Subjects with conditions associated with pleural effusion were excluded from both populations. Clinical and laboratory data as well as kidney, liver and spleen volume, pleural fluid volume, free pelvic fluid and polycystic kidney disease genotype were evaluated. Pleural effusions were observed in 56 of 268 (21%) ADPKD subjects compared with 21 of 268 (8%) in controls (p < 0.0001). In a subpopulation controlling for renal function by matching estimated glomerular filtration rate (eGFR), 28 of 110 (25%) ADPKD subjects had pleural effusions compared to 5 of 110 (5%) controls (p < 0.001). Pleural effusions in ADPKD subjects were more prevalent in females  than males  and in males were weakly correlated with the presence of free pelvic fluid . ADPKD subjects with pleural effusions were younger (48 ± 14 years old vs. 43 ± 14 years old) and weighed less  than those without pleural effusions. For ADPKD subjects with pleural effusions, the mean volume of fluid layering dependently in the posterior–inferior thorax was 19 mL and was not considered to be clinically significant. Pleural effusion is associated with ADPKD, but its role in the pathogenesis of ADPKD requires further evaluation.Autosomal dominant polycystic kidney disease (ADPKD) has cystic fluid accumulations in the kidneys, liver, pancreas, arachnoid spaces as well as non-cystic fluid accumulations including pericardial effusions, dural ectasia and free fluid in the male pelvis. Here, we investigate the possible association of ADPKD with pleural effusion. ADPKD subjects ( PKD1 gene or PKD2 gene, which encode polycystin 1 and polycystin 2, respectively. These proteins are located on primary cilia, and are important for ciliary function [Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease . It is cfunction .Bronchiectasis and increased risk for nosocomial infections have been associated with ADPKD ,11. CystThis health insurance portability and accountability act (HIPAA) compliant study of existing patient data and images was approved by the local Institutional Review Board (IRB). All ADPKD subjects enrolled in the Rogosin ADPKD Repository provided written informed consent and underwent MR imaging as outpatients. Retrospective review of existing data on control patients was also IRB approved; the requirement for informed consent was waived.Inclusion criteria for ADPKD subjects were (1) diagnosis of ADPKD based upon the Pei criteria and (2) abdominal MRI with axial T2-weighted images covering from mid thorax down to below the kidneys . The excAge, gender, and renal function matched control subjects without ADPKD undergoing abdominal MRI were identified from the electronic medical records, including Epic and picture archiving computer system (PACS). Patients with risk factors for pleural effusion were excluded from the control population, including pneumonia, thoracic tumor, heart failure or surgery within 3 months. Renal function matching was only feasible in a subset of the subjects and was performed based upon the chronic kidney disease (CKD) stage. A subset of these patients with cine images, including the heart, was reported previously .PKD1 or PKD2 mutation) was extracted from the Rogosin ADPKD Research Repository database. Cystatin C was not measured in this study.Patient demographic information and laboratory data were extracted from the electronic medical records for the date closest to the date of MRI. Kidney, liver and spleen volumes were extracted from the MRI reports prepared prospectively at the time of imaging. Genotype  using the parameters shown in 0 MR images were analyzed by 3 independent observers  blinded to all patient information. These observers had 6, 10 and 30 years of experience interpreting body MRI. Pleural effusion was designated as present when there was sufficient dependent fluid in the posterior-inferior thoracic pleural space to create a T2 bright fluid band, distinct from sub-pleural fat, at least 2 mm thick over a distance of at least 3 cm on at least two consecutive T2-weighted images. Pleural effusion was quantified using manual contouring to annotate dependent pleural fluid on every axial image using ITK-SNAP software, version 3.8.0 (downloaded 12 June 2019)  .Mean and standard deviation (SD) were reported for normally distributed continuous variables. For non-normal distributions, determined by Shapiro–Wilk test, variables were reported as median and interquartile range. Frequency and percentage were calculated for categorical variables.t-test was used to assess the significance of normally distributed variables. The Mann–Whitney test was used to assess the statistical significance of non-normally distributed continuous variables. For multi-group continuous variables, analysis of variance was used to assess the statistical significance. For categorical variables, the Chi-squared test was used to assess statistical significance. Inter-observer and intra-observer agreement for identifying pleural effusion was assessed using kappa statistic. For measuring pleural fluid volume on MRI, inter-observer and intra-observer agreement was assessed by interclass correlation coefficient (ICC).For two-group matched continuous variables, p < 0.1 in point biserial correlations as a threshold to be included in the model.Bivariate analysis was used to assess the correlation between the presence of pleural effusion and gender, age, height, weight, body mass index (BMI), blood pressure (BP), liver volume, total kidney volume, spleen volume, free pelvic fluid, aspartate aminotransferase (AST), alanine transaminase (ALT), albumin, blood urea nitrogen (BUN), creatinine and estimated glomerular filtration rate (eGFR). Multivariate linear regression models were used to predict the mixed effect of the variables found to be potentially important from the bivariate analysis using Abdominal MRI was available in 268 subjects with ADPKD including 127 males and 141 females, mean age = 47 ± 14 (range = 18–84) years A. Comparn = 91), indeterminant abdominal finding requiring further investigation (n = 77), pain (n = 49), cancer with no active disease undergoing follow-up (n = 22), liver disease (n = 15), pancreatitis (n = 7), hematuria (n = 4), small bowel obstruction follow-up (n = 1), endometriosis (n = 1), and uterine leiomyomas (n = 1).Indications for MRI in the control subjects included inflammatory bowel disease follow-up  and similar for the two groups. By contrast, the eGFR in the entire ADPKD group was lower than in the control group in 2.Matching CKD stage was possible for a subpopulation of 110 ADPKD and 110 control subjects to control for kidney function . In thisThere was good agreement among the 3 observers for identifying pleural fluid on MRI with kappa = 0.78. There was excellent agreement in the measurement of pleural fluid volume, ICC = 0.98.p < 0.0001). For those ADPKD subjects with pleural effusion identified, the mean fluid volume was 20 ± 21 (range: 2–122) ml compared to 20 ± 27 (1–102) for control subjects with pleural effusions.Dependent pleural fluid was identified on axial T2-weighted MR images in 56 of 268 (21%) ADPKD subjects , compare2). To eliminate confounding due to a potential effect that this difference in eGFR may have a risk of pleural effusion occurrence, 110 ADPKD and 110 control subjects with similar eGFR were evaluated . As with the entire cohort of ADPKD and control groups, the ADPKD subjects in the GFR-matched subgroups also had a predominance of white sub-jects with slightly higher levels of BUN, albumin and AST, although they were all within the normal range.For the entire ADPKD group, eGFR was lower than in the control group . For this subgroup of ADPKD subjects with pleural effusion, the mean fluid volume was 21 ± 14 mL (range: 2–51 mL) compared to 18 ± 30 mL (range: 3–71 mL) for the control subgroup. This similarly higher prevalence of pleural effusion in ADPKD patients compared to controls for both the entire population and the sub-study controlling for eGFR supports the conclusion that the higher prevalence of pleural effusion in the ADPKD group was not due to CKD.Pleural effusion was identified on axial T2-weighted MR images in 28 of 110 (25%) ADPKD subjects, compared with 5 of 110 (5%) controls  with a PKD1 mutation had pleural fluid compared to 9 of 56 (16%) with a PKD2 mutation (p = 0.32) (p = 0.32). For 227 subjects in whom PKD gene mutation analysis was available, 17 of 80 (21%) with truncating PKD1 mutations had pleural fluid compared to 21 of 91 (23%) with non-truncating PKD1 mutations (p = 0.77) and 5 of 34 (15%) with truncating PKD2 mutations had pleural fluid compared to 4 of 22 (18%) with non-truncating PKD2 mutations (p = 0.73).Among 227 ADPKD patients with an identified pathogenic or likely pathogenic variant in either  = 0.32) . Mean plp = 0.02), between free pelvic fluid and the presence of pleural effusions in men. For 87 female ADPKD subjects with imaging extending sufficiently into the pelvis to assess for free fluid, 72 had free pelvic fluid, which is normally expected before menopause, and female ADPKD subjects showed no significant correlation, r = 0.1 (p = 0.38), between free pelvic fluid and the presence of pleural effusion. There was no correlation of pleural fluid with height, blood pressure, liver volume, height-adjusted total kidney volume, spleen volume, AST, albumin, BUN creatinine, or eGFR. Only one subject was treated with tolvaptan at the time of MRI; that subject did not have a pleural effusion.On bivariate regression, Multivariate analysis  showed tNone of the 56 ADPKD subjects with pleural effusion had any symptoms attributed to their pleural effusion. None had thoracentesis or other procedures to further characterize the pleural fluid ,5,6,7,8.For 39 (70%) of all ADPKD subjects with pleural effusion, follow-up MRI was available ranging from 1.9 to 10.6 (mean = 5) years with a range of 2 to 7 scans (mean = 3.3) per subject. In these 39 ADPKD subjects with pleural effusions who had multiple MRIs, pleural effusion was identified on all follow-up MRIs in 35 of 39 (90%) . For theADPKD is associated with abnormal fluid collections, including cysts in many organs, free pelvic fluid, dilated cisterna chyli, dural ectasia, pericardial effusions. In the current study of 268 ADPKD subjects and matched controls, we found that the pleural space is another location of increased fluid, with pleural effusion present in ~20% of ADPKD patients, approximately 3-fold greater than in the control group of patients without ADPKD. The multivariable model found a significant association of pleural effusion with younger age. Although pleural effusion can occur in severe chronic kidney disease, it was significantly more common in a subgroup of ADPKD subjects than in a control population matched for age, sex and renal function. Moreover, the high prevalence of pleural effusion occurred in ADPKD patients with well-preserved eGFR, further supporting the conclusion that severe CKD was not a significant factor in its pathogenesis.There have been case reports of large pleural effusions in patients with ADPKD as well as in polycystic liver disease. These pleural effusions are often asymmetrical and have been attributed to cyst infections, lung cancer, cysto-pleural fistula, ascites with abdomino-pleural communications, cyst mass effect on diaphragm causing inflammation and decreased pleural capillary permeability and complicating hepatic cyst fenestration or resection ,20,21,22Bronchiectasis has been reported to have high prevalence in ADPKD ,23. DrisThere are several cardiac changes in ADPKD that might contribute to the development of pleural effusions . Mitral Incidental, physiological pleural effusions have been reported in the general population. When imaging women prone, for breast MRI, Nyguen et al. found that pleural fluid could be identified by MRI in 174 of 200 (87%) of women . This liThe correlation of pleural effusion with free pelvic fluid in males, as well as with increased pericardial fluid and the association of dilated cisterna chyli with ADPKD raises the possibility of increased extracellular fluid volume due to an increase in total body fluid or third spacing due to impaired venous or lymphatic drainage ,5. HowevNone of the patients in this study had symptomatic pleural effusion, and thoracentesis was not performed, thus, we could not determine characteristics of the pleural fluid. However, the effusions appeared simple and dark on T1, suggesting a transudative process. Exudative effusions would be expected to appear more complex, with increased signal on T1 due to the paramagnetic effects of increased protein content.The strengths of this study are the well characterized population of ADPKD subjects and the age and gender-matched control group, as well as a subgroup analysis of an eGFR-matched control group. Another strength of this study is the exclusion of subjects with heart failure, pulmonary disease and other potential clinical confounders. Limitations of this study include the relatively small sample size, retrospective design of the study, and the lack of thoracentesis data to further characterize the effusions. Although the abdominal MRIs did not cover the entire thorax, they did cover up to mid-thorax in all ADPKD and control subjects, which includes the portion of posterior pleural space where effusions accumulate in the supine position.Pleural fluid is more prevalent in ADPKD subjects compared to control subjects without ADPKD. Although this did not appear to be associated with significant clinical complications, the increased prevalence of pleural effusion, together with earlier reports of an association of ADPKD with pericardial effusion, increased cisternae chyli diameter and pelvic fluid accumulation in males suggest that increased extravascular fluid is characteristic of this disorder, independent of estimated glomerular filtration rate. The potential role of the lymphatic system in the pathogenesis of ADPKD warrants consideration.


