In this work, surface/interface effects for pull-in voltage and viscous fluid velocity effects on the dimensionless natural frequency of fluid-conveying multiwalled piezoelectric nanosensors (FC-MWPENSs) based on cylindrical nanoshells is investigated using the Gurtin–Murdoch surface/interface theory. The nanosensor is embedded in a viscoelastic foundation and subjected to nonlinear van der Waals and electrostatic forces. Hamilton’s principle is used to derive the governing and boundary conditions and is also the assumed mode method used for changing the partial differential equations into ordinary differential equations. The influences of the surface/interface effect, such as Lame’s constants, residual stress, piezoelectric constants and mass density, are considered for analysis of the dimensionless natural frequency with respect to the viscous fluid velocity and pull-in voltage of the FC-MWPENSs. Nanomechanical sensors and resonators, especially when combined with piezoelectric materials, are widely used in modern engineering, which encompasses numerous, diverse fields of science and technology, pharmaceutical, agricultural, environmental, advanced materials, chemical science, physics, electronics, information technology, biomedical and medical fields –10. Due Many studies have been carried out on the vibration and stability analysis of nanostructures with some reviews given as follows. Strozzi and Pellicano investigated the vibration analysis of triple-walled carbon nanotubes (TWNTs) subjected to the interlayer van der Waals (vdW) force in the framework of the Sanders–Koiter shell theory . Also, bI,S, µI,S), residual stress  piezoelectric constants  and mass density  are studied for analysis of dimensionless natural frequency with respect to viscous fluid velocity  and pull-in voltage  of fluid-conveying multiwalled piezoelectric nanosensors (FC-MWPENSs) subjected to direct electrostatic DC voltage with nonlinear excitation, nonlinear van der Waals force and viscoelastic foundation. As a guide to the reader, all notation and symbols are presented in To the best knowledge of the author, the surface/interface effect on pull-in voltage, viscous fluid velocity effects and dimensionless natural frequency (DNF) of multiwalled piezoelectric nanosensors conveying viscous fluid has not yet been studied. In the present study, the effect of surface/interface parameters such as Lame’s constants  for the other inner wall layers. All of the physical and geometrical properties of the mentioned nanostructures for single-walled piezoelectric nanoresonators can be seen in work done by Hashemi Kachapi et al. in reference [A schematic diagram of a multiwalled piezoelectric nanosensor with an embedded fluid-conveying inner layer, two piezoelectric layers, and a viscoelastic foundation medium in the outer layer is shown in eference ,20.The governing equations and the solution procedure can be found in I,S, µI,S), residual stress  piezoelectric constants  and mass density , are investigated for analysis of the dimensionless natural frequency with respect to viscous fluid velocity  and pull-in voltage  In order to simplify the presentation, CC, SS and CS represent the clamped edge, simply supported edge and clamped-simply supported edge, respectively. The material properties of the different layers of aluminum (Al) nanoshell and piezoelectric layers (PZT-4) are shown in A verification study is investigated in work by Hashemi Kachapi et al. –20 with The other bulk and surface geometrical parameters of FC-MWPENS are shown in R = Rout; for double-walled PENS (DWPENS): R1 = Rin, R2 = Rout; and for triple-walled PENS (TWPENS): R1 = Rin, R2 = Rmid, R3 = Rout.The value of the mid-surface radius for different PENS are presented as following: for single-walled PENS (SWPENS): I,S, µI,S), residual stress  piezoelectric constants  and mass density , of fluid-conveying multiwalled piezoelectric nanosensors are studied for analysis of the dimensionless natural frequency (DNF) with respect to viscous fluid velocity  and pull-in voltage  For this work, the material and geometrical parameters in Tables 2–4 are used. In all of the following results of the analysis of DNF on viscous fluid velocity  and pull-in voltage  respectively, the values of  = 5 and  = 0.1 are used.In this section, the effect of surface/interface parameters, such as Lame’s constants , the inertia of the shell is increased and its stiffness is reduced, which leads to a decreased DNF compared to the case without surface/interface effects. Also, with decreasing surface/interface density (case 2), the inertia of the system is increased, and with increasing stiffness, DNF increases compared to the case without surface/interface effects.It is observed that for all modes, the DNF decreases when the  and pull-in voltage In all of the following results, the lower surface/interface density (case 2) is used in the analysis of DNF on viscous fluid velocity  and direct pull-in DC voltage on the pull-in instability analysis on the DNF  of FC-MWPENS are presented in The effects of viscous fluid velocity  and  for viscous fluid velocity  and pull-in instability analysis on DNF of FC-MWPENS. It is clear that the increasing surface/interface Lame’s constants λI,S, due to increasing FC-MWPENS stiffness, DNF and critical fluid velocity increase and pull-in voltage in λI,S = 0 and λI,S = −2 has a maximum and a minimum value. and  for viscous fluid velocity  and pull-in instability analysis on DNF of FC-MWPENS are presented in I,S, it is clear that by increasing both surface/interface Lame’s constants µI,S, due to increasing FC-MWPENS stiffness, the DNF and also the critical fluid velocity and pull-in voltage increase.The effects of different surface and interface Lame’s constants,  and  for viscous fluid velocity  and pull-in instability analysis on DNF of FC-MWPENS. As can be seen in the analysis of DNF, increasing the surface/interface residual stress  leads to increasing FC-MWPENS stiffness, and as a result, the DNF and pull-in voltage increase and critical fluid velocity decreases. and  for viscous fluid velocity  and pull-in instability analysis on DNF of FC-MWPENS is presented in  and  leads to increasing FC-MWPENS stiffness, and as a result, the DNF, critical fluid velocity and pull-in voltage increase.The effect of surface piezoelectricity constants  and  for viscous fluid velocity  and pull-in instability analysis on DNF of FC-MWPENS. As it can be seen, with an increasing surface/interface mass density ρI,S, due to increasing FC-MWPENS stiffness, the DNF significantly increases and also the critical fluid velocity and pull-in voltage slightly decrease. and pull-in instability analysis on DNF of SS FC-MWPENS are presented. It can be seen that by ignoring the surface/interface density ρI,S, the inertia of the system will greatly decrease and due to increasing FC-MWPENS stiffness, the system will have a maximum DNF compared to other cases. Also when the surface/interface effects are not taken into account, due to the decreasing nanoshell stiffness, it has a lower DNF than the case with all surface/interface effects. In the cases without all surface/interface effects, the critical fluid velocity and also the pull-in voltage reach zero sooner than the rest of the parameters. In the cases without surface/interface density ρI,S and with all surface/interface effects, the pull-in voltage and critical fluid velocity reach zero later than the rest of the parameters and the system loses its stability due to the divergence via a pitchfork bifurcation.In  and pull-in voltage  The piezoelectric nanosensor is simultaneously subjected to direct electrostatic DC voltage with nonlinear excitation, nonlinear van der Waals forces and a viscoelastic foundation. For this purpose, Hamilton’s principles, the assumed mode method combined with Lagrange–Euler’s equations are used. The results demonstrated that in the case of higher (lower) surface/interface densities, the inertia of the shell is increased (decreased) and its stiffness is reduced (increased), which leads to a decreasing (increasing) natural frequency compared to the case of without surface/interface effects. Also, by increasing both surface/interface Lame’s constants, λI,S and µI,S, and the negative surface piezoelectricity constants,  and  due to the increasing FC-MWPENS stiffness, the DNF and also the critical fluid velocity and pull-in voltage increase. In addition, in the analysis of DNF, it was found that increasing the surface/interface residual stress  leads to increasing FC-MWPENS stiffness, and as a result, the DNF and pull-in voltage increase and critical fluid velocity decreases. Increasing the surface/interface mass density, ρI,S, due to increasing FC-MWPENS stiffness, it was found that the DNF significantly increases and also the critical fluid velocity and pull-in voltage slightly decrease. Finally, by ignoring the surface/interface density, ρI,S, the system will have a maximum DNF compared to other cases. In cases without all surface/interface effects, the critical fluid velocity and also the pull-in voltage reached zero sooner than the rest of the parameters. In the cases without surface/interface density ρI,S and with all surface/interface effects, the pull-in voltage and critical fluid velocity reach zero later than the rest of the parameters.In the current study, the effect of the surface/interface parameters of a fluid-conveying multiwalled piezoelectric nanosensor are studied for analysis of the dimensionless natural frequency with respect to viscous fluid velocity File 1Subsections of “Mathematical Formulation” as well as an “Appendix” section.File 2MATLAB program code for the current paper.In the ZIP file, all programs written in MATLAB software are presented for the results of the article, which is not included in the article due to the large volume of the program.


