activation can take place at the apical interface of SOP daughter cells.Notch activation requires Neur-mediated Delta endocytosis. In the wild-type situation, Delta and Neur localize at the lateral pIIa-pIIb interface C-E. Wherr (BrdR) . Althougneur promoter . In contrast, photoconversion within a ROI encompassing the lateral pIIa-pIIb plasma membrane interface led to unwanted photoconversion in the adjacent nuclei  and at the apical level over a diameter of about 3-4 cells . Therefore, the inability to ensure precise photoconversion on the lateral interface prevents us from using photoconversion to test whether Notch is activated from the apical, the lateral or from both pools of receptors in controls. However, this technical caveat is not a concern after loss of Strat and AP-1, as NimMaple3 and NiGFP signals are barely detected at the lateral interface . Following photonconversion of Notch at the apical pIIa-pIIb interface in this context, photoconverted NICD is present in the nuclei of both SOP daughter  are higher than those found in the nuclei of a neighbouring SOP that divides at the same time, but was not exposed to photoconversion . On average, the contribution of the apical pool of Notch when Strat and AP-1 are simultaneously impaired seems identical between the two nuclei, as demonstrated by the ratio of photoconverted nuclear signal pIIa/pIIa-like being close to 1 and the absence of significant differences in the amount of photoconverted nuclear signal between the two cells  are involved in apical trafficking of Spdo and Delta, respectively, and are required for Notch-Delta signalling is consistent with the proposal of apical activation of Notch . Neur haNotch gain of function despite the unequal partitioning of Numb. This phenotype can be explained by the lack of AP-1 that prevents Numb from repressing the Notch-Spdo recycling normally driving endosomal degradation of Notch-Spdo  at second and early third instar larvae stages. pnr-GAL4 was used to drive the expression of the dsRNAAP-47 and the dsRNAstrat. The following stocks were used in this study.y, hs-FLP (/w); Ubi-GFP nls, FRT40A/Ubi-GFP nls, FRT40A and y, w, hs-FLP (/−w); Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47 v24017/TM3)y, hs-FLP (/w); Ubi-GFP nls, FRT40A/Ubi-GFP nls, FRT40A and y, hs-FLP (/w); Ubi-GFP nls, FRT40A/strat, FRT40A and y, w, hs-FLP (/w); Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47 and y, w, NimMaple3 ; Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47y, hs-FLP (w); Ubi-RFP nls, FRT40A/Numb::GFPcrispr, FRT40Aneur-H2B::RFP/w; Ubi-GFP nls, FRT40A/strat, Numb::GFPcrispr, FRT40A; pnr-GAL4/dsRNAAP-47y, w, hs-FLP/NiDendra, neur-H2B::RFP; Ubi-GFP nls, FRT40A/FRT40A; pnr-GAL4/+NiDendra, neur-H2B::RFP; Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47neur-iRFP670nls , pnr-GAL4/+neur-iRFP670nls/dsRNAstrat; P [attP2 neur-GAP43-iRFP 68A4], pnr-GAL4/dsRNAAP-47NiGFP, neur-H2B::RFP; Ubi-GFP nls, FRT40A/FRT40A; pnr-GAL4/+ and y, w, hs-FLP/NiGFP, neur-H2B::RFP; Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47 and y, w, NiGFP, Par3::Scarlet; +/+; pnr-GAL4/+ and y, w, NiGFP, Par3::Scarlet;dsRNA/+strat; pnr-GAL4/dsRNAAP-47y, w, hs-FLP (/w); Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47NiDendra, neur-H2B::RFP; Ubi-GFP nls, FRT40A/FRT40A; pnr-GAL4/+ and y, w, hs-FLP/NiDendra, neur-H2B::RFP; Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47NiDendra, neur-H2B::RFP; Ubi-GFP nls, FRT40A/FRT40A; pnr-GAL4/+y, w, hs-FLP/NiDendra, neur-H2B::RFP; Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47y, w, NiGFP, Par3::Scarlet; dsRNAstrat/+; pnr-GAL4/dsRNAAP-47and y, w, NimMaple3/Y; dsRNAstrat/+; pnr-GAL4, neur-GAP43::IRFP670/dsRNAAP-47Fig. S1A,A′: NiDendra, neur-H2B::RFP; +/+Fig. S1B,B′: y, w, hs-FLP/NiDendra, neur-H2B::RFP; Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47Fig. S1C: y, w, hs-FLP (/−w); Ubi-GFP nls, FRT40A/strat, FRT40A; pnr-GAL4/dsRNAAP-47Fig. S1D: y, w, NiGFP, Par3::Scarlet ; +/+; pnr-GAL4/+ and y, w, NiGFP, Par3::Scarlet; dsRNAstrat/+; pnr-GAL4/dsRNAAP-47Fig. S2: y, w, NiGFP, Par3::Scarlet; +/+; pnr-GAL4/+ and y, w, NiGFP, Par3::Scarlet;dsRNA/+strat; pnr-GAL4/dsRNAAP-47Fig. S3A: y, w, NiGFP, Par3::Scarlet; dsRNAstrat/+; pnr-GAL4/dsRNAAP-47Fig. S3B,C: iGFP−5, neur-H2By, w, N::RFP  for SOPs and SOPs daughter cell analysis, and were aged for 24 h to 28 h APF for lineage analysis. Pupae were dissected in 1× phosphate-buffered saline (1×PBS) and then fixed for 15 min in 4% paraformaldehyde at room temperature. Dissection and staining conditions were essentially as previously described . Primaryyw; attP40(nos-cas9)/Cyo stock. The correct position of the Dendra and the DsRed cassette was verified by PCR and sequencing. The DsRed was then removed by crossings with if/Cyo, Cre, w stock. The insertion of the Dendra tag did not alter the functionality of Notch. Anti-Dendra signal is detected in the pIIa nucleus, indicating that processed NiDendra is translocated into the nucleus (data not shown).The NiDendra construct was generated using the CRISPR/Cas9 method as previously described ,b. As foneur-GAP43::iRFP670 transgene was generated by fusing the iRFP670 gene  was performed with a Zeiss Airyscan microscope (LSM 880 with AiryScan module). All images were processed and assembled using ImageJ 1.48 and Adobe Illustrator.Images of fixed nota were acquired with a Leica SPE confocal microscope and a Zeiss Airyscan microscope (LSM 880 with AiryScan module). Live imaging of NiGFP with Par3::Scarlet was performed with a Leica SPE confocal microscope. Live imaging of NiGFP in wild-type SOP and in To quantify the signal of NiGFP at the apical interface, we measured the signal in a manually drawn area on sum slices of the two apical planes where the Notch signal at the apical interface is the strongest. We also measured the signal between two epithelial cells within an equivalent drawn area and on the same sum slices. We then calculated the following ratio: average fluorescence intensity at the apical interface between SOP daughter cells/average fluorescence intensity at the apical interface between epithelial cells. This ratio was calculated for each time point. In absence of AP-1 and Strat, the manually drawn area was minimized to avoid considering the apical punctate structures positive for Notch.Fig. S2A′,B′. For each movie, we determined the time at which the largest number of NiGFP-Par3 clusters was counted (between t21 and t35 min). When a cluster was detected on two consecutive optical sections, it was considered as one cluster. To determine the intensity of NiGFP per cluster, a line two pixels in width and 20 pixels long encompassing the middle of the cluster was drawn, as shown in Fig. S2A″,B″. The corresponding plot profile was