Background and Objectives: Pelvic nonunion and malunion have been documented as rare complications in pelvic fractures and literature describing these topics is severely limited. Articles dedicated solely to pelvic malunion are nearly nonexistent. We conducted a literature search with the goal of providing a summary of the definition, causes, treatment strategies, and outcomes of pelvic malunion correction. Materials and Methods: An initial review of the literature was performed using the PubMed, ScienceDirect, and Cochrane Database of Systematic Reviews databases. Search terms used were “malunion” AND “pelvic” OR “pelvis”. Duplicate articles, non-English language articles without translations available and non-human subject studies were excluded. Results: Eleven original publications were found describing experiences with pelvic malunion. Seven of the articles were exclusively dedicated to the topic of pelvic fracture malunion, and only two reported on a series of patients treated for malunion with variably staged procedures. Most reports define pelvic pain as the main indication for surgical correction, along with gait disturbance, standing or sitting imbalance, and urinary or sexual dysfunction. Radiographically, vertical displacement of one to two centimeters and rotation of the hemipelvis of fifteen degrees or more have been described in defining malunion. No treatment algorithms exist, and each patient is treated with a unique work-up and operative plan due to the complexity of the problem. Only one series reported a patient satisfaction rate of 75% following malunion treatment. Conclusions: Pelvic malunion is a rare complication of pelvic ring injury and is seldom discussed in the literature. We found two small case series reporting exclusively on malunion treatment and complications. While some of the combination studies made the distinction in the diagnosis of malunion and nonunion, they rarely differentiated the treatment outcomes between the two categories. This paper describes pelvic malunion and highlights the need for more research into surgical outcomes of treatment specifically regarding functionality, patient satisfaction, and recurrence of preoperative symptoms. Pelvic ring fractures are commonly caused by high-energy trauma, typically motor-vehicle collisions and falls from a height ,2,3,4. IWe performed a systematics review of online scientific databases to find articles describing pelvic malunions, treatment algorithms or case reports. PubMed, Cochrane library and ScienceDirect were searched for articles on malunion with the keywords “malunion” AND “pelvic” OR “pelvis” to be included in the title. One hundred and seven articles were initially found dating between 1981 and 2022. Duplicate articles were excluded. Articles describing treatment of non-human subjects were excluded based on their title or upon review of the abstracts. Articles were also excluded due to their being published in non-English language journals with no available translations. Referential analysis of the remaining articles provided additional material. Articles were included according to the following criteria: (1) population included in the publication all sustained pelvic ring fractures that resulted in a malunion, (2) underwent surgical treatment for a malunion, (3) outcomes and/or patient satisfaction was discussed in acceptable manner. Case reports were included in the review to include all relevant literature. Review papers were excluded.Eleven publications were found describing pelvic malunions using the methodology described.The literature reporting on pelvic malunion and its causes is sparse. Several authors attribute the cause of pelvic malunion to inappropriate initial treatment of pelvic fractures ,6,10,11.Minimal literature is available defining pelvic malunion. Clinical assessment for symptoms as well as radiographic examination are key in developing the diagnosis. Utilization of plain radiography and computed tomography (CT) can help assess the pelvic deformity and state of the osseous union. The symptoms of pelvic malunion are myriad and include sitting discomfort, standing imbalance, sexual issues, urinary and bowel dysfunction, dyspareunia, insomnia due to pelvic instability, vaginal impingement, gait disturbance, and low back pain ,4,5,6,12Symphysis disruption can be measured on anteroposterior radiograph of a pelvis as the distance between the vertical aspects of the pubis bones. Anterior to posterior displacement can be measured on an inlet radiograph as the distance between two horizontal reference line drawn along the posterior aspect of each iliac crest . RotatioA dichotomy exists between acceptable initial fracture reduction compared to malunion definition. Anatomic fracture reduction at the time of injury is deemed necessary. However, clinically relevant malunion definitions vary. Malunion and nonunion have been described as a spectrum of post-traumatic deformity . Cano-LuChronic pain, cosmetic asymptomatic disfigurement, pelvic instability, and deformities that affect physiologic function are the main indicators for surgical intervention ,17,18,19The main treatment goals of pelvic malunion are deformity correction, pain relief and achieving union. Operative malunion deformity correction is technically demanding, and patients are at risk of intraoperative and postoperative complications. These challenging patient situations are typically approached with methodical preoperative imaging, planning and multiple operations to achieve satisfactory results . Unions Pelvic fractures often present with severe life-threatening traumatic injuries that require immediate intervention and can result in delayed or inappropriate imaging and treatment. Inadequate management methods including nonoperative treatment of displaced fracture patterns and/or insufficient or short-term fixation of unstable fractures are the main causes of malunion. Malunion of pelvic ring injuries create deformities, such as leg length discrepancy and rotational abnormalities that negatively affect physical function. Aside from physical disability the most reported symptom of pelvic malunion is severe disabling pain ,4,12. PaPelvic malunion is a panful, function-limiting, stable deformity and is a rare complication of pelvic ring injury. This is seldom discussed in the literature. We found two small case series and several case reports discussing malunion treatment and complications. The large series frequently cited for malunion definitions present nonunion and malunion patients in the same cohort. While some of the combination studies made the distinction in the diagnosis of malunion and non-union, they rarely differentiated the treatment outcomes between the two categories. The most common definition of malunion includes vertical displacement of the injured hemipelvis by one centimeter or more and internal or external malrotation of the hemipelvis by more than 15 degrees resulting in symphysis overlap or widening. Keshishyan Index is commonly used to evaluate rotational deformity correction. This paper describes pelvic malunion and highlights the need for more research into surgical outcomes of treatment specifically regarding functionality, patient satisfaction, and recurrence of preoperative symptoms.