A high expression of PRR at the tumour centre and at the infiltrative front of CCRCC tissues is significantly associated with high grade, tumour diameter, local invasion and stage, and with high mortality risk by UCLA integrated staging system (UISS) scale. (4) Conclusions: These findings indicate that PRR is associated with the development and progression of renal tumours. Its potential as a novel biomarker for RCC diagnosis/prognosis and as a promising therapeutic target should be taken into account in the future.(1) Background: Renal cancer is one of the most frequent malignancies in Western countries, with an unpredictable clinical outcome, partly due to its high heterogeneity and the scarcity of reliable biomarkers of tumour progression. (Pro)renin receptor (PRR) is a novel receptor of the renin–angiotensin system (RAS) that has been associated with the development and progression of some solid tumours by RAS-dependent and -independent mechanisms. (2) Methods: In this study, we analysed the immunohistochemical expression of PRR at the centre and border in a series of 83 clear-cell renal cell (CCRCCs), 19 papillary (PRCC) and 7 chromophobe (ChRCC) renal cell carcinomas, and the benign tumour renal oncocytoma (RO,  Renal cell carcinoma (RCC) is one of the most common malignancies in Western Countries ,2,3. NeaClear-cell renal cell carcinoma (CCRCC) is by far the most frequent histological subtype of RCCs, accounting for approximately 75–80% of cases, followed by papillary renal cell carcinoma (PRCC) (10–15%) and chromophobe renal cell carcinoma (ChRCC) (5%) . CCRCC iThe RAS has been traditionally described as a circulating hormone system that regulates cardiovascular and renal function . HoweverThese phenomena are also part of neoplastic processes and, therefore, the study of RAS and the potential of RAS-targeting therapies has received considerable attention in research into renal cancer ,11. ThusThe (pro)renin receptor (PRR) is a novel component of the RAS that was first described in the past decade  and thathttps://www.proteinatlas.org (accessed on 5 February 2021)) described PRR staining in RCCs; however, the analyses were limited to only 11 cases, which was insufficient to understand the association between this protein and tumour progression.These RAS-dependent and -independent signalling pathways contribute to cancer initiation, so it was expected that PRR expression could be altered in tumour tissues . Thus, iIn this study, we analysed PRR immunohistochemical expression in a series of 120 kidney tumours. The series included three subtypes of RCC  and the benign tumour RO. Both the centre of the tumour and the infiltration front was analysed to test the possible heterogeneity of PRR expression in these tumours. Since CCRCC is the most frequent RCC , we analwww.biobancovasco.org (accessed on 5 February 2021)) was the source of samples, and the data from employed patients could possibly be used for research purposes. Each patient signed a specific document which was approved by the Ethical and Scientific Committees of the Basque Country Public Health System (Osakidetza) (PI + CES-BIOEF 2018-04).The present study, including all its experiments, comply with current Spanish and European Union legal regulations. The Basque Biobank for Research (OEHUN) , 19 PRCCs , 7 ChRCCs  and 11 ROs . Samples from the tumour centre and the infiltrating front were included in tissue microarrays (TMAs) for further immunohistochemical analyses. One sample of the uninvolved kidney was also included in each TMA.Rabbit polyclonal antibody specific for PRR  was used for the immunostaining of formalin-fixed and paraffin-embedded tumour tissues. The antibody’s specificity was tested previously  and the Slides were reviewed under light microscopy for staining evaluation. Two observers independently evaluated the slides and, in the event of discrepancies, samples were re-evaluated to arrive at a final conclusion. As previously reported , stainin® 24.0.The statistical analysis was performed with SPSSWe applied a Kolmogorov–Smirnov test to assess if data obtained from tumour tissues followed a normal distribution. Based on this information, data were further analysed using parametric or non-parametric tests.The correlation between PRR expression in tumour centre and front and patient age and gender was evaluated using Spearman Rho tests. Associations between categorical PRR expression (weak/intense) in renal tumour tissues’ pathological and clinical variables were tested by Chi-square (χ2) test. Cancer-specific survival (CSS) analysis was performed by long-rank test with a cut-off point based on the categorical expression of PRR.The uninvolved part of the kidney showed more intense staining in tubules from the distal nephron than from proximal ones . PRR staSame as has been observed in normal renal tubules, the different subtypes of renal cell carcinomas  and RO expressed PRR in the cytoplasm and plasmatic membrane of tumour cells a. Only oNearly half of CCRCCs and PRCCs showed weak staining of PRR, whereas in ChRCC and RO, the staining was mainly intense. Significant differences were found between tumours from distal and proximal nephron both in the centre and tumour front b,c.p = 0.6), PRCC (p= 0.64), ChRCC (p = 0.3) and RO (p = 0.31).We also compared differences in the expression of PRR between the tumour centre and front in each kidney tumour subtype. Both tumour locations showed a similar staining pattern in CCRCC , the presence/absence of affected lymph nodes (N) and metastases (M), TNM stage and tumour progression . The association between PRR and clinical variables such as patients’ sex, age, mortality risk , and 5-year cancer-specific survival was also analysed.p = 0.58; tumour front: r = −0.01, p = 0.99) and age .The non-parametric Rho Spearman test was performed to assess if PRR protein expression varies according to the gender or age of the patients. Results showed no statistically significant correlation between PRR and sex  and high histological grade (G3–G4). High-grade CCRCCs expressed significantly stronger PRR staining than low-grade tumours, both at the centre and at the infiltrating front .CCRCCs were classified in three groups following previously reported classifications ,32: tumoThe series had a limited number of pT4 cases and we stratified local invasion in three groups: pT1 (organ-confined tumours ≤7 cm), pT2 (organ-confined tumours >7 cm) and pT3-pT4 (non-organ-confined tumours). PRR expression at the centre and at the infiltration front of pT2 tumours was significantly higher than in pT1 ones. Non-organ-confined tumours also showed stronger staining of PRR than pT1 ones, with significant results at the tumour centre c,d.p = 0.23; at tumour front, p = 0.33)   showed a similar trend, towards more intense PRR staining than tumours without lymph node invasion (N0), but data were not statistically significant  e,f. Simi = 0.39) g,h.CCRCCs were stratified as low-stage (Stage I–II) and advanced- or high-stage (Stages III–IV). PRR staining at the centre of tumours was stronger in advanced than in low-stage CCRCCs i,j.p = 0.026) and UISS scales  and 5-year CSS was statistically significant.We used two validated scales that predict tumour progression and mortality risk of CCRCC patients: the Mayo Clinic stage, size, grade, necrosis (SSIGN) model  and the p = 0.23; PRR at tumour front, p = 0.12) .PRR at the tumour centre and front showed more intense PRR staining in patients with intermediate and high mortality risk (UISS) than in those with low risk a,b. Howe = 0.12) c,d, or tRCC is a complex and