obtained on Image J and allowed us to determine the fluorescence intensity within the cluster and the fluorescence intensity of the background . After removing the background value, the integration of the signal under the curve gave us the value of the NiGFP signal present in the cluster. For each organ, we have determined the number of clusters and their intensity. By adding the intensity of each cluster, we obtained the total NiGFP intensity at the lateral pIIa-pIIb interface.To quantify the signal of NiGFP at the basal interface of SOP daughter cells, we first manually identified each individual NiGFP and Par3::Scarlet-positive cluster present in every confocal section (thickness 0.5 μm) as shown in The fluorescence intensity was calculated with ImageJ, at the two-cell stage, as previously described . For SpdPhotobleaching of NiGFP/NiGFP5 was performed using a Zeiss AiryScan (LSM 880 with AiryScan module) with the 488 nm laser wavelength (20 mW) at 90% of the maximal power and by using the 63× oil objective (NA 1.4). Two consecutive iterations were performed. The FRAP area was defined by the pIIa/pIIb interface. A control area corresponding to an adjacent epidermal cell interface was measured to obtain the general photobleaching of the sample over the period of acquisition. All FRAP data were analysed using the easyFRAP software tool (https://easyfrap.vmnet.upatras.gr/?AspxAutoDetectCookieSupport=1). Half-time (t1/2) and mobile fraction were then extracted with GraphPAD Prism software using a one-component equation.neur promoter. Because GAP43::IR is excluded from the nuclei, nuclei were defined using an area within cells where the GAP43::IR signal background is the lowest. Nuclei measurements  with a 405 nm diode (30 mW) at 1.8% power (40 iterations and pixel dwell time: 1.52 µs) according to pnr-GAL4>dsRNA, AP-47dsRNAstrat SOP daughter cells were performed at 15, 20, 25 and 30 min after anaphase transition. Before photoconversion, z-stacks were performed to localize the apical interface using GAP43::IR only first, and then with green NimMaple3. A z-stack was then acquired 35 min after the anaphase onset to quantify nuclear NimMaple3 in photoconverted SOP , and leads to higher photoconversion efficiency . A normalization factor (1.59) was applied on nuclei photoconversion assays .The NimMaple3 signal at the apical interfaces between epidermal cells in the wild-type situation is, on average, 1.59-fold higher than upon silencing of t-test, if the data followed a normal distribution, or an unpaired Wilcoxon test or an unpaired Mann–Whitney test, if the data did not follow a normal distribution. The normal distribution was tested by a Shapiro test. Statistical significances are represented as follows: not significant (ns)≥0.05; *P<0.05; **P<0.01; ***P<0.001 and ****P<0.0001.Statistical analyses were carried out using the GraphPad Prism 6.07 software. A two-way ANOVA was performed for the quantification of the enrichment of Notch at the apical and at the basal pIIa-pIIb interface with a multiple comparison Bonferroni test. For other quantifications, we performed a


In acute respiratory distress syndrome (ARDS), increased pulmonary vascular permeability makes the lung vulnerable to edema. The use of conservative as compared to liberal fluid strategies may increase the number of ventilator-free days and survival, as well as reduce organ dysfunction. Monitoring the effects of fluid administration is of the utmost importance; dynamic indexes, such as stroke volume and pulse pressure variations, outperform static ones, such as the central venous pressure. The passive leg raise and end-expiratory occlusion tests are recommended for guiding fluid management decisions. The type of intravenous fluids should also be taken into consideration: crystalloids, colloids, and human albumin have all been used for fluid resuscitation. Recent studies have also shown differences in outcome between balanced and non-balanced intravenous solutions. In preclinical studies, infusion of albumin promotes maintenance of the glycocalyx layer, reduces inflammation, and improves alveolar-capillary membrane permeability. Fluids in ARDS must be administered cautiously, considering hemodynamic and perfusion status, oncotic and hydrostatic pressures, ARDS severity, fluid type, volume and infusion rate, and cardiac and renal function. Of note, no guideline to date has recommended a specific fluid composition for use in ARDS; most physicians currently follow recommendations for sepsis. Acute respiratory distress syndrome (ARDS) is a multifactorial syndrome caused by different etiologies, which can be pulmonary or extrapulmonary . A majorMitigating lung edema, accelerating its resorption, and maintaining systemic perfusion and distal organ function through modulation of fluid intake seem to be beneficial in ARDS. Several issues should be considered during fluid infusion in this patient population: (1) the etiology, severity, and phase of ARDS; (2) static and dynamic methods to monitor fluid administration; (3) the type of fluids ; and, (4) if albumin is used, its concentration. The present review will discuss recent studies on different fluid types and strategies and their effects on the aforementioned aspects of ARDS.Early ARDS is characterized by increased alveolar-capillary barrier damage, which results in lung edema, as well as reduced peripheral perfusion . IncreasChoosing the optimal fluid strategy (both regarding quantity and type of fluid) is one of the greatest clinical challenges in ARDS management, since both impaired hemodynamics and electrolyte disturbances must be corrected , 7, but Starling’s classic model states that transvascular fluid exchange depends on a balance between hydrostatic and oncotic pressure gradients Fig.  8]. How. How8]. Conservative fluid replacement strategies have been recommended to prevent lung edema. These strategies are associated with less invasive mechanical ventilation, less organ dysfunction, and a trend toward less renal replacement therapy , 23. A rTo date, no ideal method has been developed to continuously monitor the response to fluid replacement in ARDS. Four questions have been described to guide fluid strategy in patients with septic shock: “When to start intravenous fluids?”, “When to stop intravenous fluids?”, “When to start de-resuscitation or active fluid removal?”, and, finally, “When to stop de-resuscitation?” . These qThe passive leg raise test (PLR)  consistsVT) ≥ 8 ml/kg, regular heart rhythm, and respiratory system compliance ≥ 30 ml/cm H2O [SVV and PPV, derived from the arterial pressure signal, represent the influence of the periodic increase in intrathoracic pressure due to invasive mechanical ventilation. For SVV and PPV to