Looking for an optimal flow shape for culture growth in vortex bioreactors, an intriguing and impressive structure has been observed that mimics the strong swirling flows in the atmosphere (tornado) and ocean (waterspout). To better understand the flow nature and topology, this experimental study explores the development of vortex breakdown (VB) in a lab-scale swirling flow of two immiscible fluids filling a vertical cylindrical container. The rotating bottom disk drives the circulation of both fluids while the sidewall is stationary. The container can be either sealed with the still top disk (SC) or open (OC). As the rotation strength (Re) increases, a new circulation cell occurs in each fluid—the dual VB. In case SC, VB first emerges in the lower fluid at Re = 475 and then in the upper fluid at Re = 746. In case OC, VB first emerges in the upper fluid at Re = 524 and then in the lower fluid at Re = 538. The flow remains steady and axisymmetric with the interface and the free surface being just slightly deformed in the studied range of Re. Such two-VB swirling flows can provide efficient mixing in aerial or two-fluid bioreactors. This lasting interest from researchers is due to important applications and apparently enigmatic nature of VB. The VB is recognized to influence aircraft wings' lift and drag forces, flame stabilization in combustion chambers, mixing and mass transfer in chemical and biological reactors, and weakens tornadoes2.A core of a vortex can abruptly expand, and its axial velocity can reverse. This phenomenon is referred to as vortex breakdown (VB). After the pioneer publication3 and Escudier4, many fundamental studies of VB have been performed using flow in a sealed cylindrical container whose one end-disk rotates while all other walls are stationary. Such a flow is free of unpredictable ambient disturbances and has well-defined boundary conditions and control parameters. These favorable features allow for a meaningful comparison between precise experimental4 and numerical5 results that help understand the VB physics. In VB studies before 2015, only one fluid filled the container.Starting with works by Vogel7 led to interest in two-fluid or multiphase flows. A propeller drives an air flow, which in turn drives a water flow. The air flow transports oxygen, required for tissue growth to the interface. The oxygen diffuses through the interface and dissolves in water. The meridional circulation of water enhances the mixing of the dissolved oxygen with other ingredients. Thus, the aerial vortex bioreactor provides the gentle, fine, and nonintrusive mixing of ingredients required for the efficient growth of tissue cultures.The development of aerial vortex bioreactorsA useful model of an aerial vortex bioreactor is a sealed vertical cylindrical container filled with two immiscible fluids having remarkably different density and viscosity. For instance, the air–water flow can be driven by the rotating lid while the other walls of the container are stationary. The simple geometry and well-defined boundary conditions are convenient for detailed experimental and numerical studies and can aid understanding some intriguing features of two-fluid swirling flows. One of these striking features is the development of dual VB.8 VB in the upper fluid in a water/soybean–oil flow. Initially, the experimental studies of two-fluid flows used only photo and visual observations9. Numerical simulations revealed that a few changes in the flow topology occur in the lower fluid before the VB occurrence10 and significant deformation of the interface11 observed in the experiments9.Tsai et al. first observed12, considering different velocities and phase ratios. Another set of problems, also related to oil production are flows in flat mini-channels, which simulates oil–water motions in a porous medium14.It is obvious that the transition from one-fluid to multi-fluid flows significantly complicates hydrodynamics, but two-fluid flows have a wide range of technical applications, mainly related to heat and mass transfer at the interface between different fluids. The study of immiscible-multi-fluid flows attracts considerable interest due to a number of technical problems arising in oil production and associated with the transport of a mixture of oil and water over long distances through pipesIn our experimental study, the interaction between two liquids is controlled by the centrifugal force. This way has applications in bioreactors, providing smooth and fine mixing of ingredients that is beneficial for the growth of biological culture. In addition, the use of two immiscible liquids, having different viscosities and densities, makes it possible to significantly expand the range of realizable velocities with a stable and pronounced interface.15 of the particle-image-velocimetry (PIV), the experimental and numerical results agreed in terms of velocity distribution, including the VB appearance10 in the upper fluid (oil) contacting the rotating disk. However, velocity could not be measured in the lower fluid (water), because the velocity magnitude was too small to be well detected.Measuring two-fluid swirling flows is a challenge because the maximal velocity of the meridional motion is drastically smaller than the maximal swirl velocity, especially in the fluid remote from the rotating disk. In the first application16 this limitation by replacing water with a water–glycerin solution whose viscosity was close to that of oil. This modification and the averaging of 200 PIV images  allowed them to measure velocity and experimentally to identify flow patterns in the lower fluid as well. This helped reveal a paradoxical feature of two-fluid swirling flows—a discontinuity in radial velocity (slip) at the interface caused by the centrifugal force and a jump in density.Naumov et al. overcame17 and experimentally18. Our work extends the experimental study of the VB development to the two-fluid flows. For comparison, we first consider the dual VB development in the sealed container, and then in the open one. Such approach helps highlight the important differences in the VB scenarios in these two cases.The one-fluid open flow was studied both numerically22. The discovery of slip means that all numerical results, which were obtained under the continuity condition for all components of velocity at the interface, must be revised. As the slip makes problematic numerical simulations, importance enhances for further improvement of the experimental technique.The slip was confirmed in the further studies of VB in two-fluid flowsThis study does advance the PIV experimental technique (Sect. 2) to better examine the VB development in the sealed (Sect. 3) and open (Sect. 4) containers. The obtained results are discussed (Sect. 5) and summarized.R, which rotates with angular velocity ω. The entire cylindrical container was placed inside a rectangular transparent box made of high-quality glass and filled with water to minimize thermal changes and optical aberrations which take place due to the cylindrical surface. The upper fluid is polymethyl siloxane oil of density ρo = 920 kg/m3 and viscosity νo = 8·10−6 m2/s. The lower fluid (glycerol) is a 65% distilled water and 35% glycerol solution of density ρg = 1070 kg/m3 and kinematic viscosity νg = 70·10−6 m2/s. The flow is kept isothermal at 22 °C. The swirling motion of flow is characterized by the Reynolds number Re = ωR2/νg. The harmonic control signal is generated using a PC sound card and passing through the low-frequency amplifier to match the level, is fed to the input of the stepper motor driver. Rotational frequency after multiplying by the gear ratio of the stepper motor is transmitted by frequency modulation to the stepper motor driver SMD-4.2PL, which controls the stepper motor rotation providing required Re number.Figure The height of the lower and upper layers was chosen in such a way as to obtain bubble vortex breakdown in both liquids even in the laminar regime; with an increase in the height of the liquid layers, more energy is required. Thus, for the given ratio of the viscosities of the lower and upper liquid, it was possible to obtain the simultaneous onset of a bubble breakdown far from the Re numbers corresponded to an unsteady flow.23, which was focused on another interesting phenomenon—the slip of the radial velocity component at the interface.Due to the remarkable difference in the fluid densities, the interface remained stable during the experiment and did not have significant deformations for Re values required for the formation of VB in both fluids. The exact configuration of liquids was considered earlier24. Digital filtering based on averaging with a sliding window and subtraction of the static background were used to increase the signal-to-noise ratio, making it possible to observe low-contrast movements of light-scattering particles in a wide dynamic range even in the presence of significant background illumination. Used CMOS camera MC023MG-SY Ximea .Since the bubble VB onset maps for two fluids are unknown, a new visualization technique was used to determine the boundaries of the realized regimes. The track visualization system implements the adaptive real-time track visualization technique developed and tested by the authorsm (Am—8-bit 1936 × 1216 matrix of intensities).Gathering images for background image calculation Am from each collected image Ai.In real-time, extraction of the background matrix of intensities Ab images from the camera are stored, Nb is a current number of images in a buffer.The last NThe matrices of intensities in the buffer are summed, and the average result image A is calculated:When a new image is transmitted from the camera, steps 2 to 4 are repeated.Visualization processing algorithm propose next steps:b. The larger buffer size provides longer tracks. This system allows for a precise tune of the observed regimes in real-time to get information about the Re number at which PIV measurements are required.The length of the tracks in the resulting visualization is influenced by the frame exposure time and the size of the buffer N3 and diameter around 10 μm for lower fluid (0 < z < hg), and polyethylene microspheres, of density 950 kg/m3 and diameter around 5 μm for upper fluid (hg < z < ho), to minimize the buoyancy effects, were employed as seeding light-scattering tracers for the PIV measurements. For each regime, 500 images were accumulated to perform velocity field averaging that increases the signal-to-noise ratio. Since the velocities in the lower and upper liquid are significantly different, measurements for each of them were carried out separately in order to find a suitable time delay between frames.The velocity fields in vertical cross-section were measured using the Particle Image Velocimetry (PIV). Polyamide seeding particles, of density 1030 kg/m23. It consists of a double-pulsed Nd:YAG Beamtech Vlite-200 laser with a repetition rate up to 10 Hz, pulse energy 200 mJ, a CCD-camera IMPERX IGV-B2020, 8 bits per pixel, 4Mp matrix, and Polis synchronizing processor. Two-dimensional velocity fields were calculated using the ActualFlow software. The laser sheet thickness in experiments was about 1 mm in the measurement area. The camera was placed on a special mount system at 800 mm from the laser sheet plane for the vertical section.This PIV system is well approved in our early works for velocity measurements in a cylindrical containerWe calculated the velocity fields using the iterative cross-correlation algorithm (3 inner iterations) with a continuous window shift and deformation and 50% overlap of the interrogation windows. In order to have a relatively large dynamic range , the size of the interrogation window initially was 64 × 64 pixels. Time delays between the two images varied from 50 to 200 ms depending on Re and fluid type. The background image averaged over 500 accumulated images was subtracted to reduce the noise level. The sub-pixel interpolation of a cross-correlation peak was performed over three points, using a one-dimensional approximation by the Gaussian