heterogeneous tumour with only two first-line therapeutic approaches with some efficacy at present: the inhibition of angiogenic pathways by tyrosine-kinase inhibitors (TKIs) and the improvement in anti-tumour immunity by recently included immune checkpoint inhibitors (ICIs) . ClinicaACE2 mRNA levels in CCRCC are associated with better survival of RCC patients and better response to immunotherapy [The second most-known axis of the RAS, the ACE2/Ang1-7/Mas axis, counterbalances the actions of AngII. Anti-tumour effects of this axis have been demonstrated in several solid cancers ,11; howeotherapy .The place of PRR in the complex scheme of RAS and renal cancer remains to be determined. As previously reported , we obseThe mechanism by which PRR upregulation could affect the progression of kidney could be RAS-dependent or -independent ,21. It iThe binding of renin or prorenin to PRR can activate the PI3/AKT/mTOR pathway independently of the action of angiotensins ,21. ThisPRR can be found in the cell-surface but also in the cytoplasmic organelles. The protein is expressed in the membranes of the endoplasmic reticulum and the Golgi apparatus, from which it can be released as a soluble isoform (sPRR) after cleaving by ADAM19, furin and Site-1 protease ,42. The PRR has also been described in benign tumours, such as adrenal ,47 and cIn summary, this study describes PRR protein in a series of kidney tumours with different topographic origins in the nephron. Furthermore, the results in CCRCCs show that the protein expression is increased in both the centre and the invasive front of the most advanced tumours, as has been observed in other solid tumours ,27,28,29


Fluid in interstitial spaces accounts for ~20% of an adult body weight and flows diffusively for a short range. Does it circulate around the body like vascular circulations? This bold conjecture has been debated for decades. As a conventional physiological concept, interstitial space is a micron‐sized space between cells and vasculature. Fluid in interstitial spaces is thought to be entrapped within interstitial matrix. However, our serial data have further defined a second space in interstitium that is a nanosized interfacial transport zone on a solid surface. Within this fine space, fluid along a solid fibre can be transported under a driving power and identically, interstitial fluid transport can be visualized by tracking the oriented fibres. Since 2006, our data from volunteers and cadavers have revealed a long‐distance extravascular pathway for interstitial fluid flow, comprising at least four types of anatomic distributions. The framework of each extravascular pathway contains the longitudinally assembled and oriented fibres, working as a fibrorail for fluid flow. Interestingly, our data showed that the movement of fluid in a fibrous pathway is in response to a dynamic driving source and named as dynamotaxis. By analysis of previous studies and our experimental results, a hypothesis of interstitial fluid circulatory system is proposed. This active ISF transport is of distinguished characteristics below: (a) It is long‐ranged instead of short‐ranged. (b) It is systemic instead of regional. (c) It is ordered instead of random. (d) It is structuralized, multilayered, multi‐levelled, multiscaled, fibre‐oriented and interfacial‐constrained. (e) It is power‐controlled, that is heart‐beating‐driven or periodic‐power‐source‐driven. (f) It is a faster transport process rather than a slower diffusion process, that is the measured peak flow speed is approximately 2 cm/s. (g) It is systemically cycled, that is the exchanges with vascular circulations and the dynamic cycled patterns are universally exist. Finally, a hypothesis of interstitial fluid circulatory system is proposed.2The total body fluids in adult can be divided into three compartments: (a) the fixed intracellular fluid compartment that accounts for about 32% of body weight, (b) the systemically circulatory fluid compartment  for about 8% of body weight, and (c) the local‐transport interstitial fluid compartment for about 20% of body weight. It is noted that the word “local‐transport” or “diffusive transport” is used when ISF is mentioned in literatures.In physiology, ISF is thought to be entrapped within the interstitial/extracellular matrix and transports diffusively for a very short‐ranged.Roughly speaking, a few key words about the characteristics of traditional ISF diagram may be summarized: entrapped, localized, short‐ranged, disordered, permeated, single‐level structured, single‐size scaled, and concentration‐gradient driven or pressure‐gradient driven, Darcy's law controlled. However, such localized diagram of ISF flow has been debated for several decades.3Could the ISF transport systematically throughout the whole body? As far as we know, there are three representative studies on the ISF transport beyond vascular circulations in history.The earliest record of a long‐distance ISF transport is “the fluid in perivascular spaces” described in 1851 by Rudolph Virchow, which is an extravascular channel for ISF flow between the outer and inner lamina of brain vessels or around fenestrated capillaries. This diagram is further studied recently in researches on the clearance pathways of the brain. A perivascular drainage of ISF along the basement membrane of smooth muscle cells of intracerebral and cerebral arteries and capillaries has been observed by identifying the deposits of amyloids.The second is the interstitial tissue channel. Around the 1970s, by injection of ferrocyanide ions into blood vessels from which they leak into the interstitial tissues, it was found that the precipitate deposits of ferric ions were in the connective tissues near vessel wall or in the skeletal muscle tissues under the electron microscope, light microscope and in the mesentery of rabbit and cat by dark‐field transilluminations.The third is the explorations on the Meridian and Collateral Channel network described in traditional medical literatures. Around the 1980s, instead of intravenous injection, an isotopic tracer was hypodermically injected into an acupuncture point in hands or feet of humans.The above experiments are heuristic: (a) By comparison with the complicated measurements of the interstitial pressure gradients, it is more convenient to identify interstitial fluid flow by an imaging tracer. (b) To visualize the extravascular ISF transport pathways, the imaging tracer shall be injected into a certain region of interstitial tissues outside the vascular vessels. (c) The strategy to identify a tubular structure for fluid flow may be ineligible. Accordingly, a few essential questions need to be answered: (a) How to clarify the anatomical and histological structures of the long‐distance ISF transport pathways in humans? (b) What are the mesoscopic and microscopic structures conducting the ISF flow? (c) What is the transport mechanism of the long‐distance ISF flow? (d) What is the role of the beating heart in ISF flow?4Since 2006, our research group has made a series of progresses exploring the ISF transport. Different from the isotopic tracers, we use several techniques with higher spatial resolution including contrast‐enhanced MRI and have visualized a long‐distance extravascular transport pathway from a hypodermic point in the extremity endings of volunteers, usually an acupuncture point.4.1Another valuable progress from our research group is the discovery of the anatomical and histological structures of the long‐distance extravascular pathways from an acupuncture point, especially in physiological