be accurate predictors of fluid responsiveness, at least four conditions are necessary: sedation and paralysis, tidal volume  was designed to assess the impact of 20% albumin with crystalloid with a target albumin level of > 30 g/L in patients with septic shock, with 28-day mortality as the primary endpoint. Another ongoing clinical trial (NCT03654001) is comparing 20% albumin plus balanced solutions, 20% albumin plus saline, and balanced solutions alone in septic shock. No clinical trial has evaluated albumin or other types of fluids specifically in ARDS, and none is ongoing.Crystalloids can also be categorized as balanced or non-balanced  solutions. Balanced solutions contain a precursor anion of bicarbonate in their composition: lactate in Ringer’s lactate, acetate in Ringer’s acetate and Plasma-Lyte®, and others such as gluconate, malate, citrate, and succinate in less commonly used solutions . Non-balBalanced solutions provide some advantages, not least the choice of anion: they (1) replace bicarbonate, (2) are rapidly metabolized, (3) are nontoxic, and (4) maintain pH at 4–8, minimizing hemolysis and endothelial cell damage in peripheral circulation . HoweverHowever, clinical studies comparing balanced versus unbalanced crystalloids in intensive care have shown conflicting results regarding renal outcomes and mortality –48. The Colloids are water-based solutions containing permeable ions and non-permeable molecules, which can be plasma-derived  or synthetic  and are unable to cross intact semipermeable biological membranes. The intravascular duration of colloid-derived volume expansion depends on the rate of metabolism and clearance of its constituent molecules . ColloidHuman albumin is commercially available in several concentrations: iso-oncotic (5%) and hyper-oncotic (20% and 25%) . HoweverAlbumin has many other properties in addition to its effects on intravascular volume, including antioxidant activities and molecular transport . ClinicaLung tissue endothelial VE-cadherin expression is reduced in ARDS . In vitrActivation of endothelial cells  leads to the production of mediators (such as angiopoietin-2) and neutrophil infiltration, thus increasing lung vascular permeability. Neutrophil-mediated epithelial injury has been associated with multiple mechanisms: tumor necrosis factor (TNF)-α, neutrophil extracellular traps (NETs), and TNF-related apoptosis-inducing ligand (TRAIL) , 66. In Activation of the coagulation cascade is also involved in the pathophysiology of ARDS . AlbuminAlbumin reduces lung edema and restores/maintains glycocalyx integrity in experimental ARDS . PerhapsARDS is characterized by lung edema and damage to alveolar epithelium, endothelium, and extracellular matrix. Fluid administration is thus very challenging in these patients, since it can increase cardiac output and peripheral perfusion—thus improving tissue oxygenation and organ function—but may also result in serious adverse events. Fluid overload may be particularly dangerous in ARDS due to the possibility of worsening pulmonary edema.Different methods have been used to assess the appropriateness of fluid infusion. However, clinical parameters frequently employed at bedside, such as mean arterial pressure, urine output, oxygen consumption, lactate, and central/mixed venous oxygen saturation, are actually of little use. Static monitoring parameters  are unreliable markers of fluid responsiveness; thus, dynamic parameters  have been recommended instead.In short, fluids in ARDS must be administered cautiously, considering hemodynamic and perfusion status, oncotic and hydrostatic pressures, ARDS severity, fluid type, volume and infusion rate, and cardiac and renal function. To date, there is no published evidence to recommend any specific fluid composition over another in ARDS, and most physicians follow recommendations designed for sepsis instead. Further research is needed to help elucidate this issue with a view to improving clinical management of ARDS.


Proton-pump inhibitors (PPIs), frequently prescribed to lower gastric acid secretion, often exert an effect on the absorption of co-medicated drug products. A previous study showed decreased plasma levels of the lipophilic drug ritonavir after co-administration with the PPI Nexium (40 mg esomeprazole), even though duodenal concentrations were not affected. The present study explored if a PPI-induced decrease in gastrointestinal (GI) fluid volume might contribute to the reduced absorption of ritonavir. In an exploratory cross-over study, five volunteers were given a Norvir tablet (100 mg ritonavir) orally, once without PPI pre-treatment and once after a three-day pre-treatment with the PPI esomeprazole. Blood samples were collected for eight hours to assess ritonavir absorption and magnetic resonance imaging (MRI) was used to determine the gastric and duodenal fluid volumes during the first three hours after administration of the tablet. The results confirmed that PPI intake reduced ritonavir plasma concentrations by 40%. The gastric residual volume and gastric fluid volume decreased by 41% and 44% respectively, while the duodenal fluid volume was reduced by 33%. These data suggest that the PPI esomeprazole lowers the available fluid volume for dissolution, which may limit the amount of ritonavir that can be absorbed. Although additional factors may play a role, the effect of PPI intake on the GI fluid volume should be considered when simulating the absorption of poorly soluble drugs like ritonavir in real-life conditions. By targeting the final and universal step in the complex and highly regulated acid secreting pathway, they outperform their predecessors in terms of efficacy and safety. However, as PPIs are so frequently (over)prescribed, concern has recently been risen regarding the adverse effects of prolonged PPI treatment. Adverse effects include C. difficile-associated diarrhoea and increased risk of acute and chronic kidney disease, and myocardial infarction [Since their introduction three decades ago, proton-pump inhibitors (PPI) have taken over as the leading drug class in the treatment and prevention of various gastric acid related diseases , peptic ulcer bleeding and non-steroidal anti-inflammatory drug (NSAID) induced ulcers) . Their mfarction . In addifarction .With respect to the absorption of orally administered drugs, the change in gastric pH due to PPI treatment may affect poorly soluble basic drug compounds that rely on the gastric acidity to dissolve and remain in a supersaturated state upon entering the small intestine creating the driving force for absorption . Drug-drP 4.3) weak base (pKa 2.6) with pH-dependent solubility, formulated as an amorphous solid dispersion (Norvir 100 mg) [In a recent