function. The obtained instantaneous velocity vector fields were validated with the two subsequent procedures: the peak validation with the threshold 1.8 and the adaptive median filter over 8 × 8 nodes. The resulting velocity fields spatial resolution was about one vector per 1.5 mm.Thus, combining the traditional PIV method and the adaptive real-time track visualization technique allows us to record both statistical fields and velocity distributions Figs. , 3, 4, 5In this section, our experimental results are presented on the simultaneous bubble vortex breakdown onset in upper and lower liquids for sealed and open container flows. The flow patterns are provided via plotting of velocity streamlines in the vertical cross section for each Re values.23 resulting in the flow pattern shown in Fig. z = 0; see Fig. z/(ωR) = − 0.0029 at z/R = 0.49. At Re = 500, the maximum value is positive, Vz/(ωR) = 0.0029 at z/R = 0.55. Interpolation yields that the maximum is zero at Re = Rel = 475.As Re increases starting from zero, three changes in the topology of the upper fluid occurAt this value of the Reynolds number, a new cell of anti-centrifugal circulation emerges – vortex breakdown occurs near the center of the lower fluid. Figure As the Reynolds number (Re) increases, VB emerges first in the fluid, contacting the rotating bottom, and then in the upper fluid. The dark spot denotes the region between the fluids not resolved by PIV.z(z) develops as Re increases. Figure u = 746. Thus, the dual vortex breakdown occurs for Re > 746. The difference in Rel = 475 and Reu = 746 is rather large. It becomes very small in the case where the top disk is absent, as shown next.In the upper fluid (oil), the velocity at the axis remains negative in Fig. z = 0; see Fig. z/(ωR) = − 0.00295 at z/R = 0.439. At Re = 600, the maximum value is positive, Vz/(ωR) = 0.00529 at z/R = 0.558. Interpolation yields that the maximum is zero at Re = Rel = 538. The new cell is well developed at Re = 550  develops as Re increases. Figure u = 524 and z/R = 1.81. Therefore, in the open container case, vortex breakdown in the upper fluid occurs at the significantly smaller value of Re (524) that in the sealed container case (746) and even smaller than in the lower fluid (538) in the open-container case. Thus, the dual vortex breakdown occurs for Re > 538 nearly simultaneously in both fluids filling an open container.In the upper fluid (oil), the velocity at the axis also is negative at Re = 450 and 500 in Fig. 25. In a few words, SDM is the following. In high swirling flows, the azimuthal velocity is much larger than other velocity components in cylindrical coordinates. This feature allows for approximation reducing the radial momentum equation to the cyclostrophic balance, ∂p/∂r = ρvθ2/r, between the radial gradient of pressure and the centrifugal force. It follows from the balance that pressure at the vortex axis is smaller than at the vortex periphery and the pressure difference grows as the centrifugal force increases. If the swirl decays downstream, then pressure grows along the vortex axis and a local minimum of pressure develops. The lower pressure sucks ambient fluid, particularly along the axis, reducing and even somewhere reversing the axial velocity and thus producing recirculation regions (VB bubbles).To better understand the physics behind the obtained results, we use and test the swirl decay mechanism (SDM) of vortex breakdown developmentConsider whether SDM works in the current case. The rotating bottom disk feeds the attached fluid with angular momentum. The generated centrifugal force pushes the fluid from the axis to the periphery near the bottom, thus, driving meridional circulation of heavy fluid as shown at Re = 450 in Fig. The flow converging near the interface transports angular momentum close to the axis that according to the cyclostrophic balance, develops a local minimum of pressure near the interface-axis intersection. The low-pressure suction decelerates the near-axis jet and can reverse it forming a local counter-circulation (VB bubble), as Fig. As Re increases, the bubble expands axially and moves up due to the suction, as Fig. Therefore, SDM well works here like in the one-fluid case, despite the different boundary conditions at the top: wall there and the interface here. A difference is in Re values at which VB emerges. Here this Re value is twice smaller than there due to less friction at the top. The SDM scenario of VB works in the lighter fluid as well, as Fig. t = 17523. For Re < Ret (weak swirl) the upper fluid circulates clockwise driven by the convergent motion of the heavy fluid—the radial friction force.Paradoxical is the velocity jump at the interface: the heavy fluid moves to the axis while the light fluid moves away from the axis. as Fig. t, the centrifugal force dominates and first develops a small centrifugal circulation area near the upper fluid's interface-axis intersection. Then this area expands and occupies entire volume of the lighter fluid for Re > 250 resulting in the flow topology as shown in Fig. The effect of rotation is opposite: the centrifugal force pushes fluid away from the axis. The circulation direction depends on what force dominates: radial friction or centrifugal. For Re > RePatterns of oil-glycerol immiscible two-phase rotating flow were experimentally studied. For the first time, a simultaneous dual bubble-like vortex breakdown has been observed in a container with a rotating bottom disk. The two cases with sealed (SC) and open (OC) container were explored. The case OC, with the free surface, in fact, corresponds to a flow of three fluids: glycerol, oil, and air.In case SC, the shape and location of VB cells are similar for both glycerol and oil flows. In case OC, the shape of VB cell in the oil flow is thinner and axially more elongated than those in the glycerol flow. The VB develops in the oil flow at Re = 524 (OC) and 746 (SC). This difference is due to the upper still lid consumes the angular momentum, while the free surface does not. In the glycerol flow, the VB develops at Re = 538 (OC) and 475 (SC). This difference is relatively small because the lower-fluid flow is driven by the rotating bottom and weakly depends on the upper-fluid flow.The large Re = 746 for the case SC is because the still disk consumes the angular momentum that must be compensated by addition supply from the rotating bottom disk, i.e., by larger Re than that (Re = 524) in an open container. The upper fluid motion is driven by the lower fluid motion at the interface, which serves as a liquid bottom for the upper fluid. In the open container the top surface is nearly stress-free that promotes the upper fluid circulation. We observed no visible effect of air on the liquid flow. It seems due occur to the thousand times difference in density.The fact that in the glycerol, the VB develops at larger Re for the open container than that in sealed container indicates that the transfer of angular momentum from the lower to the upper fluid increase in the OC case. Since the OC case involves three fluids—glycerol, oil, and air—there is an open question: whether a triple VB occurring in each fluid can be observed?The obtained results are important both for practical technological applications of multi-fluid flows (bioreactors) and for fundamental knowledge on the topology of swirling multi-component systems.Supplementary Information 1.Supplementary Information 2.


The association between cancer and a hypercoagulatory environment is well described. Thrombotic complications serve not only as a major mortality risk but the underlying molecular structure and function play significant roles in enhancing tumour progression, which is defined as the tumour’s capacity to survive, invade and metastasise, amongst other hallmarks of the disease. The use of anticoagulant or antiplatelet drugs in cardiovascular disease lessens thrombotic effects, but the consequences on tumour progression require interrogation. Therefore, this review considered developments in the management of platelet activation pathways , focusing on the use of Aspirin, Clopidogrel and Atopaxar, and their potential impacts on tumour progression. Published data suggested a cautionary tale in ensuring we adequately investigate not only drug–drug interactions but also those unforeseen reciprocal interactions between drugs and their targets within the tumour microenvironment that may act as selective pressures, enhancing tumour survival and progression. The earliest documented association between cancer and the vascular system was by the surgeon Sushruta, circa 1000 BC , in whicCancer is a heterogeneous disease. Differences observed within a single tumour, termed intratumoral heterogeneity ,8, deterIn understanding the biology that underwrites the relationship between vascular events and cancer, it is becoming increasingly evident that dysregulation of vascular homeostasis facilitates tumour progression . In thisIn cancer patients, the thrombotic state is characterised by reciprocal interactions between endothelial dysfunction, blood flow stasis and hypercoagulation, which is known as Virchow’s Triad  2]. Thr. Thr2]. Arterial thrombosis typically leads to myocardial infarction and stroke, whereas venous thrombosis is associated with venous thromboembolism (VTE) and pulmonary embolism (PE) ,19. PhenPlatelets are key mediators in vascular homeostasis; furthermore, they play major roles in immunity and inflammation. Their role in vascular injury is via the processes of activation, adhesion and aggregation, thereby forming a clot to prevent bleeding and promote healing . These s2 and thrombin are agonists that drive platelet activation, leading to the expression of surface glycoproteins that facilitate adhesion [Platelets present with a range of integrins and glycoproteins that mediate adhesion, as well as coagulation factors that drive a positive feedback loop that enhances platelet activation and the coagulation cascade to stabilise the developing thrombus . Adenosiadhesion . These gadhesion . Coupledadhesion .There are three main types of granules in platelets, namely, α-granules, dense granules and lysosomes, which vary in terms of their formation, cargo and secretory kinetics ,29. α-GrNot all proteins are synthesized and packaged within megakaryocytes prior to budding off the proplatelet and subsequent maturation. Some proteins are endocytosed from plasma either via receptor-mediated or receptor-independent pathways . As such2+ stores to enhance platelet activation and granule exocytosis. Notably, under thrombin stimulation, α-granules rapidly release their contents, with dense granules secreting their contents afterwards. Dense granules are the second most common granule within platelets and contain calcium, ADP, ATP and serotonin that enhance platelet activation and aggregation [2+ stores, the small GTPase known as Ral was implicated in regulating dense granule exocytosis, even in sub-maximally activated platelets [Tumour-secreted thrombin activates platelets via PAR1 and PAR4 receptors, promoting the release of intracellular Caregation . While glatelets . That thlatelets . Traditilatelets , are notlatelets  and to sFlow cytometric assessment of, for example, CD62P (p-selectin), which is contained within α-granules and translocates to the platelet membrane surface during early activation and degranulation, can be used as a marker of these early events ,41. Howe2 and collagen [Platelet shape changes were also identified to occur despite a significant reduction in degranulation in platelets deficient in the G-protein α subunit Gαq in response to thrombin, TXAcollagen . Deficiecollagen . This hicollagen ,46,47. Tcollagen ,48. Platcollagen ,49.CD63 is a marker that is linked to a period of later platelet activation , wherebyA refractory response to agonists was also suggested to reflect platelet exhaustion, whereby platelets that are in a higher state of activation and are unable to upregulate CD62P or CD63 expression and aggregate are nevertheless prone to increase the risk of thrombosis, as