conditions. One subject was recruited who had severe gangrene foot due to arteriosclerosis obliterans and would receive the amputation of his right lower leg. BEFORE the amputation, he took the hypodermic injection of the fluorescent tracer into an acupuncture point in ankle. Around 90 minutes after the amputation, tissue samples from the lower leg were histological analysed. It was clearly visualized by the fluorescein that four types of the extravascular pathways from the ankle to the amputated end of leg were consistently formed by fibrous connective tissuesIn addition to physiological conditions, ISF transport pathways in non‐physiological conditions were also investigated. Three amputated lower legs received the injection of the fluorescent tracer into the same acupuncture point were investigated AFTER their amputation. In these cases, periodic “mechanical compressions” on the amputated end of the legs were covered by a sphygmomanometer cuff with a systolic pressure of 50‐60 mm Hg and a compression‐relaxation frequency of 18‐20 times/min. After around 90 minutes of manipulation, the same four types of the fluorescently stained fibrous pathways were observed.The ISF transport pathways discovered above in both physiological and non‐physiological conditions may lead to the following judgements: (a) The long‐distance ISF transports include at least four types of anatomical structures in living human body or the cadavers. (b) The histological structures of each ISF transport pathway are consistently formed by fibrous connective tissues which suggest that ISF flow universally exists in fibrous matrices throughout the whole body. (c) The transport mechanisms of a long‐distance ISF transport were not fully understood yet but might be periodically power‐sources‐driven or multi‐driving‐centres‐driven. The to‐and‐fro movements of the dynamic source are of vital importance in ISF transport, like the pulsation of the heart and the vasomotions of vascular vessels.We have disclosed some optical imaging features of the long‐distance ISF transport as well. Using two‐photon confocal laser microscopy (TPCLSM), all the extravascular pathways were found to contain thousands of the fluorescently stained and micron‐sized fibres being distributed longitudinally along the long axis of the transport pathways.To further confirm the universal existence of the above discoveries in the whole body, the human cadavers were used to testify the extravascular pathways by the simulated heart pulsation using a mechanical chest compressor.4.2in situ imaging evidence of a fluid film paved on the fibre by the optical microscope. There are no other visible features in this transport region except for the visualized fibres and gel under the submicron resolution. From the viewpoint of interface science, an interfacial space would be formed physically between two phases of matters. In interstitial matrix, the only possible transport space for ISF flow must be an interfacial transport zone (ITZ) between the fibre (a solid phase) and the gel/liquid substance (a liquid phase).In interstitial connective tissue, neither the fibre nor the gel themselves can flow.interfacial dynamic transport (IDT).” According to the anatomical positions, the IDT pathways can be classified into different types, such as a cutaneous IDT pathway, a neural IDT pathway, a facial IDT pathway, an adventitial IDT pathway, an extracellular IDT pathway, a tumorous IDT pathway, a neoplastic IDT pathway, an interosseous IDT pathway, a glandular IDT pathway, etcIn macroscopic scale like the anatomical structures, the microscopic ITZs can be topologically connected along the intrinsic fibres and comprise a macroscopic transport pathway of long‐ranged characteristics throughout the whole body. When ISF comes into the topologically connected ITZs, ISF may continuously flow via these oriented interstitial connective tissues by a driving power and present two types: ISF flow along the embedded, oriented and ordered fibrorails and in the meanwhile diffuse/exchange between the ITZs and the surrounding tissue gel along the long‐distance extravascular pathways.4.3in vivo.In rabbits, a continuous fluid flow in both the adventitial pathway and its surrounding fibrous connective tissues as well as their topographical connections with the heart has been observed in vivo rabbits in our laboratory. Firstly, when an incision was made in a segmental venous adventitia, continuous ISF flow can be observed in the incision area and exhibit a continuous bulk‐like emerging flow of ISF in the ITZs along the fibres of the adventitial IDT pathway. Secondly, when the fluorescein in the IDT pathways began to fade away, a few fluorescent plaques formed by dozens to over one hundred micrometres were found to flush away, that probably represented a bigger ITZ on layers of smooth muscle cells between the tunica adventitia and tunica media of vascular vessels  Fluid transport via vascular vessels comprises two types: an intravascular blood flow and an adventitial ISF transport. (Ⅱ) For venous vessels, the direction of adventitial ISF transport is the same as that of blood flow. (Ⅲ) For arterial vessels, the direction of adventitial ISF transport is opposite to that of blood flow.In addition, we define that an adventitial ISF/IDT transport includes three parts Figure B,C: (1) For the arteriole, venule and capillaries in the diverse visceral organs including the brain, the ISF flow directions of each part of an adventitial ISF/IDT pathway need to be identified. According to our data, the to‐and‐fro movements of the beating heart play a special role as a driving centre or transferring centre in regulating fluid flow in an adventitial ISF/IDT pathway. Within these driving centres, like the beating heart and the respirometric lung, ISF flow in each part of an adventitial ISF/IDT pathway needs to be clarified, respectively. The lymphatic vessel, structured like blood vessels, comprises endothelial cells, a thin layer of smooth muscle and fibrous adventitia that binds the lymphatic vessels to the surrounding tissues. We predict that there is an adventitial ISF/IDT transport in the lymphatic adventitia as well.6interfacial matrix driven by an active dynamic mechanism. (c) Fluid flow through fibrous matrices is of multiscale characteristic. An ITZ on a fibrorail is microscopic but the topologically connected ITZs on the multilayered, highly ordered and cross‐linked fibrorails are macroscopic and long ranged. In an extravascular pathway with a diameter of millimetres and a length of centimetres or even over one metre, fluids in the ITZs would be a bulk‐like continuous flow under a driving power. (d) At macroscopic scale or a topographical anatomy, fluid flow through fibrous matrices constitutes an extremely complex network of a long‐distance IDT pathway from the extremity endings to visceral organs or among organs or tissues or cells throughout the whole body. (e) The transport processes of the IDT pathway network are “active and dynamic.” Without the powers, fluid in matrices would diffuse locally. The movement of fluid in an IDT pathway is in response to a dynamic driving source or centre, which is named as dynamotaxis. Usually, the dynamotactic direction of fluid flow in an IDT pathway is towards a driving source or centre. (f) It is noted that the driving sources and centres are various other than one unique driving centre. One of the driving sources in an IDT system has been proposed to be the dynamic to and fro “gel pump.”In lights of the revealed second space in interstitium and structural framework of fibrous