study in healthy human volunteers, Van Den Abeele et al. assessed the effect of the PPI esomeprazole on the luminal and systemic concentrations of the BCS class IV drug ritonavir, an HIV protease inhibitor often used as a booster with other HIV protease inhibitors . Ritonav 100 mg) . In norm 100 mg) . It can In the present study, an exploratory cross-over clinical trial was performed similar to the one described above . Systemi6H14, >99%, p.a.). Hanks’ Balanced Salt Solution without phenol red (HBSS) was ordered from Lonza . Merck  supplied sodium hydroxide  and disodiumtetraborate decahydrate . Ritonavir (RTV) powder was purchased from Sigma-Aldrich  for analytical purposes. Lopinavir (LPV) powder was donated by Hetero Drugs Ltd. . Water was purified using a Maxima System .Acetonitrile (ACN) and methanol (MeOH) (LC-MS grade) were purchased from Biosolve . Methanol (HPLC grade) and dimethylsulfoxide  were ordered from Acros Organics . Ethyl acetate  were purchased from VWR , while Chem-Lab  supplied hexane  and Nexium  tablets were ordered via the hospital pharmacy of the University Hospitals Leuven .Control condition: Oral intake of one Norvir tablet (100 mg ritonavir) with 240 mL of tap water after a fasting period of 12 h.PPI condition: Oral intake of one Norvir tablet (100 mg ritonavir) with 240 mL of tap water after a fasting period of 12 h preceded by a once-daily dose of the PPI esomeprazole  for three days .In this exploratory clinical trial, five healthy volunteers  participated in a two-arm crossover study (washout period: at least one week) comprising the following test conditions:Possible volunteer candidates (aged 18–35 years) were excluded from the trial in the case of disease, chronic use of medication (birth control excluded), gastrointestinal pathology or prior surgery, hepatitis B/C and/or HIV infection, and (possible) pregnancy. The clinical trial was approved by the competent authority in Belgium  and the Ethics Committee Research UZ/KU Leuven (S59578—amended), and was performed in accordance with the rules in the Declaration of Helsinki.All volunteers provided written informed consent before participating in the trial. Each trial day, the participant came to the radiology department at the University Hospitals Leuven after an overnight fast (at least 12 h). Water was allowed until the night before the trial except for the volume needed to swallow the PPI tablet. For the PPI condition, participants were pre-treated with a once-daily dose of Nexium (just before breakfast) starting two days prior to the trial day. On the morning of the trial, the final Nexium tablet was taken 2 h prior to the arrival time at the hospital, with as little water as possible to swallow the tablet. A daily dose of 40 mg esomeprazole for three days has shown to elevate and sustain the gastric and duodenal pH to values around 7.1 and 7.5, respectively , indicatUpon entering the MRI system, a rectangular dStream FlexCoverage Anterior coil  was placed on the torso of the subjects. A scouting sequence was used in the coronal and transversal plane to allow correct sequence positioning. At each time point, three standardized sequences were used to acquire images; sequence parameters are depicted in Images were analysed using Horos™ , a free open source medical images viewer. Gastric and duodenal fluid volumes reported in this study were acquired with sequence 1. Although gastric fluid volumes determined with sequences 2 and 3 were similar, sequence 1 was chosen because (i) no artefacts were present  and (ii) both the stomach and duodenum were completely visible (in contrast to sequence 3 where the duodenum of some volunteers was not visible). Images made with sequence 2 yielded a superior detail of the small intestine; however, due to the ultra-rapid acquisition, both the vena cava and aorta have a similar contrast as the fluid in the intestines. This made the sequence unsuitable for fluid determination as the duodenum passes these blood vessels which would interfere with the outlining of the fluid present in the duodenum. Sequence 2 was therefore used as a localizer for the duodenum . Plasma was collected and stored at −20 °C pending analysis. For analysis, a liquid-liquid extraction was performed in which 50 µL of plasma was added to 450 µL HBSS and vortexed for a few seconds. Lopinavir was used as internal standard: 100 µL of a 200 nM stock solution in 25% v/v DMSO-water was added to the mixture. Before extraction, the pH was adjusted by adding 500 µL of 40 mM borax buffer (pH = 9), followed by 5 mL of the extraction medium hexane—ethyl acetate  and shaken for 1 min. The mixture was centrifugated at 2880× g, for 5 min at 4 °C and the organic layer was transferred to a new test tube and evaporated to dryness under a gentle airflow. The residue was redissolved in 1 mL of methanol and vortexed for 30 s before another evaporation cycle. The resulting residue was reconstituted in 200 µL methanol—water  awaiting analysis.Blood samples were stored on ice during the remainder of the study day, before centrifugation at 2880× ® column  using isocratic elution with methanol—0.1% formic acid  followed by a wash gradient and re-equilibration (2) flow 700 L/h and temperature 500 °C, and cone gas (N2) flow 400 L/h. Fragmentation was induced by collision with argon. The system was operated in multi-reaction monitoring (MRM) mode with the mass transitions according to For the analysis, a Waters ACQUITY UHPLC H-Class system consisting of a quaternary pump was used combined with a Waters Xevo TQ-S micro mass spectrometer . Separation took place on a Kinetexibration . Column The method was validated according to ICH guideline M10 on bioanalytical method validation and passed on selectivity, linearity and range (1 nM–4000 nM), intra- and interday accuracy (bias < 10%) and precision (relative standard deviation < 5%) tested on 4 QC levels , recovery (95%) and freeze-thaw stability (100%). In addition, no matrix effect or degradation was observed during the experiments. Carryover was observed when higher concentrations were injected but was mitigated by blank injections. QC samples were included in each run to show method suitability.The explorative nature of the study did not allow for a detailed statistical analysis. Results are presented as median (range) unless otherwise specified. Wherever appropriate, individual profiles are given to further clarify the observations made in this study.max, tmax and AUC0-480min in both control and PPI conditions, and from the median profiles depicted in To confirm the effect of pre-treatment with the PPI esomeprazole on the absorption and systemic disposition of ritonavir, its plasma concentrations were determined after intake of a Norvir