identified in cases of traumatic brain injury and viral haemorrhagic diseases ,43,54. MThe TME acts as a double-edged sword by facilitating tumour progression and hypercoagulation. In the TME, platelets interact with tumour cells within the tumour bed and the vasculature. Fibrin or platelet plugs in and around many types of tumours were observed by histological analyses, implying local activation of the coagulation process . ExtravaExtravasated CD42b+ platelets were found in primary breast tumours, with definitive co-localisation with tumour cells at the invasive front and in the perivascular tissue . These tPlatelet aggregation in the tumour bed has been associated not only with inducing and maintaining key EMT genes in tumour cells  but withCD97, which is a G-protein coupled receptor, involved in adhesion is upregulated on several solid tumours, whereby it facilitates trans-endothelial migration of tumour cells by inducing the release of platelet-derived lysophosphatidic acid that enhances tumour cell invasiveness and ATP that enhances vascular permeability . Such biThrombin was also shown to not only be involved in the coagulation cascade but also promote tumour survival through PAR-1 and fibrinogen-mediated pathways . FibroblPotent angiogenic factors, including VEGF, PDGF, bFGF and EGF, and antiangiogenic factors  are primarily stored in platelet alpha granule stores . Tumour-Bi-directional signalling between breast tumour cells and platelets was also investigated in vitro, revealing that breast cancer cell lines, including MCF-7, MDA-MB-231 and T47D, are able to induce platelet activation and aggregation and that platelets, in turn, are able to induce more aggressive tumour phenotypes, promoting EMT and metastasis ,80,81. C2, ADP and thrombin, bind to P2Y1 and P2Y12 receptors on platelets  by thrombin [The role of antiplatelet therapies on the incidence of cancer is reviewed here by presenting three major pathways that are involved in platelet activation during thrombosis, namely, cyclooxygenase (COX) function in TXAthrombin ,89. Whil2 and other prostaglandins [2 binds to TP receptors, causing platelet activation and aggregation and facilitating thrombosis [Aspirin , along with other classical non-steroidal anti-inflammatory drugs (NSAIDs), competes with arachidonic acid to bind irreversibly to COX enzymes ,91, therglandins ,93,94. Tglandins , but is glandins . Under prombosis ,97. Evenrombosis . Similarrombosis . Prostagrombosis . High lerombosis ,100,101.rombosis . Studiesrombosis ,103,104.2 production; however, the inhibition of COX2/PGE2 was not associated with anti-metastatic effects [2 generated through the COX-1-dependant pathway is associated with metastasis, hence Aspirin usage is shown to reduce metastasis. TXA2 signalling was also shown to activate the endothelium, promoting tumour cell survival by mediating the recruitment of pro-metastatic monocytes in proximity to tumour cells through the release of the chemotactic factors CCL2/MCP-1 and CCL5 [2 have a great impact on the development of the intravascular metastatic niche. While Aspirin effectively reduces these effects, it adversely affects the gastrointestinal tract with prolonged usage. As such, other selective TXA2 inhibitors, such as Picotamide, may be used as an alternative treatment, as it preserves the production of gastroprotective PGI2 [In a murine model, the effect of Aspirin on cancer was shown to be dose-dependent, with anti-metastatic effects induced by low-to-medium dosages of Aspirin inhibiting TXA effects . TXA2 geand CCL5 . Overallive PGI2 .Several studies pointed to an association between low-dose Aspirin use and reduced breast cancer risk and breast-cancer-related mortality; however, the results are inconsistent, with some studies indicating no association ,107,108.2 synthesis, which is also produced directly by tumour cells [2 biosynthesis) and TBXA2R (TXA2 receptor) both of which are overexpressed in breast tumours, reduced the colony formation and proliferation of hormone-dependent breast tumour cell lines [2-dependent platelet activation and aggregation, platelets may still retain their responsiveness to other agonists in healthy individuals [To achieve the inhibition of COX signalling in nucleated tumour cells, which, unlike platelets, have the capacity to synthesise de novo COX-1/2 enzymes, much higher and more frequent doses of Aspirin need to be administered ,96. Howeur cells ,112, mayividuals . For thiividuals ,114. In ividuals , highligThienopyridines are a family of antiplatelet agents that bind irreversibly to platelet purinergic receptors and consist of three clinically approved drugs, namely, Ticlopidine, Clopidogrel and Prasugel ,117, andClopidogrel is a prodrug that is metabolised in the liver and converted to its active metabolite whereby it blocks the ADP P2Y12 platelet receptor in an irreversible manner ,123. CytClopidogrel is the most commonly used P2Y12 antagonist and is an effective agent against cardiovascular disorders, including peripheral artery cerebrovascular and coronary artery diseases . While bBreast tumours have the capacity to metabolise Clopidogrel into its active components via the expression of CYP450 family members. In vitro, breast cancer cells were shown to modulate the expression of purinergic receptors, suggesting a possible protective effect of this receptor against cancer . StudiesMouse models have highlighted that an Aspirin and Clopidogrel cocktail induced pro-tumorigenic alterations in primary tumours, increased angiogenesis through vascular mimicry and was associated with high mortality rates . SimilarThrombin is the main extracellular serine protease responsible for activating PAR1, PAR3 and PAR4 receptors ,140. PARThrombin is a potent agonist for platelet activation and is an essential component of the coagulation cascade. Thrombin activates PAR1 receptors in two stages: first by binding to the cleavage site on either side of the PAR1 receptors, then cleaving the PAR1 receptor between Arg and Ser, exposing the N-terminal tethered ligand domain SFLLRN ,144,145.Cancer patients often present with elevated levels of thrombin, and this is associated with a poor prognosis . In the PAR-1 receptor expression in various cancer types, including breast, lung and ovarian cancer, is associated with a poor prognosis . PAR1 inAtopaxar and Vorapaxar are small non-peptide molecules that interact with PARs through ECL2, and thus, inhibit receptor binding . There aAtopaxar is metabolised by the cytochrome P450 enzyme CYP3A4 . Breast Bi-directional signalling between platelets and tumour cells creates favourable circumstances for tumour progression, with thrombotic complications being necessary side effects by virtue of the associations between the cell types. While more is known about the role of Aspirin in preventing metastasis, less is known regarding the prevention of early tumour processes and what effects drugs targeting other activation pathways, including Clopidogrel and Atopaxar, may have. What is becoming clearer is the complexity of platelet activation and the plethora of growth factors, cytokines and coagulation agonists that may be released in response to indirect or direct tumour signals. That these factors impact tumour progression is increasingly evident, with the need to prevent platelet activation, if not aggregation, also evident. However, the drugs used, as well as their combination with standard cancer therapeutics, may not yield the desired beneficial effects against thromboembolic disease but may act as a selective pressure for tumour survival. It is increasingly evident that further research to recapitulate the clinical tumour microenvironment more faithfully in vitro and in in vivo animal models is necessary to provide systems in which to unpack these complex interactions.


The high reaction rate constant of NBD-TM with HOCl makes this probe the most reliable tool to monitor HOCl formation in the presence of compounds showing HOCl-scavenging activity.MPO-derived oxidants including HOCl contribute to tissue damage and the initiation and propagation of inflammatory diseases. The search for small molecule inhibitors of myeloperoxidase, as molecular tools and potential drugs, requires the application of high throughput screening assays based on monitoring the activity of myeloperoxidase. In this study, we have compared three classes of fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid, including boronate-, aminophenyl- and thiol-based fluorogenic probes and we show that all three classes of probes are suitable for this purpose. However, probes based on the coumarin fluorophore turned out to be not reliable indicators of the inhibitors’ potency. We have also determined the rate constants of the reaction between HOCl and the probes and they are equal to 1.8 × 10 Hypohalous acids are powerful oxidants and are crucial reactive species that immune cells use to eradicate invading pathogens. Nonetheless, excessive or misplaced formation of HOCl and/or other hypohalous acids is associated with inflammatory pathologies2. Strong association of halogenating activity of MPO with initiation and propagation of inflammatory diseases resulted in the interest in developing small molecule inhibitors of the MPO enzyme as potential drugs and protective agents in various pathologies involving an inflammatory component3. While several compounds interfering with the peroxidase activity of MPO 10 showed protective effects in various animal models of inflammation14, there is a need for more potent inhibitors, which would irreversibly block HOCl generation by the enzyme. The search of potent MPO inhibitors demands application of high throughput screening (HTS)-compatible rigorous methodology(ies) to detect and quantify MPO-derived HOCl, with the aim to screen large libraries of compounds.Myeloperoxidase (MPO) is a mammalian heme peroxidase that produces hypohalous acids  to a colorless product22 or by oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of potassium iodide, to a strongly absorbing blue product23. Another compound used for this purpose is tyrosine which forms in the reaction with HOCl 3-chlorotyrosine that can be subsequently quantified by HPLC or LC/MS25. Those methodologies are, however, relatively complex and/or not compatible with HTS. Most HTS assays are based on reactions yielding easily detectable luminescent  products, due to high detection sensitivity, and compatibility with multiwell plate readers.Numerous probes and detection approaches have been developed over the last decade to measure the production of HOCl by MPO35. While the number of recent reports on the development of fluorescent probes for HOCl is relatively high, comparing with probes for other cellular oxidants, only few classes of probes gained wider interest and application, due to their reliability and commercial availability. Those include probes containing thiols, aminophenyl and boronate groups as the sensing moieties for HOCl. It has been shown that boronate probes react with peroxynitrite (ONOO‒), amino acid hydroperoxides, and H2O2 with the rate constants of 106, 101, 100 M−1s−1 order of magnitude, respectively38. We have also shown that simple aromatic boronates react directly and stoichiometrically (1:1 ratio) with HOCl yielding the corresponding phenolic derivatives with the second-order rate constant of 104 M−1s−1 order of magnitude38. O-substituted p-aminophenyl derivative of fluorescein has been shown to release fluorescein upon reaction with HOCl, while it also respond to HRP/H2O2, •OH and ONOO–-derived radicals40. The kinetic data for this class of probes are, however, very limited. The third class of probes is based on a rapid reaction of a thiol group with HOCl, producing a sulfenyl chloride intermediate, which undergoes further reactions to produce a fluorescent product, via a mechanism depending on the chemical design of the probe43.Recent progress in the development and use of profluorescent probes for detection of reactive oxygen species enabled specific detection of various oxidants of