connective tissues throughout the whole body, the diagram of fluid flow through fibrous matrices are renewed Figure : (a) Theinterfacial transport zone paved on a solid surface, like a fibrorail or a layer of cells. Fluid in an ITZ can transport along the solid surfaces under an active dynamic driving power, the transport pattern of which is named as an interfacial dynamic transport. Secondly, the intrinsic structural framework of interstitium is NOT irregular but ordered. By contrast to the bulky and irregular interstitial space between cells and vasculature, the fine ITZ provides a constrained, ordered and oriented space for fluid flow. Molecules and solutes in interstitial spaces would execute not only “random walks” by diffusion but also “oriented transport” along ordered fibrorails. (2) Based on the fundamental definitions of ITZ and IDT/IDC, systemic fluid flow in fibrous matrices may be reconstructed. (3) The conventional definition of interstitial fluid may refer to fluid in the interstitial compartment that accounts for ~ 20% of body weight as well as fluid in the interstitium between the cells and vasculature. Parts of interstitial fluid, which is fluid in an ITZ of interstitial matrices, may be redefined as interfacial fluid. (4) Like the above analysis, interfacial fluid can transport or flow systemically around the whole body at multiscales, multi‐levels and multi‐layers, such as an adventitial IDT network, a neural IDT network, a cutaneous IDT network, an facial IDT network, etc (5) The movements of ISF is from and back to blood circulation. Parts of ISF enter the ITZs and are transported by an IDT pathway. Fluid in an IDT pathway can quickly and easily interchange with ISF and eventually exchange with vascular circulations by capillary beds or other unidentified ways, that might be the adventitia of an arterioluminal vessel or thebesian veins. (6) Here, we make a hypothesis: (a) Besides diffusive transport in short range, interstitial and interfacial fluid circulate through all parts of the body, which is named as an interstitial/interfacial fluid circulatory system (IFCS) and abbreviated as interstitial fluid circulation. (b) The interstitial and interfacial fluid in extracellular/interstitial matrices, the blood and lymph in vascular circulations comprise three principal compartments of the circulatory system in animals and humans, which together constitute a multiplex circulatory system. (7) Like blood and lymph flow under the actions of the heart, fluid in an adventitial ISF/IDT system  The interstices of interstitium or interstitial space may refer to a steric space between the cells. In this bulky space, the majority components of interstitial space are an extracellular matrix that is mainly composed of types of collagenous and elastic fibres, and glycosaminoglycans which form a gel‐like substance. In other words, the matrix in interstitial space contains a structural three‐dimensional scaffolding embedded in the gel‐like interstitium. Firstly, we have further defined a fine space within this structuralized interstitium as an rorails.  Based onParticularly, further anatomic studies are indispensable. We propose an international cooperation project for Human Interstitial Fluid Connectome Atlas (HICA) to identify the detailed anatomical and microscopic structures of the entire IFCS and IDT system by a method of dynamic topographical anatomy and a guidance of traditional medicine. The medical applications might benefit from any progress in the understandings on the IFCS and HICA, such as medical imaging technique, a novel pathway for drug delivery, therapeutic instruments, etc. Future studies will need an interdisciplinary cooperation to reveal the regulating mechanisms of IFCS and its relationships with therapeutics. Our pilot studies may provoke a new field of fascinating and fruitful researches in life science.The authors declare no competing financial interests and have no conflicts to disclose.HyL conceived the original ideas, conceptions and designed the experiments and analysis. HyL, QcY, CM and YyK performed the gross human anatomic and animal experiments and the histological analysis. HyL, MC, FW, FsJ and HL analysed the MRI data. HyL, YjY and JH analysed the interfacial transport pattern. HyL wrote the manuscript. All authors contributed to scientific discussions of the manuscript.


However, the disturbance effects of drilling on the stability of NGH-bearing sediments are unclear. There are still few experimental apparatuses on this issue, and existing experimental apparatuses cannot comprehensively simulate the drilling process as well. In order to fill this gap in prior studies, an experimental drilling apparatus used for evaluating drilling risks related to NGH was developed. The apparatus consists of a high-pressure vessel with a drilling system, a drilling fluid injection system, a drilling fluid treatment system, and a data acquisition system. Hydrates can form in the high-pressure vessel placed inside a walk-in cold room. The drilling fluid can be cooled to the desired temperature by the drilling fluid treatment system and be injected into the drilling system by the drilling fluid injection system. The drilling system can simulate the comprehensive drilling process, including drilling feed, trip up & down operations, drilling fluid circulation, etc. 48 thermometers were inserted into the high-pressure vessel from the bottom. The thermometers uniformly distribute in the high-pressure vessel, and they could quickly and accurately measure the hydrate phase change process under high-pressure and low-temperature conditions.  Specifications TableNatural gas has increasingly become a profitable alternative energy resource The hydrate is formed in the hydrate sediment simulation system. As shown in The drilling system is used to simulate the drilling process. The drilling system consists of the support, a high-pressure rotary connecting device, two electric motors, and a drilling device. As shown in The drilling fluid injection system, including a drilling fluid tank, a drilling fluid cooling unit, and a drilling fluid pump, is used to provide the drilling fluid within a temperature range of 0 - 20 °C and a flow range of 0 - 1 L/min. The drilling fluid tank is provided with a cooling unit and a stirring unit. The drilling fluid with the desired temperature and flow can be injected into the drilling rod from the drilling fluid tank via the drilling pump; then the drilling fluid flows out from the drilling bit through a one-way valve, and finally flows out from the mud outlet via the annular space between the drilling rod and the wellbore, as shown in The drilling fluid flowing out from the drilling system can be gathered through the drilling recovery system. The drilling fluid recovery system consists of a high-pressure desander, a backpressure regulator, a gas-liquid separator, a gas flowmeter, a balance, and a conductivity meter. The high-pressure desander can reduce the sand content in the drilling fluid flowing out from the drilling fluid outlet. The backpressure regulator, connected to the outlet of the high-pressure desander, is used to control the pressure of the high-pressure vessel. The balance and gas flowmeter are used to measure the mass of liquid and gas produced from the vessel, respectively. Besides, the conductivity meter  can measure the conductivity of the drilling fluid flowing out from the drilling fluid outlet.The data acquisition system includes the matching software and several sensors. The data measured by each sensor, including