tablet. max decreased from 1129 nM (range 833.0 to 2194 nM) in the control condition to 826.6 nM (329.6 to 1302 nM) in the PPI condition. In addition, median tmax slightly delayed from 193 min (range 175–240 min) to 225 min (175–270 min). Despite a relatively large intersubject variability, both the median and individual plasma concentration-time profiles (0-180min) decreased upon PPI pre-treatment   b. OveralTo explore a possible link between the observed PPI effect on ritonavir absorption and gastrointestinal fluid volumes, we first determined the gastric residual volume (GRV), i.e., prior to ritonavir intake. Compared to the control condition, PPI pre-treatment lowered the GRV in all volunteers, with a relative decrease ranging from 14.6% to 81.1% .After intake of the ritonavir tablet with 240 mL of water, the median gastric fluid volume over time profiles depict generally reduced fluid volumes in the PPI condition compared to control a. The gaIn comparison to the gastric fluid volume, the duodenal fluid volume appeared more stable, as can be seen in the median fluid volume over time profile b. Again,max (median 39.0% vs. 49.6%) and AUC0–480min (median 40.5% vs. 39.5%). The delay in tmax differed between the studies (median 10% vs. 50%) but was highly variable in both. Due to the aberrant ritonavir plasma concentration observed after 270 min in HV03 (PPI condition), plasma concentrations from this volunteer were omitted from the calculations of Cmax, tmax and AUC0-480min. Irrespective of the aberrant point, however, the effect of PPI co-intake in HV03 was in line with the effect observed in the other volunteers, as can be seen in max or AUC of ritonavir. Chiu et al. [The effect of the PPI esomeprazole on the absorption of ritonavir has previously been investigated by Van Den Abeele et al., who first discovered that a decreased systemic exposure to ritonavir upon PPI pre-treatment could not be explained by a reduced driving force for absorption as the duodenal concentrations of ritonavir were unaffected . By usinu et al.  did obseu et al. . In addi+/K+ ATPase of the parietal cells, thus reducing the gastric acid secretion. In addition, it has been shown that pre- and postprandial gastric fluid volume is decreased [Esomeprazole inhibits the Hecreased ,11,25. Tecreased  . Using TrueFISP, however, the aorta and vena cava interfere as they light up like fluids. For this reason, it was decided to use sequence 1 (turbo spin-echo) with a slower acquisition but without interference of the blood vessels, to determine gastric and duodenal fluid volumes. To accurately outline the duodenum and account for the intestinal movement, however, the TrueFISP (sequence 2) was used as a localizer for the duodenum and duodenojejunal flexure. Due to the inherent slicing  of the MRI, a slight under- or overestimation is easily made when the slice thickness is large compared to the intestinal distention. However, by considering these minor flaws  and by standardization of the sequence protocol and data analysis, one can obtain accurate and comparable results in a non-invasive manner .The present study confirmed that pre-treatment with the PPI esomeprazole substantially lowers the rate and extent of ritonavir absorption. By combining the PK study with MRI, the study further brought forward reduced gastric and duodenal fluid volumes as a viable mechanism behind the decreased exposure. The performance of amorphous solid dispersions, like Norvir, is highly depending on the composition and volume of the dissolution media . Duodenamax in the present study. The more gradual exposure of ritonavir to the metabolic enzymes may reduce the saturation of the metabolic pathways leading to a more extensive metabolism, which could strengthen the PPI effect on the systemic exposure to ritonavir. It should be noted that the PPI esomeprazole is mainly metabolized by CYP2C19 and to a lesser extent by CYP3A4. Direct drug-drug interactions due to competition for CYP3A4 have been investigated, but no clinically relevant interactions have been reported [It is important to note that the PPI-induced decrease in duodenal fluid volume is relatively limited as compared to the decrease in ritonavir plasma concentration . In addition, no direct correlation could be made between the gastric or duodenal fluid volume and the ritonavir plasma concentration in this small-scale study, raising the question if more factors are in play. Of course, one must take into account that, in this study, only duodenal volumes are reported, while drug absorption takes place along the entire small intestine. Fluid volumes in more distal parts  may also decrease with PPI treatment, possibly strengthening the overall effect. Another factor which must be considered is the first-pass metabolism of ritonavir, which may play an important role in the observed difference. Ritonavir is mainly metabolized in the liver by the CYP3A family and is, in itself, a potent inhibitor of CYP3A4; a smaller fraction is metabolized by CYP2D6. Studies have shown that the metabolism of ritonavir has at least one saturable process that is quickly saturated even after a single dose . Followireported . MoreoveAlthough the exploratory design of the present study does not allow formal conclusions, the observed median and individual data clearly suggest that intake of the PPI esomeprazole may reduce fluid volumes in not only the stomach but also the duodenum. A decrease in the fluid volume available for dissolution may contribute to the observed reduction in systemic exposure of the poorly soluble drug ritonavir upon PPI co-administration. The precise impact of the observed fluid volume changes, in combination with the PPI-induced drop in gastric pH, on the absorption of ritonavir will be further evaluated using in vitro dissolution testing in combination with physiologically-based pharmacokinetic (PBPK) modelling. PBPK modelling has previously been utilized to predict in vivo drug-drug interactions between weakly basic compounds and PPIs or other acid-reducing agents ,32,33. F