interestp-aminophenyl)-fluorescein (APF), 7-(p-aminophenyl)-coumarin (APC), and the newly developed thiol based probe, 4-thiomorpholino-7-nitrobenz-2-oxa-1,3-diazole (NBD-TM)  and sodium azide. The obtained IC50 values by means of CBA, FLBA, APC, APF, and NBD-TM probes were compared to the IC50 values for these inhibitors determined using the Amplex Red based peroxidative assay. Finally, the Z’ parameter reflecting the quality of the assay was determined for each probe.In this work, we characterized and compared the HOCl detection performance of the boronate-, aminophenyl- and thiol-based fluorogenic probes, with special emphasis on their applicability for HTS-compatible screening for MPO inhibitors. The probes used include coumarin boronic acid (CBA), fluorescein boronic acid (FLBA), 3’-(2O2 (10 µM), NaCl (100 mM) and phosphate buffer   in the incubation mixtures completely quenched the fluorescence signal regardless of the composition of a sample.In the first set of experiments, the performance of the boronate probes for monitoring of MPO-mediated HOCl production was determined. The CBA probe 20 µM) was incubated in the mixture containing MPO (1.2 nM), low concentration of H.4) Fig. a. During0 µM was 2O2 unless catalase was present in the reaction mixture. Both boronate probes were oxidized in the MPO/H2O2/Cl– system .The analogical experiment was performed using fluorescein boronic acid (FLBA) with similar set of incubation mixtures Fig. b. In thitem Fig. a,b. Whil2O2/Cl– system were p-aminophenyl derivatives of fluorescein and 7-hydroxycoumarin: 3’-(p-aminophenyl)fluorescein (APF) and 7-(p-aminophenyl)coumarin (APC), respectively   Fig. e. SimilaSO) Fig. e.2O2/Cl–, the fluorophores were incubated under the same reaction conditions, as described above for the five probes used in the study , as evidenced by the stable fluorescence signals of the products observed within 1 h of incubation . The determined value of the rate constant for NBD-TM is consistent with the reaction rate constants reported for other thioethers with HOCl46.The rate constants of 1.8 × 10ely Fig. . The hig50) values for two well-established MPO inhibitors: 4-aminobenzoic hydrazide (ABAH) and the azide anion (N3–). The dose–response curves for ABAH and N3– are presented in Fig. 50 values are shown in Table 50 values for ABAH are in the range of 2.6 to 40.6 nM and for the azide ion are between 0.5 and 2.4 µM. In the case of the ABAH inhibitor, the CBA and APC probes yielded significantly lower IC50 values than the other three probes. As both probes share the same oxidation product, COH, these discrepancies can be attributed to the interference of COH with the mechanism of MPO inhibition by ABAH. Therefore, we recommend the IC50 value of 40 nM, obtained consistently for all four other probes used. In the case of the azide anion the discrepancies between the IC50 values for different probes were less noticeable. While similar values were obtained using boronate and the NBD-TM probes, more than two-fold higher IC50 values were determined for the probes that undergo o-dearylation, APC and APF (Table 3•), to produce the fluorescent products. Azidyl radical is known to be formed in the presence of MPO, H2O2 and azide47. Therefore, we recommend the average IC50 value of 0.7 µM, obtained from the three probes resistant to one-electron oxidation by the N3• radical: CBA, FLBA and NBD-TM. In order to ensure the accurate data interpretation, the competition experiment between the inhibitors and the probes for HOCl was performed and the effect of the inhibitors on the fluorescence intensity of the products formed (fluorophores) was examined. At a 20 µM concentration of the probes their reaction with HOCl and the observed fluorescence intensities were not influenced by the inhibitors in the concentration range used.To establish the applicability of the probes to screen for novel MPO inhibitors, all the probes under study were used to determine the half-maximal inhibitory concentration IC0 values PF Table . Our ten2O2 and peroxidases  of large chemical libraries. For the probes tested, the Z’ parameter was calculated on the basis of end-point measurements (fluorescence measured at a specific time of incubation) and kinetic measurements (rates of fluorescence increase), according to the Eq. −1s−1 and it has been shown that it may interfere with oxidation of boronate probes by HOCl49. Thus, the influence of DMSO additive on the observed fluorescence signal generated in the reaction between HOCl and the studied probes was evaluated. In this experiment, the incubation mixtures contained the chosen probe (20 µM), phosphate buffer , and DMSO in the amounts between 0–5% v/v (0–0.7 M). The reaction with HOCl was initiated by the bolus addition of 1 mM sodium hypochlorite to obtain the final HOCl concentration of 10 µM. In the case of NBD-TM that reacts rapidly and stoichiometrically (1:1 ratio) with HOCl44 about 10 µM of NBD-TSO was formed  or 0.1% v/v (14 mM) DMSO had negligible effect on the HOCl-induced oxidation of NBD-TM to NBD-TSO, while at of 0.5% (0.07 M), 1% (0.14 M), and 5% (0.7 M) concentration, DMSO diminished the NBD-TSO concentration about 5, 10 and 35%, respectively. In case of the other probes tested , the addition of even 0.05% v/v (7 mM) DMSO led to a strong inhibition of the fluorescence signal  is in reasonable agreement with the reaction rate constant of thioether L-methionine and HOCl (2k = 3.4 × 107 M−1s−1)41.The rate constants determined for the boronate probes are consistent with those previously determined for this class of compounds50 values for ABAH and the azide anion and the values obtained using different probes are convergent, but some deviations are observed  of DMSO is a condition to be met for its application in an HTS campaign. Among the studied probes, NBD-TM was the most resistant probe to the DMSO additive Fig. . The dat22. A more sensitive assay based on iodide catalyzed oxidation of TMB in the presence of taurine chloramine is five times more sensitive23. Substitution of TMB with dihydrorhodamine increases the sensitivity of this assay about 10 times shifting the HOCl detection limit to hundreds of nanomoles23. Similar limits of detection exhibit NBD-TM and other fluorescent probes (approx. 70–100 nM)44. Taking into account the rapid reaction between taurine and HOCl (2k = 4.8 × 105 M−1s−1)52 the assays based on formation N-chlorotaurine may be sufficient for many purposes, where HOCl detection is needed. However, a requirement of the use of developing reagents and/or the need of a time-dependent probing of a reaction mixture to continuously monitor the MPO activity, in combination with their lower sensitivity in comparison to fluorescent assays  and aminophenyl (APF) probes for the secondary/orthogonal assays in such HTS campaigns.This study provides a comparison of three different classes of fluorescent probes for HOCl, with the goal of establishing a platform for high throughput screening of compounds in search of new inhibitors of MPO. We demonstrate that all three classes of the probes  can be used for monitoring the chlorinating activity of MPO, but based on the ICCorynebacterium, hydrogen peroxide, sodium hypochlorite, 4-aminobenzoic acid hydrazide (ABAH), NaN3, umbelliferone (COH), fluorescein, N,N,N’,N’-tetramethylbenzidine, NaCl, taurine, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich and were of the purest grade available. Fluorescent probes were synthetized according to the procedures described in the literature. The synthesis of fluorescein pinacol boron ester (Fl-B) was performed based on the procedures published by Dickinson et al. and Rios et al.59. The obtained boronate was then hydrolyzed to fluorescein boronic acid (FLBA). The synthesis of APF was performed based on the procedure published by Setsukinai and cooworkers39. The APC probe was synthesized according to the procedure published by Kavani and cooworkers60. The detailed description of synthetic procedures can be found in the Supplementary Information. The CBA synthesis is described elsewhere61.MPO from human neutrophils was obtained from Athens Research and Technology . Catalase form The one millimolar stock solutions of CBA, and APC were prepared in acetonitrile. In the case of FLBA and APF, the two millimolar probe solutions were prepared in the water/acetonitrile mixture (1:1 v/v). Such prepared stock solutions were stable at least one-month and stored ready to use at 6 °C in the dark. The concentration of probes was checked during the day of plate reader measurements using an UV–Vis spectrophotometer (Varian Cary 300 Conc) and the determined molar absorption coefficients Table .Table 2A−1cm−1. The changes of fluorescence intensity were measured by Varioscan LUX (Thermo Fisher Scientific) equipped with double monochromators based on diffraction gratings and controlled by SkanIt Software 6.0.2. The second plate reader used in the measurements was CLARIOstar (BMG Labtech) equipped with Linear Variable Filter (LVF) monochromators.The lyophilized powder of MPO was dissolved in distilled water accordingly to the supplier recommendation and, if required, stored at 6 °C by the day of analysis. Such prepared solution was then diluted with phosphate buffer, pH 7.4, to the appropriate concentration and the volume of 0.5–2 µL/well depending on the activity was pipetted directly to 96-well plate (Costar 3840). Then, the enzyme was further diluted on a plate with volume of 100 µl of already prepared solutions containing phosphate buffer (100 mM) and other necessary components depending on the experiment or control, such as NaCl (0.2 M), the selected probe (40 µM) or taurine (20 mM), and catalase (200 U/ml). Reactions were stared adding 100 µl/well of 20 µM hydrogen peroxide solution. The concentration of hydrogen peroxide stock solution was determined spectrophotometrically at 240 nm using the molar absorption coefficient equal to 43.6 MKinetic measurements of the reaction of boronate probes, APC, and APF with HOCl were performed using a SX20 stopped-flow spectrometer (Applied Photophysics) equipped with 150 W Mercury-Xenon arc lamp. During the monitoring of COH formation the excitation monochromator was set at 330 nm and the emission monochromator was set at 450 nm. The formation of fluorescein was followed using the excitation wavelength of 492 nm and emission wavelength of 518 nm. During measurements, the slits width in the excitation and emission monochromators were set at 2.5 mm. The measurements were carried out under the pseudo-first-order conditions, where the concentration of a probe was at least five times higher than the concentration of HOCl. The observed pseudo-first-order rate constants were determined by the fitting of a curve described by the equation below (Eq. 2kCBA+HOCl = 1.8 × 104 M−1s−1) and concentrations of the formed NBD-TSO, determined by LC/MS, the unknown second-order rate constant for the NBD-TM/HOCl reaction was calculated according to the equation below  equilibrated with an acetonitrile/water mobile phase containing 0.1% trifluoroacetic acid (TFA). The samples containing NBD-TM and CBA, and their oxidation products after mixing with HOCl were separated using a flow rate of 0.3 ml/min and a gradient method with the initial concentration of the organic phase equal to 20%. This concentration was kept constant for 0.5 min after the sample injection. Then, the linear increase of acetonitrile from 20 to 100% over 1.5 min was applied.LC/MS analyses were performed using an Acquity UPLC (Waters Ltd.) system combined with a LCT Premier XE (Waters Ltd.) mass spectrometer and a reversed-phase C57.control+ and S.D.control- are the standard deviation values, and meancontrol+ and meancontrol- are the mean values for the positive and negative control, respectively. The value of Z ' above 0.5 means good quality of the assay. The higher value of Z ' parameter means the better quality of the assay. This parameter cannot reach value higher than 1.The Z’ factor is a dimensionless coefficient and was calculated on the basis of data presented in Fig. Supplementary Information.