temperature, pressure, drilling depth, drilling torque, total quality of the cumulative liquid and gas produced from the vessel, and conductivity of drilling fluid flowing out from the outlet, can be recorded and saved at a regular interval by the matching software.The experiment procedure essentially comprises two stages: a) sample preparation; b) drilling in hydrate-bearing sediments.Since it is hard to recover core samples from actual NGH-bearing sediments and maintain their intact status, artificial cores are designed to simulate the porous nature of NGH reservoirs. There are two kinds of artificial cores in experiments. One is the unconsolidated core, which has high permeability but low mechanical strength. Another is the consolidated core made by mixing quartz sands and epoxy resin. The consolidated core has high mechanical strength, and its permeability can be controlled.The artificial core is tightly packed into the high-pressure vessel. After the artificial core is prepared, the gas or saline solution is injected into the high-pressure vessel by a booster pump until the pressure in the high-pressure vessel reaches a desired value. Meantime, the temperature of the cold room is set to the desired experimental temperature. Subsequently, hydrates will form gradually in the artificial core.Take the cases in this study for example, for the unconsolidated sediments, quartz sands with particle sizes from 40 – 70 mesh number (0.425mm-0.212 mm) without any additive were tightly packed into the high-pressure vessel. The weight of the quartz sands packed into the vessel is approximately 8.2 kg. For the consolidated sediments, the quartz sands with particle sizes from 40 – 70 mesh number (0.425mm-0.212 mm) were firstly mixed thoroughly with epoxy resin and then packed into the high-pressure vessel. The mass ratio of epoxy resin to quartz sands used in experiments is 1:10. In the experiments, the weight of the cementation packed into the high-pressure vessel is approximately 10.0 kg.1)Turn on the drilling fluid cooling unit, the drilling fluid in the mud tank is cooled to the desired temperature;2)Adjust the set pressure value of the back-pressure regulator to the pressure required by experiments, and control the pressure inside the high-pressure vessel to reach the experimental pressure;3)Set the values of drilling fluid flow, drill rod rotation speed, and drill rod movement speed;4)Inject the drilling fluid in the drilling fluid tank into the drilling system by the mud pump at a constant rate;5)At the same time, turn on the electric motors, making the drill rod to move downward at a set speed and rotate at a set rotating speed;6)When the drill bit reaches its maximum drilling depth, the drill rod stops moving downward and continues to rotate until the experiment is ended;7)The data required are recorded at certain time intervals during the experiment process by the software;8)At the end of the experiments, the high-pressure vessel can be opened to observe the condition of hydrate-bearing sediments directly.After hydrate-bearing sediments are prepared, the drilling experiments are carried out following these steps:Tetra-n‑butyl ammonium (TBAB) hydrate was formed in the unconsolidated or consolidated cores to simulate hydrate-bearing sediments because TBAB hydrate can form at atmospheric pressure and near room temperature and has some similar physical and structural properties with NGH. Therefore, it is a practical and easier method to carry out drilling experiments in TBAB hydrate-bearing sediments.The phase equilibrium condition of TBAB hydrate is decided by the TBAB solution concentration. Normally two types of TBAB hydrate can grow simultaneously in aqueous solution, type A and type B The TBAB solution was injected into the unconsolidated core in the high-pressure vessel to form the TBAB hydrate-bearing sediments at 4 °C, and the temperatures of drilling fluid are 4 °C and 8 °C for experiments 1 and 2, respectively. In summary, the drilling apparatus in this study can experimentally simulate the dynamic drilling process in hydrate-bearing sediments with different drilling fluid properties, rod rotation speeds, and rod movement speeds. The temperature distribution around the wellbore, the pressure change in the high-pressure vessel, the water production, the gas production, and the conductivity change of drilling fluid during the drilling process can be obtained by the apparatus. Therefore, this experimental drilling apparatus can provide an analogy between drilling operations in a field and drilling experiments in an indoor laboratory, and investigate the hydrate dissociation characteristics around the wellbore and wellbore stability during the drilling process.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


It is thought that extensional structures  generated during the post-seismic period create fluid pathways that enhance the drainage of the subducting plate interface, thus reducing the pore pressure and increasing fault strength. However, it remains to be elucidated how much pore fluid pressure decreases by the extension crack formation. Here we examined (i) the pore fluid pressure decrease, and (ii) the degree fault strength recovery by the extension crack formation during the post-seismic period by analyzing extension quartz veins exposed around the Nobeoka Thrust, southwestern Japan. The Nobeoka Trust is an on-land analog of the modern splay fault at shallow depths (~ 8 km) in the Nankai Trough. The poro-elastic model of extensional quartz vein formation indicates that the formation of extensional cracks only releases up to ~ 7–8% of the total pore fluid pressure at ~ 8 km depth. The pore pressure around the Nobeoka Thrust was close to lithostatic pressure during the entire seismic cycle. The estimated effective frictional coefficient along the Nobeoka Thrust after this small fluid-loss by the extensional crack formation does not exceed 0.15. Hence, the pore fluid pressure reduction due to the post-seismic extensional cracks contributes little to increase the fault strength of the megasplay fault. Pf) is of great importance in understanding earthquake mechanics. The temporal buildup of pore fluid pressure during the seismic cycle may promote temporal changes in fault strength1. In the co-seismic period of the seismic cycle, high pore fluid pressure close to lithostatic is observed around faults . The distribution of high pore fluid pressure is likely to be time-dependent, thus varying over the seismic cycle1. The time-varying seismic reflectivity may be used to detect the porosity changes related to the development of pore fluid overpressure along the subduction interfaces4. The porosity changes are controlled by the temporal stress changes during the seismic cycle. In forearc regions, the near complete stress release that occurs during huge trench-type earthquakes  induces a change from reverse-faulting type stress regime to a non-Andersonian stress regime including normal faulting earthquakes5. Previous hydrological research has suggested that the fluid loss by the formation of these extensional deformation structures  in the post-seismic period increases the fault strength and creates drainage asperities along the plate interface6. Here, we focus on the fluid migration in the hanging wall by the extension crack formation. The key question arises: To what degree is pore fluid pressure reduced by the extension crack formation? The temporal change in pore fluid pressure given by Skempton's relationship8 is of the same order as the stress drop related to trench type earthquakes6. However, crustal