Mechanistically, transforming growth factor‐β1 (TGF‐β1) transcriptionally activated PSG9 expression through enhancing the enrichment of Smad3 and Smad4 onto PSG9 promoter regions containing two putative Smad‐binding elements (SBEs). Mutation of both SBEs in the PSG9 promoter, or knockdown of TGF‐β receptor 1 (TGFBR1), TGFBR2, Smad3, or Smad4 impaired the ability of TGF‐β1 to induce PSG9 expression. Consequently, PSG9 contributed to TGF‐β1‐induced epithelial‐mesenchymal transition (EMT) and breast cancer cell migration and invasion. Moreover, PSG9 enhanced the stability of Smad2, Smad3, and Smad4 proteins by blocking their proteasomal degradation, and regulated the expression of TGF‐β1 target genes involved in EMT and breast cancer progression, thus further amplifying the canonical TGF‐β/Smad signaling in breast cancer cells. Collectively, these findings establish PSG9 as a novel player in breast cancer progressionvia hijacking the canonical TGF‐β/Smad signaling, and identify PSG9 as a potential plasma biomarker for the early detection of breast cancer.Pregnancy‐specific glycoprotein 9 (PSG9) is a placental glycoprotein essential for the maintenance of normal gestation in mammals. Bioinformatics analysis of multiple publicly available datasets revealed aberrant PSG9 expression in breast tumors, but its functional and mechanistic role in breast cancer remains unexplored. Here, we report that PSG9 expression levels were elevated in tumor tissues and plasma specimens from breast cancer patients, and were associated with poor prognosis. Gain‐ or loss‐of‐function studies demonstrated that PSG9 promoted breast cancer cell proliferation, migration, and invasion via hijacking the canonical TGF‐β/Smad signaling and identify PSG9 as a potential plasma biomarker for the early detection of breast cancer.Our findings establish PSG9 as a novel player in breast cancer progression  ANGPTL4angiopoietin‐like 4EMTepithelial‐mesenchymal transitionIL‐11interleukin 11PSG9pregnancy‐specific glycoprotein 9SBESmad‐binding elementTGFBRTGF‐β receptorTGF‐βtransforming growth factor‐β1Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer‐related death among females worldwide.The pregnancy‐specific glycoproteins (PSGs) are the most abundant placental proteins in the maternal bloodstream during pregnancy in rodents and primates.Analyses of multiple publicly available databases22.1Human breast cancer cell lines , normal human mammary epithelial cell lines (HMEC and HBL100), and human embryonic kidney 293T cell line (HEK293T) were obtained from the Cell Bank of the Chinese Academy of Sciences . MDA‐MB‐231 (MDA‐231)‐derived LM2‐4173 and LM2‐4175 cells were kindly provided by Guohong Hu . Both cell lines have enhanced lung metastasis potential compared to their parental counterpart.MCF10A sublines were cultured as described in detail previously.2.2PSG9 promoter region was amplified by PCR and cloned into a pGL3‐basic luciferase reporter vector . Mutations of the SBEs in the PSG9 promoter were performed by PCR‐based methods. All construct sequences were verified by DNA sequencing. The primers used for molecular cloning are provided in Table S1. Transient plasmid transfection was performed using Neofect DNA transfection reagent  according to the manufacturer's protocol. Lentiviral infection and generation of stable cell lines were carried out as described previously.cDNAs for PSG9 (#CH879913), Smad2 (#CH809987), Smad3 (#CH881773), and Smad4 (#CH889836) in pEnter vector were obtained from Vigene Biosciences, and then subcloned into the lentiviral vector pCDH‐CMV‐MCS‐EF1‐Puro  to generate HA‐PSG9, Flag‐Smad2, Flag‐Smad3, or Flag‐Smad4, respectively, using CloneEZ PCR Cloning Kit . Expression vector encoding V5‐ubiquitin has been described previously.2.3Specific siRNAs targeting TGFBR1, TGFBR2, Smad2, Smad3, Smad4, and corresponding negative control siRNAs (siNC) were purchased from GenePharma , and their targeting sequences were listed in Table S2. The siRNA duplexes were transfected into cells using Lipofectamine 2000 transfection reagents  following the manufacturer's instructions. Knockdown efficiency was determined by immunoblotting analysis after 48 hours of transfection.2.4http://rsb.info.nih.gov/ij/), and was normalized to the internal control vinculin.All primary antibodies used in this study are listed in Table S3. The secondary antibodies for immunoblotting assays and immunofluorescent staining were purchased from Cell Signaling Technology. Immunoblotting analysis and immunoprecipitation assays were performed following standard protocols as described previously in detail.2.5−ΔΔCt method and was normalized to internal control GAPDH. Chromatin immunoprecipitation (ChIP) assays were performed using SimpleChIP Enzymatic Chromatin IP Kit (magnetic beads)  according to the manufacturer's instructions. Quantitative results are displayed as corresponding fold change, and antirabbit IgG was used as a negative control. Primers used for qPCR and ChIP assays are listed in Tables S4 and S5.Total RNA was isolated using TRIzol reagent , and 1 μg of RNA was subjected to cDNA synthesis using PrimeScript RT Master Mix . Quantitative real‐time PCR (qPCR) was performed using SYBR Premix Ex Taq  following the manufacturer's instructions. The expression levels of the indicated mRNAs were calculated using the 22.6PSG9 promoter activities were normalized to the corresponding values of the cotransfected Renilla luciferase gene.Cells were transfected with 200 ng of pGL3 or pGL3‐PSG9 expression vector using Lipofectamine 2000. Renilla luciferase expression vector (pRL) (5 ng) was also transfected into cells as a transfection control. After 48 hours of transfection, luciferase assays were performed using a Dual‐Luciferase Reporter Assay System  according to the manufacturer's instructions. The 2.73 cells were plated into 96‐well plates in triplicates and cultured for 5‐7 days. Total 10 μL CCK‐8 solution  was added to each well. The plates were incubated in an incubator at 37°C for 3 hours, and then absorbance at 450 nm (A450) was determined. For colony formation assays, 1 × 103 cells were plated into six‐well plates in triplicates and allowed to grow out for 10‐14 days. Cells were fixed by methanol, stained with 0.2% crystal violet solution and photographed. Colonies consisting of >50 cells were counted.For cell counting kit‐8 (CCK‐8) assays, a total of 1 × 102.8For wound‐healing assays, cells were plated into six‐well plates and allowed to grow to 90% confluence. Then, a 200 μL pipette tip was used to generate the wound. Cells were then cultured in serum‐free medium, and the wound healing was observed at the indicated times with inverted microscope, and photographs were taken. Transwell migration and invasion assays were performed using Boyden chambers with 8 μm pores  and Matrigel Invasion Chambers , respectively, as described previously.2.96 MDA‐MB‐231 cells stably expressing pCDH and HA‐PSG9 and LM2‐4175 cells stably expressing shNC and shPSG9 were injected into the mammary fat pad of 6‐week‐old BALB/c female mice . The mice were killed after 8 weeks of the inoculation, primary tumors were harvested, and tumor weight was determined. For experimental metastasis assays, 2 × 106 MDA‐MB‐231 cells stably expressing pCDH and HA‐PSG9 as well as LM2‐4175 cells stably expressing shNC and shPSG9 in 200 μL of PBS were injected in the tail vein of 6‐week‐old BALB/c female nude mice . After 6 weeks of injection, the lungs were excised, fixed in Bouin solution overnight, and lung colonies were counted under a Nikon SMZ1500 stereomicroscope .All animal studies were approved by the Institutional Animal Care and Use Committee of Shanghai Cancer Center, Fudan University. For tumorigenesis assays, 2 × 102.10All procedures were conducted in accordance with the Declaration of Helsinki and International Ethical Guidelines for Biomedical Research Involving Human Subjects, and approved by the institutional ethics review board of Fudan University Shanghai Cancer Center. All breast tumor samples and plasma samples were obtained from patients with breast cancer who underwent surgery at Fudan University Shanghai Cancer Center. These patients did not receive any therapies before surgical operation.2.11g for 10 min at 4°C. The plasma was stored at −80°C for further analysis. PSG9 concentrations in plasma samples were determined using PSG9 human ELISA kit  according to the manufacturer's instructions. All experiments were conducted in duplicate.Peripheral blood samples were collected into vacuum tubes with EDTA anticoagulant and spun at 1000  ×  2.12A total of 161 primary breast cancer specimens were obtained from the Department of Pathology, Fudan University Shanghai Cancer Center. IHC staining was performed as previously described,2.13The expression and purification of GST‐tagged or His‐tagged proteins were performed as described previously.2.14t‐test was used to compare data between two groups using SPSS20 . The probability of survival was estimated with the Kaplan‐Meier method and differences between groups were evaluated by the log‐rank test. P‐values of less than .05 were considered statistically significant.All data are presented as the mean ± standard deviation from at least three independent experiments. The unpaired two‐tailed Student's 33.1To investigate the potential role of the members of PSG gene family in breast cancer progression, we first analyzed their mRNA levels in breast tumors and normal breast tissues using RNA‐sequencing data from The Cancer Genome Atlas (TCGA) and Genotype‐Tissue Expression (GTEx) databases.P = .026). Moreover, subclassified analyses revealed that the expression levels of PSG9 were positively correlated with the presence of LN metastasis (P = .023) and with HER2 positivity (P = .036) (Table S6). In contrast, there was no significant correlation between PSG9 expression levels and other clinicopathological factors, including age, menopausal status, tumor grade, and ER/PR status in the case of the limited sample size (Table S6).To validate these results, we examined PSG9 protein levels in nine pairs of matched adjacent normal breast tissues, primary breast tumors, and metastatic lymph node (LN) tissues by immunoblotting with an anti‐PSG9 antibody. Results showed that PSG9 levels were elevated in primary breast tumor tissues, particularly in metastatic LNs, compared to matched normal breast tissues Figure . Metastavs 84 cases, respectively). As expected, high plasma PSG9 levels were associated with poor DFS of breast cancer patients . In addition, plasma PSG9 levels were positively correlated with the presence of LN metastasis, but not other clinicopathological factors (Table S7). Collectively, both bioinformatic and experimental data suggest that PSG9 is upregulated in breast tumors and is associated with poor prognosis of breast cancer patients.As PSG9 is a secreted glycoprotein, we next measured plasma levels of PSG9 in 20 healthy, nonpregnant females and 60 breast cancer patients by ELISA. As shown in Figure 3.2in vivo in light of subsequent mouse xenograft experiments, we stably expressed HA‐PSG9 in MDA‐231 cells and knocked down PSG9 in LM2‐4175 and Hs578T cells by infection with lentiviral vectors encoding HA‐PSG9 and shRNA targeting human PSG9 (shPSG9), respectively. The expression status of PSG9 in the resultant cell lines was verified by immunoblotting , and BT549. Results showed that PSG9 was frequently upregulated in breast cancer cell lines compared to normal HMEC control Figure . Furtherg Figure . CCK‐8 ag Figure  and geneg Figure  as compag Figure  and colog Figure .in vivo, MDA‐231 cells stably expressing pCDH and HA‐PSG9 were injected into the mammary fat pad of 6‐week‐old female BALB/c nude mice. Consistent with in vitro results, ectopic expression of PSG9 significantly promoted tumor growth in mice implanted with MDA‐231 cells stably expressing HA‐PSG9 as compared with mice implanted with pCDH expressing control cells PSG9 promoter were found, located at −996 to −991 and −95 to −90 , indicating that PSG9 may be regulated by the TGF‐β/Smad signaling pathway. To test this notion, MDA‐231 and Hs578T cells were treated with or without 10 ng/mL TGF‐β1, the most abundant and ubiquitously expressed TGF‐β isoform, for the indicated times, and the expression levels of PSG9 were evaluated by immunoblotting and qPCR analyses. Results showed that TGF‐β1 treatment induced PSG9 expression at both protein , and then treated with or without 10 ng/mL TGF‐β1 for the indicated times. Luciferase reporter assays revealed that TGF‐β1 significantly enhanced the activity of the PSG9 promoter in a time‐dependent manner  and three Smad proteins (Smad2/3/4).s Figure . ChIP ass Figure . Furtherd Figure . These r3.5TGF‐β is a potent inducer of EMT both during development and in cancer. To understand the role of PSG9 in TGF‐β1‐induced EMT, we stably expressed HA‐PSG9 in human mammary epithelial cell line MCF10A, a commonly used cell model for TGF‐β‐induced EMT.3.6To further explore the impact of PSG9 on TGF‐β signaling, we next examined the protein expression levels of the key components of the TGF‐β signaling, including TGFBR1/2 and Smad2/3/4 in MDA‐231 cells stably expressing HA‐PSG9 by immunoblotting. Results showed that ectopic expression of PSG9 resulted in a significant increase in the protein levels of Smad2/3/4 but not TGFBR1/2 Figure . ConversTo address the possible mechanism for the above observations, we next examined the subcellular localization of PSG9 by immunofluorescent staining. Results showed that PSG9 was localized in both cytoplasm and nucleus . In agreement with our observations, another study recently reported that PSG9 is distributed in the cell membrane, cytoplasm, and nucleus of human umbilical vein endothelial cells and colorectal cancer cells.4In this study, we uncovered several interesting findings concerning the emerging role of PSG9 in breast cancer progression Figure . First, PSG9 promoter regions containing SBEs Table S7 Characterization of clinicopathological features of 161 patients with primary breast cancer (detection of plasma PSG9 levels by ELISA)Click here for additional data file.