Cytomegalovirus (CMV) disease is associated with increased morbidity and mortality following solid organ and hematopoietic cell transplantation. Currently, standard of care CMV treatments often have significant myelosuppressive or renal toxicities. Given letermovir’s favorable safety profile when used prophylactically, it may be a safe and efficacious alternative agent for CMV treatment.A proof-of-concept, open-label trial was conducted in patients who lacked effective therapeutic options or presented with baseline organ dysfunction. Participants were eligible for enrollment if they were ≥12 years old and had documented CMV infection refractory to treatment defined as failure to achieve >1 log reduction in CMV viral load (VL) (when VL > 500 IU/mL) or lack of clinical improvement for CMV end-organ disease after ≥14 days of standard CMV treatment. Alternatively, patients with severe myelosuppression and renal dysfunction at baseline or genotypic antiviral resistance were also eligible. Participants were excluded if their current CMV infection developed while receiving letermovir for CMV prophylaxis. Letermovir was administered daily (480 mg PO/IV) for up to 12 weeks, with optional additional 12 weeks of treatment for secondary prophylaxis if clinically indicated.Ten patients met eligibility criteria and were enrolled. Reasons for enrollment included ganciclovir resistance (1/10), refractory CMV infection (6/10), renal dysfunction (7/10), and myelosuppression (7/10). The median baseline CMV VL was 1272 IU/ml . Six patients completed the study, three died due to complications of primary disease, and one discontinued due to diarrhea. Five patients (50%) had documented CMV viremia clearance, with a median time to first unquantifiable/undetectable CMV VL of 13 days  and a median treatment duration of 53 days . Infections and GI disorders were the most common adverse events (AE), none considered related to study drug. No unexpected AE were observed during letermovir treatment.Letermovir may be a safe and tolerable alternative for patients with treatment refractory CMV infection or for patients with severe baseline myelosuppression and renal dysfunction.Matthew Cheng, MD, AstraZeneca: Honoraria|Cidara Therapeutics: Grant/Research Support|Scynexis Inc.: Grant/Research Support Sandra Burchett, MD, MSc, merck: Grant/Research Support Sarah P. Hammond, MD, F2G: Advisor/Consultant|F2G: Grant/Research Support|GSK: Grant/Research Support|Merck: Grant/Research Support|pfizer: Advisor/Consultant|Scynexis: Grant/Research Support Nicolas C. Issa, MD, AiCuris: Grant/Research Support|Merck: Grant/Research Support Francisco M. Marty, MD, SM, AlloVir: Advisor/Consultant|Amplyx: Advisor/Consultant|Amplyx: Grant/Research Support|Ansun: Grant/Research Support|Avir: Advisor/Consultant|Chimerix: Grant/Research Support|Cidara: Grant/Research Support|F2G: Advisor/Consultant|F2G: Grant/Research Support|Gilead: Grant/Research Support|Janssen: Advisor/Consultant|Kyorin: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Regeneron: Advisor/Consultant|Regeneron: Grant/Research Support|ReViral: Advisor/Consultant|Scynexis: Grant/Research Support|Symbio: Advisor/Consultant|Takeda: Grant/Research Support|United Medical: Advisor/Consultant|WHISCO: Grant/Research Support.


The mechanisms of permeability and friction evolution in a natural fault are investigated in situ. During three fluid injection experiments at different places in a fault zone, we measured simultaneously the fluid pressure, fault displacements and seismic activity. Changes in fault permeability and friction are then estimated concurrently. Results show that fault permeability increases up to 1.58 order of magnitude as a result of reducing effective normal stress and cumulative dilatant slip, and 19-to-60.8% of the enhancement occurs without seismic emissions. When modeling the fault displacement, we found that a rate-and-state friction and a permeability dependent on both slip and slip velocity together reasonably fit the fault-parallel and fault-normal displacements. This leads to the conclusion that the transient evolution of fault permeability and friction caused by a pressure perturbation exerts a potentially dominant control on fault stability during fluid flow. The mechanisms of permeability and friction evolution in a natural fault are investigated in situ. The authors conclude that the transient evolution of fault permeability and friction caused by a pressure perturbation exerts a potentially dominant control on fault stability during fluid flow Friction and permeability of faults are commonly measured in the laboratory using rock mechanics experiments on samples with sizes ranging from centimeters to a meter6. These laboratory-sized experiments are very useful to derive constitutive laws describing the evolution of friction and fluid flow along faults. The friction laws (i.e. rate-and-state or slip-weakening friction) are then used in physics-based models to explain the spectrum of slip behavior on faults7, while permeability laws (i.e. stress- or strain-dependent permeability) are used to model fluid pressure diffusion9.Fault friction and permeability are central to understand the mode of fault slip, including seismic and aseismic response, and fluid flow, with major interest to subsurface reservoirs engineering and earthquake hazard mitigation10. Part of the difficulty is that simultaneous measurements of fluid pressure and deformation into real-world faults at depth are rare, and consequently, hydromechanical processes and properties are not well constrained. However, recent in situ experiments of fault activation caused by fluid injection at shallow depths (from 0.3 to 1.5 km) have proved to be well adapted for studying at the decametric scale how a fault responds to a known fluid pressure perturbation15. Such experiments provide quantitative information on the hydromechanical processes18, and significant insights have been gained into the physics of fault slip19. Yet, to date, constraints on the evolution of permeability and friction during fluid pressurization in a natural fault are rare.Although laboratory-scale experiments are important for precise examination of the mechanisms in controlled environments, questions remain on the extrapolation of laboratory-derived frictional and hydraulic properties to faults at large scale in natural conditionsHere, we explore the hydromechanical behavior of a fault zone in response to controlled fluid injection combining the analysis of simultaneous and continuous measurements of fluid pressure and fault displacements in the injection borehole and seismicity in the rock volume that surrounds it. We report on three injection tests that activated the fault in three different zones. The data are used to infer the evolution of fault permeability and friction during increasing fluid pressure, and its coupling with slip.https://lsbb.cnrs.fr) in a horizontal gallery intersecting a seismically-inactive kilometer-scale fault zone in limestone rocks representative of sedimentary reservoirs . Inside the injection chamber, the pressurized fault plane is located at the middle of the interval between two inflatable packers. Tests 1 and 2 are situated along a secondary minor fault plane, while Test 3 is in a fractured zone. In Test 1, the rock matrix around the pressurized fault plane contains three subparallel bedding planes in the sealed borehole interval, while in Test 2, the rock matrix contains micro-fractures without bedding planes. In Test 3, the pressurized fault plane is surrounded by a fractured matrix with a fracture density higher than in Tests 1 and 2. In the three tests, the selected planes are not filled with clay material. The pressurized fault segments have similar dip angles . Among the three tests, the injection duration varies from 1400 s (Test 1), 460 s (Test 2) to 1000 s (Test 3), and fluid pressure is increased from near zero to maximum values ranging from 4.8 (Test 1), 5.02 (Test 2) to 5.88 MPa (Test 3). For each test, the injected fluid volume is 3.8, 0.52, 0.33 m3, respectively. The evolution of fluid pressure, fault-normal (opening) and fault-parallel (slip) displacements were recorded at the injection point with a specially designed hydromechanical probe “SIMFIP” based on high-precision fiber-optic sensors  in a 2.4 m long packer-isolated borehole interval22 . Three injection tests were performed at different places in the damage zone on selected secondary fault segments in order to explore the variety of fault hydromechanical response. Test 1 was performed 7.5 m below the gallery floor, 9.5 m for Test 2, and 3.75 m for Test 3 , σ2 = 5 ± 0.5 MPa and σ3 = 3 ± 1 MPa 11. Thus, the maximum injected pressures into these experiments represent an extreme pressurization level relative to the stress state, facilitating pronounced opening and slip. Based on laboratory velocity-step friction experiments on powdered (grain size < 125 μm) simulated gouge material collected from rock samples drilled from the fault zone, the frictional behavior evolves with increasing fluid pressure from rate-weakening at slow slip rate (<10 μm/s) to rate-strengthening at greater slip rate, and the initial friction coefficient was measured between 0.53 and 0.6519.Prior to the experiment, the average permeability of the fault zone and intact rock matrix were estimated from in situ hydraulic pulse tests at ∼7 × 10Figure 21 and with laboratory tests conducted on core samples drilled from the fault19. Indeed, seismic events are triggered after a certain amount of fluid pressurization and slip is accumulated over the fault, that is after 1391 s (Test 1), 177 s (Test 2), 679 s (Test 3) of injection into these experiments  compared to that of the fault (∼7 × 10−12 m2) and that injections are of relatively short duration, we assume that the fluid flow is mostly through the fault plane. During slip, the apparent fault permeability is governed by the evolution of fault aperture associated with dilation or compaction24. Consequently, the change in fault permeability relative to the initial fault permeability (k/k0) can be related to the change in hydraulic aperture relative to the initial fault aperture (Δb/b0) as proposed by Zhang et al.25:Considering the average permeability of the intact rock matrix is small (1 × 10b0) of 10 μm, consistently with previous permeability estimations20.This equation assumes a Darcy’s flow with an incompressible fluid of uniform density along a single smooth fault (i.e. no roughness). Permeability changes are estimated from the steady-state fluid pressure conditions, that is when the pressure is stabilized towards the end of the step-increasing phase and the fault displacement velocity is near zero  analogous to the dilatancy relationship of Van Den Ende et al.26 defined from laboratory-derived microphysical model:b0 is the initial hydraulic aperture before the fault is reactivated, bmax is the maximum attainable aperture, β is a constant that represents how much dilatancy is involved when the fault slips, γ is a geometric constant, us is the slip, and L is a characteristic thickness of the fault. In the model, us is sensitive to the change in effective normal stress, and so slip increases upon an increase in the fluid pressure as observed experimentally . We find a best-fit solution  with values of b0 = 7.35 μm, bmax = 62.9 μm, β = 2.1, γ = 8.12 × 10−5 and L = 191.25 