stresses and pore fluid pressures at depth are difficult to quantify directly, and downhole measurements of in situ pore fluid pressure are generally limited to depths of a few kilometers9. Sibson6 estimated that the change in frictional strength at 10 km depth is 6–64 MPa, increasing to 26–256 MPa at 40 km depth. Given the large range of estimates at each depth range, we seek to more tightly constrain the change in pore fluid pressure and the resulting change in frictional strength.Pore fluid pressure  the pore fluid pressure loss and (ii) the amount of fault strength recovery by the extension crack formation during the post-seismic period in a subduction zone.To answer this question, we examine temporal changes in pore fluid pressure during seismic cycles by analyzing extension mineral veins that are exposed along an ancient megasplay fault. The Nobeoka Thrust, southwestern Japan, which is an on-land example of the megasplay fault at shallow depths in the Nankai Trough, contains datasets of quartz veins that enable an understanding of fluid pathways in the hanging wall of the subduction zone1 > σ2 > σ3) plays a critical role in the containment of pore fluid overpressure, with overpressures being much more easily sustained in compressional stress fields20. Brittle-rock failure depends on the coefficient of internal friction and the balance between differential stress (Δσ = σ1 − σ3) and rock tensile strength (Ts)23. In particular mode I cracks, which we focused on this study, is formed under low differential stress of Δσ < 4Ts21. At the same time, the type of failure affecting an intact rock mass is strongly dependent on Pf, which controls the principal effective stresses24. For fluid to generate a crack, Pf must exceed the normal stress (σn) acting on the crack wall25. Extensional veins are formed when pore fluid pressure exceeds the sum of the minimum principal stress (σ3) and Ts26. Hence, the extensional veins are formed whenThe character of the tectonic stress regime  defined asThe mechanics of crack opening can be represented in three dimensions using the Mohr circle construction, with the conditions for opening being represented by the shaded area in Fig. Po. To calculate the ΔPo is possible to use the vein aspect ratio (W/L), where W represent vein aperture width and L the vein length, assuming that it is linearly related to pore fluid overpressure ΔPo and to the elastic rock properties of the host rock19:Here, we employed the poro-elastic model to estimate Δν is the Poisson’s ratio and E the Young’s modulus. Hence, W/L of veins is depended on ΔPo and E19.where P*) is the ration between the pore fluid overpressure ΔPo and Δσ as follows:The driving pore fluid pressure ratio  under lower pore pressure than the pore pressure to newly form the cracks. Therefore, to estimate the upper limit of the pore pressure, considering the formation of the crack is appropriate and we use the equation with Ts on P*. P* varies from 0 (no opening of cracks) to 1 (forming new cracks), and describes the equilibrium between Pf and the minimum and maximum stresses 10. Its total estimated displacement of ~ 8.6–14.4 km, based on a 70 °C temperature difference between the hanging wall (phyllite that include thin coherent sandstone layers of the Kitagawa Group) and footwall , is comparable to the deeper part (~ 8 km) of the modern megasplay fault in the Nankai subductione zone12. Around the Nobeoka Thrust, we observed ~ 800 quartz veins that filled mode I crack  mode I crack veins with no appreciable shear displacement; and (ii) veins that do not intersect other cracks, veins or rock discontinuities (unrestricted veins). W and L of the quartz veins around the Nobeoka Thrust were estimated from the thin sections and field surveys, respectively; both measurements are log-normally distributed, with W = 10–400 μm (geometric mean 52 μm) and L = 1–50 cm (geometric mean 7.4 cm), with a few veins being longer than 50 cm18. Overall, W/L ratios of all veins vary between 0.00002 and 0.04 with an average of 0.0007.We observed discrete extensional veins around the Nobeoka Thrust, filled mainly with quartz and lesser calcite3) axis being sub-horizontal and trending roughly NNW–SSE in both the hanging wall and footwall13. The reverse faulting type stress regime in pre-seismic period, combined with the σ3 axis sub-parallel to the slip direction of the Nobeoka Thrust (top to the SSE)31 . The following elastic parameters were derived from the literature: ν = 0.25 and Ts = 15 Ma  and 10 MPa 36. The resulting average ΔPo values range between ~ 9 and ~ 19 MPa and between ~ 6 and ~ 11 MPa for the hanging wall and footwall veins, respectively  at 8 km depth (z = 8 km), the Pf derived from our vein data range between ~ 189 and ~ 196 MPa and between ~ 200 and ~ 211 MPa for the hanging wall and the footwall veins, respectively. Hence, the maximum case of Pf for the hanging wall and footwall are 196 MPa and 211 MPa, respectively.Δely Fig. . The aveely Fig. . Here, bλv = Pf/σv) for the formation of the mode I crack exceeds 0.95. To compare with the pore fluid pressure inferred from the seismic velocity structures, this ratio can be converted to the normalized fluid pressure ratio, λ*, which is calculated as follows:We show that the pore fluid pressure at the mode I crack formation is close to the lithostatic pressure around the Nobeoka Thrust. In the maximum case of footwall of the Nobeoka Thrust at 8 km depth, the pore fluid pressure ratio , ~ 7% and ~ 8% of the total pore fluid pressure, ~ 12 MPa and ~ 11 MPa, are reduced by the formation of the mode I cracks in the hanging wall and footwall of the Nobeoka Thrust, respectively. When the pore fluid pressure falls below the sum of σ3 and Ts, the cracks are closed and the reduction of pore fluid pressure is stopped26. Here, for the Eq.  changes from 0.75 to 0.66 (from 0.84 to 0.79) and from 0.92 to 0.80 (from 0.95 to 0.87) in the maximum cases of hanging-wall and footwall of the Nobeoka Thrust, respectively . Our results indicate that the present pore fluid pressure around the megasplay fault in the Nankai Trough has not recovered to the value of the pre-seismic period.We compare our results with the pore fluid pressure around the currently-active megasplay fault in the Nankai Trough. The fluid pressure ratio 40. In the subduction zone, the fluid can be supplied to the upper plate by the dehydration from subducting sediments and altered oceanic crust44. Saishu et al.18 proposed a novel model of quartz vein formation associated with fluid advection from the host rocks and silica precipitation in a crack. In the extension quartz veins around the Nobeoka Thrust, the fluid pressure drops of 10–25 MPa from the lithostatic pressure facilitates the formation of the extension quartz veins under a period of 50–100 years18. When the cracks are closed by the silica, the porosity of the host rock decreases. The occlusion of the porosity promotes the build-up of the pore fluid pressure. Hence, the sealing time of silica in the cracks around the Nobeoka Thrust18 suggests that the onset of increasing of pore fluid pressure around the megasplay fault is delayed from the start of tectonic stress accumulation.Cracks opened in the post-seismic period are closed by the precipitation of silica in the pore fluid6. Approximating the frictional strength of the megasplay fault to τf = μ0σv′ = μ0ρgz(1 − λv) and adopting a frictional coefficient (μ0) of 0.6 (Byerlee's friction law23) and a density (ρ) of 2,700 kg/m3 , any increases in frictional strength (Δτf), can be related to decreases in Pf. When the pore fluid pressure decreases by ~ 11–19 MPa due to the extension crack formation, frictional strength can increase by ~ 7–12 MPa. The order of pore fluid pressure change (ΔPf) and increasing Δτf is less than the stress drop in large trench type earthquakes (20–40 MPa45). The temporal change in fault frictional strength is at the low end of the estimates of Sibson6 for 10 km depth. Here, the effective frictional coefficient (μ′) of the megasplay fault is estimated by μ′ = μ0(1 − λv). Previous studies showed that the frictional coefficient (μ0) of illite-rich shales ranges from ~ 0.4 to ~ 0.746 at room temperature. In this study, we employed a frictional coefficient (μ0) of 0.4–0.7 along the Nobeoka Thrust. In the case of the Nobeoka Thrust, the effective frictional coefficient (μ′) before the fluid-loss by the formation of mode I cracks is smaller than 0.12  increases up to 0.15