The impacts of entropy generation and Hall current on MHD Casson fluid over a stretching surface with velocity slip factor have been numerically analyzed. Numerical work for the governing equations is established by using a shooting method with a fourth-order Runge–Kutta integration scheme. The outcomes show that the entropy generation is enhanced with a magnetic parameter, Reynolds number and group parameter. Further, the reverse behavior is observed with the Hall parameter, Eckert number, Casson parameter and slip factor. Also, it is viewed that Bejan number reduces with a group parameter. The study of magnetohydrodynamic flows with Hall currents has evinced interest attributable to its numerous applications in industries, such as MHD power generators, Hall current accelerators, Hall current sensors, and planetary fluid dynamics. Sato  was the Some different fluids are termed as non-Newtonian fluids such as Jeffrey fluid, viscoelastic fluid, power-law flow, Williamson fluid, micropolar fluid, and Casson fluid. Casson  was the e, In this paper, the magnetohydrodynamic flow of incompressible Casson fluid is considered. The flow is generated owing to the stretching surface with linear velocity th component of the deformation rate, According to Refs ,13, the Due to the above-mentioned assumptions and the boundary layer approximations, the governing equations of Casson fluid and generalized Ohm’s law with Hall current influence are given by: Subject to the boundary conditions: k, ρ, b, c, L, f, and h, are the kinematic viscosity, thermal diffusivity, thermal conductivity, fluid density, specific heat, temperature at the surface, ambient temperature, positive constants, characteristic length, dimensionless stream function, and dimensionless transverse velocity, respectively. By invoking Equation (10), Equation (5) is automatically satisfied whereas the other equations and the boundary condition take the following form:The following non-dimensional variables are defined as: x-direction z-direction x-and z-directions, respectively, and Here, prime denotes differentiation with respect to Using the similarity and dimensionless variables (10), we get:The local entropy generation rate is defined as Bejan ,21):(18):(18)21])In the entropy equation, the first term represents the heat transfer irreversibility, second term the fluid friction, and the last term due to the impact of the magnetic field. The characteristic entropy generation rate is expressed as:The dimensionless entropy generation can be expressed as follows:From Equation (22), Bejan number is in the range The emerging differential Equations (11)–(13) along with the relevant boundary conditions (14) are tackled numerically using the Runge-Kutta fourth order procedure with shooting technique. Numerical calculations were performed in the ranges m on the velocity components and the temperature field for two different cases. From m for both the cases. Physically, decreasing the conductivity m generates a magnetic damping force which boosts the velocity components of the fluid. It is also revealed that the velocity components are greater in the case (m. For the no-slip boundary case (m and slip factor m and m augments effective electric conductivity which in turn depreciates Be against magnetic parameter Be reduces with increasing η from the stretching sheet for both cases. Physically, an increase in Be is more pronounced with the rise in Be with various values of the Hall parameter η from the boundary for both cases. It is observed that increasing Be is more significant in the sense of magnitude for the slip case as compared to the no-slip case (η from the boundary for both cases of slip and no-slip. For the case (m which eventually dwindles the magnetic damping force. This serves to boost Ec retard the fluid flow adjacent to the stretching surface. This agrees with the fact that the temperature distributions are enhanced with an increase in Ec as shown in 1-The primary velocity 2-The secondary velocity 3-m whereas the opposite scenario is observed for the temperature field Both the velocity components 4-Enhancing the values of 5-Both the velocity components 6-Entropy generation 7-Entropy generation 8-Be reduces with rising η from the stretching sheet.Bejan number 9-Be enhances with rising η from the stretching sheet.Bejan number 10-Be is a decreasing function of Bejan number 11-Be diminishes with a rise in η from the stretching sheet.Bejan number 12-The impact of 13-The magnitude values 14-The values of 15-The values of 16-Both the magnitude values of In the present work, a numerical study of entropy generation on MHD Casson fluid with Hall current and slip factor has been addressed. The skin-frictions


Solitary fibrous tumours (SFTs) mainly originate from the visceral pleura and may protrude to the thoracic cavity, but intrapulmonary SFTs are extremely rare. We describe a rare case of SFT arising in the right lung of an 83-year-old man who underwent surgical excision. Chest computed tomography (CT) revealed a 10-mm tumour in the lower lobe of the right lung. The size of tumour gradually increased and reached 17 mm 2 years after the first radiologic examination. Considering the possibility of malignancy, wedge resection of the right lower lobe was performed via video-assisted thoracic surgery. Microscopically, the tumour consisted mainly of spindle-shaped cells. Immunohistochemical staining indicated the tumour was positive for CD34, STAT6, vimentin and bcl-2, but negative for cytokeratins, D2–40 and S-100. Based on the histological findings, the tumour was diagnosed as SFT. The patient has been in good health for 6 months since the surgery. Solitary fibrous tumours (SFTs) usually arise in the visceral pleura and less frequently in a wide variety of extra-pleural locations. Although SFTs can occur in the pulmonary parenchyma , intrapu18F-fluoro-deoxy-glucose positron emission tomography (FDG-PET) showed slight uptake in the right lung tumour (maximum standardized uptake: 1.71). Since malignancy could not be completely ruled out, surgery was scheduled. Wedge resection of the right lower lobe was performed using video-assisted thoracic surgery. Intraoperative frozen section diagnosis revealed a tumour with spindle cells that did not contain a malignant component.An 83-year-old man with a 60-pack-year history of smoking was found to have an abnormal shadow on chest radiography. He was asymptomatic and laboratory test results, including tumour markers, were all normal. Chest CT revealed a 10-mm-diameter tumour in the lower lobe of the right lung . Since tMacroscopically, the tumour was a well-circumscribed, yellowish-white mass measuring 17 mm in diameter. Microscopically, the tumour showed mainly spindle-shaped cells  and did SFTs arising from the visceral pleura may rarely grow inward into the peripheral lung . HoweverSeveral previous reports have included radiological findings of intrapulmonary SFTs and described them as well-demarcated tumours . Benign Immunohistochemical examination is useful for differentiating SFTs from other neoplasms. Although CD34 immunoreactivity has traditionally been used to differentially diagnose SFTs, it is not specific. Nuclear staining of STAT6 has been more recently established as a highly sensitive and specific marker of SFTs and is currently widely used for differential diagnosis of SFTs. STAT6 staining is also useful to reveal from other neoplasms revealing spindle cell proliferation , 8.Although SFTs are usually slow-growing and have a favourable prognosis, some are malignant and eventually result in death because of local recurrence or metastatic disease . The hisIn conclusion, we experienced an extremely rare case of intrapulmonary SFT that was resected after 2 years of follow-up on CT. Although preoperative diagnosis of SFT is difficult, we should be aware that slow-growing, well-circumscribed pulmonary nodules may be anSFT.None declared.None.