μm. The model solution fits well the whole dataset with reasonable values. This shows that the permeability variation related to the complex in situ fault hydromechanical response to reducing effective normal stress can be reasonably figured by relating the hydraulic aperture to dilatant slip as observed at the laboratory scale28.Using Eqs. 30 or during fluid pressurization31. Upon a step change in normal stress, the transient slip response was traditionally reproduced with the rate-and-state friction law32. Here, we examine whether our in situ data collected in decametric scale experiments can fit this type of friction law during a stepped-pressure increase. We evaluate the data using a 1D spring-slider model with a uniform fluid pressure to retrieve frictional parameters. This assumption is valid for high initial permeability fault26, such that the characteristic timescale of fluid diffusion is smaller than the timescale of deformation. We selected three independent sequences in Tests 1, 2, 3  was interpreted with the rate-and-state friction law32:μo is the friction coefficient at a reference slip velocity (V0). The parameter a quantifies the direct effect of a change in slip velocity (V). The parameter b describes the effect of the state variable θ. The characteristic slip distance, dc, governs the evolution of the state variable. We use the Dieterich law  extended by Linker and Dieterich29 to account for variable effective normal stress in the formulation of the state variable as:σn is the normal stress, p is the fluid pressure, and α describes the evolution of shear stress associated with changing effective normal stress (σn-p). The choice of this evolution law is motivated by previous studies showing a good fit to experimental data34.The velocity dependence of friction (Ks) and its elastic coupling with the fault, Eqs.   Ks) and a, b, dc, μ0, α and Ks were obtained by an iterative grid search inversion approach (Supplementary Table Vlp = 0). We impose the initial normal and shear stresses previously estimated in the field21.We modeled the fault slip data by solving Eqs. , 4), an, an4), a5 and Ishibashi et al.6 showed from the analysis of laboratory experiments of stepped-velocity friction tests with fluid infiltration in fractures that the evolution of aperture is dependent on both the effects of slip and slip velocity. Friction and permeability of faults are known to change during slip due to the rearrangement and destruction of asperities. Permeability may increase due to shear induced dilation6 or decrease as a result of gouge formation35 or irreversible changes in surface roughness36. During slip, frictional strength is affected by the state of the contact area between the displacing surfaces33. Previous field experiments on faults have also demonstrated that the contribution of slip on the evolution of permeability and friction is central37. This gives a degree of confidence about the applicability of slip- and slip velocity-dependent models inferred from laboratory data for the analysis of in situ data. Analogous to the shear dilation relationship of Samuelson et al.38, Fang et al.5 proposed the following two equations to represent the evolution of aperture (bevo) due to a dilation parameter dependent on the slip velocity (Δbslip):i refers to the ith velocity step, ψ is a dilation factor, and t is the time. We use the best-fit solution to fault slip, together with slip velocity, as input of the aperture model to estimate the resulting permeability using Eqs.  of 0.054 followed by a slow decay to a new steady state value, μss = 0.478. For the secondary phase of slower slip, the model reproduces the general evolution; however, some differences appear between model and data. Indeed, the measured slip oscillations could reflect the interplay between different fault segments within the damage zone, an effect that is not considered in our single fault model. This best-fit solution (χr2 = 0.594) is obtained with rate-weakening frictional properties (Supplementary Table k/k0 = 2.5) that is poorly coupled with slip and slip velocity at the considered time scale. The exact process responsible for such a difference remains subtle. It could be related to additional hydromechanical effects or geometrical complexities ignored in the model we use. In such well-developed fault zone, it could also reflect heterogeneous fluid flow (i.e. channeling) over the pressurized fault segment or a fluid leakage from the pressurized fault into the surrounding connected fractures or bedding planes. As the injected fluid volume in Test 1 is much larger than for the other tests, it is likely that an extended network of faults is pressurized, leading to more complex hydromechanical response.For Test 1 Fig. , overallχr2 = 0.018) with rate-weakening frictional parameters exhibiting a direct friction increase (Δμ = 0.057) followed by a slower decay to μss = 0.422  of 1.36.For Test 2, the fit to the slip is excellent χr2 = 0.08 with raχr2 = 0.061) and permeability data. Friction obeys to a rate-strengthening behavior with a sudden friction increase (Δμ = 0.048) immediately at the pressure step and then follows by a slower decay to μss = 0.523. The fit to the transient change in permeability is excellent  of 1.76.For Test 3 Fig. , the nument Fig.  and resuIn summary, models tend to show two sequences for the permeability change Fig. . First, Although some differences in the fault response observed between the tests can be due to the different levels of fracturing of the rock matrix that surrounds the pressurized fault plane, the exact mechanical process responsible for permeability increase or decrease after the sudden acceleration remains elusive. Based on geological observations of the borehole intervals before injection, they could reflect geometrical changes of contact areas, gouge content and void space that comprise the fault during deformation at low effective stress for Tests 1 and 2, whereas the hydromechanical response observed in Test 3 may be related to the overall permeability of the fractured damage zone surrounding the tested fault segment.μ0 = 0.5, a–b = −0.028 to 0.01, dc = 5 to 50 μm, and α = 0.091 to 0.1, that are reasonably consistent with previous laboratory measurements of rate-and-state friction parameters  on centimeter-sized gouge samples collected in the same fault zone19.Overall, the best-fit model parameters  are comprised between 0.54 to 9.74 m for Test 1, and 2.06 to 37.3 m for Test 2, which prevent seismicity to occur near injection. Additionally, the small values of critical slip distance estimated at the injection points favor rapid strength drop at this place. As a result, stress concentration in the surrounding is large enough to trigger seismicity away from the pressurized zone. Thus, the seismicity observed in these experiments represents a remote response of the fractured rock volume to the forcing caused by aseismic deformation of the pressurized plane.24. Thus, for Tests 1 and 2, the difference between observed and calculated permeability enhancement may be explained by a combined results of both slip-induced dilation and fluid flow-driven particle mobilization.At the short time scale of a fast-pressure increase, we also observed that in some cases the transient change in permeability is well coupled to slip, and, in other cases, it cannot be explained only by slip and/or slip rate. Nevertheless, although we obtain a reasonable fit to data with a simple model, other coupled processes, not considered here, can also influence the fault response, such as the deformation associated (1) with geometrical irregularities heterogeneously distributed along natural faults, (2) with the fractures network in the volume surrounding the pressurized fault segment, or (3) with the fluid pressure gradient developing from the injection point toward the outer fault boundaries. Indeed, owing to the interplay between permeability and friction, the fault hydromechanical response may result in heterogeneous fluid pressure distribution, channeling flow, stress interaction between neighboring asperities or fractures, and a variety of slip behavior. Moreover, in some conditions, the permeability enhancement is not well coupled with aperture changes and can rather be linked to flux-driven particle mobilization that induces clogging and unclogging within the fault41. Overall, the frictional parameters derived from the in situ experiments are in good agreement with those measured in the laboratory, despite the scale difference at which the parameters were estimated, from centimeters in the laboratory to meter in the field. Thus, the measurements of fault displacement during in situ fluid injection experiments allow to validate with direct observations the physical laws and processes derived at the laboratory scale, and hence, allow for their extrapolation to geological reservoirs and seismogenic zones.Although, fault slip is a complex process dependent on different factors, including fault type, host rock lithology, physical properties and stress conditions, our observations and models demonstrate that the transient evolution of fault friction and permeability is both linked to effective normal stress and slip. This co-evolution can be explained consistently in the framework of laboratory-derived rate-and-state friction law together with a permeability model considering the effects of both slip and slip velocity43. Thus, we used our direct measurements of the fault-normal displacement , and selected a stabilized value at near steady-state fluid pressure conditions, that is when the fluid pressure stops varying towards the end of the transient step increase, and before the next pressure step  once the slipping region reaches a critical size (Lc)32:G is the rock shear modulus . For the estimated rate-weakening parameters at the injection point, dc ranges from 5 μm (Test 1) to 48.75 μm (Test 2), (b–a) ranges from 0.011 (Test 1) to 0.028 (Test 2), and (σn-p) ranges from 0.35 MPa for the maximum pressures to about 6.35 MPa at the initial zero pressure. For this range of values, our estimates of the critical nucleation lengths are comprised between 0.54 to 9.74 m for Test 1, and 2.06 to 37.3 m for Test 2.To estimate the nucleation length for seismic rupture to start along a rate-and-state fault, the nucleation theory indicates that an earthquake occurs within rate-weakening regime (L) greater than the critical nucleation length L > Lc are susceptible to trigger seismicity whereas those with radii L < Lc are not. This analysis is consistent with the slip being aseismic in the pressurized area as observed in situ with seismic events located between 0.76 and 10.7 m in the volume around the injection point. Moreover, the source radius of the seismic events (0.19 to 0.57 m) estimated in21 are smaller than the theoretical minimum length over which earthquake nucleation can occur.Over the fault, slipping zones with radii (It is also important to note that the frictional and elastic properties in the zones of seismicity observed at a distance of injection points are not evaluated in the present study. A future characterization of the frictional parameters within the whole fault zone may help for better defining the seismic potential of fault segments and fractures around the injection.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesDataset 1Dataset 2Dataset 3Dataset 4Dataset 5Dataset 6