The MRI showed the presence of a mediastinal mass suggestive of a lymphoma, which was diagnosed as Hodgkin’s lymphoma following a lymph node biopsy. This finding confirmed the suspicion of a paraneoplastic glomerulonephritis (PG).2.Paraneoplastic glomerulonephritis needs to be distinguished from idiopathic kidney disease. The recognition of PG before the detection of malignancy requires a high index of suspicion and needs to be included in the differential diagnosis of secondary NS, even in children and adolescents. A work-up for malignancy should be considered in all patients presenting an apparently idiopathic NS showing a poor response to steroid or immunosuppressive therapy. An atypical age of onset, the presence of systemic symptoms , persistent positive acute reactant phase laboratory tests and persistent proteinuria without other features of NS should further strengthen the suspicion of an occult lymphoma , 7.3.The effective treatment of the tumour is reported to result in the remission of the NS without the need for a specific therapy directed at the kidney lesion . Hence, Paraneoplastic glomerulonephritis is an uncommon complication of both solid and haematological malignancies, with glomerular lesions induced by products from tumour cells rather than being directly related to tumour invasion or metastasis . Though The remission of NS was attained within 3 months after the initiation of chemotherapy. During the last 4 years of follow-up, proteinuria did not relapse and neither did the lymphoma.Paraneoplastic glomerulonephritis is a rare occurrence in children with solid and haematological tumours. Prolonged proteinuria, an atypical age-onset for minimal change disease, ineffective steroid, and/or immunosuppressive therapy, presence of systemic symptoms, and persistence of laboratory alterations should all suggest a paraneoplastic syndrome. Adequate and effective treatment of the tumour will result in remission of the NS. Even if the diagnosis of PG does not run parallel to the tumour, the reappearance of NS correlates directly with the tumour’s relapse. For this reason, the monitoring of proteinuria becomes relevant to ensure early detection of malignancy recurrence.


Total leg volume and limbTBW were correlated (r = 0.635). %TBW (p = 0.001) and %ECF (p = 0.007) decreased over time. The maximum effects were seen within one week of CDT. LimbICF (p = 0.017), %TBW (p = 0.009) and %ICF (p = 0.003) increased post-MLD, whereas ECF/ICF decreased due to MLD. Plasma volume increased by 1.5% post-MLD, as well as albumin and the albumin-to-globulin ratio . Our results indicate that physical therapy leads to fluid shifts in lymphedema patients, with the greatest effects occurring within one week of therapy. Fluid shifts due to physical therapy were also reflected in increased plasma volume and plasma protein concentrations. Perometry, in contrast to bioelectrical impedance analysis, does not seem to be sensitive enough to detect small fluid changes caused by manual lymphatic drainage.Complete decongestive therapy (CDT), a physical therapy including manual lymphatic drainage (MLD) and compression bandaging, is aimed at mobilizing fluid and reducing limb volume in lymphedema patients. Details of fluid shifts occurring in response to CDT are currently not well studied. Therefore, we investigated fluid shifts before, during and after CDT. Thirteen patients  diagnosed with stage II leg lymphedema participated. Leg volume, limb and whole-body fluid composition , extracellular (limbECF/%ECF) and intracellular (limbICF/%ICF fluid), as well as ECF/ICF and limbECF/limbICF ratios were determined using perometry and bioelectrical impedance spectroscopy. Plasma volume, proteins, osmolality, oncotic pressure and electrolytes were assessed. Leg volume ( The lymphatic vasculature is a second network of vessels, adjacent to the blood vasculature. It plays a crucial role in normal physiology ,2. The dLymphedema is characterized by a chronic, progressive and disfiguring form of regional swelling. The disease is usually diagnosed in peripheral extremities ,6. The aThis study was conducted at the Clinical Center for Lymphatic Disorders, State Hospital, Wolfsberg, Austria. All collected data were analyzed at the Medical University of Graz. Both ethics committees—Medical University Graz, Austria (EK: 29-090 ex 16/17) and Carinthia (EK: A 03/17)—approved the study. Good clinical practices and the WMA Declaration of Helsinki (2013) were followed throughout the study. Each patient received detailed information about the study protocol before giving written consent.Patients with stage II  primaryThe complete decongestive therapy protocol used in this study strictly followed the recommendations by Döller (2013) ,17 and wData collection took place in the morning, between 08:00 a.m. and 11:00 p.m., in a dimly lit quiet room. The temperature was set between 22–25 °C and the humidity remained constant between 50–55%. Fluid shift assessments  to determine time-course effects over physical therapy were performed on the following days of CDT: day 1, day 2, day 7, day 14 and day 21. Additionally, in order to investigate the effect of MLD, all assessments were performed directly before and after 30 min of MLD on each of the indicated days .g, 15 min, 4 °C). Plasma was stored in aliquots at −80 °C until further measurements. Hematocrit (Hct) was determined in duplicates . Plasma density (PD) was assessed using a high-precision mass densitometry device  on 0.2 mL samples via the mechanical oscillator technique. Density determinations were performed at 37.00 ± 0.02 °C controlled by an ultrathermostat . Mass density (FD) was calculated from the corresponding plasma density (PD) and hematocrit (Hct) values. For further details, please see Hinghofer-Szalkay and colleagues (2008 and 2011) [Patients were asked to remain seated for blood collection. Fifty milliliters of venous blood was collected from the ante-cubital vein. EDTA blood samples were immediately cooled and then centrifuged (1500× nd 2011) ,19. ProtFor limb volume assessment of the legs, a perometer type 550 T together with the PeroPlus 2000 software  was used. Assessment of fluid composition was performed using the SFB7 device  and the manufacturer’s software . Patients were asked to empty their bladder prior to the measurement. Electrodes were positioned on previously disinfected skin of the patients in supine position. Total body composition analysis as well as segmental measurements in the lower limbs were performed . PercentThree different equations were used to calculate relative plasma volume changes (%) pre and post MLD based on a combination different plasma parameters: (1) plasma density (PD) and hematocrit (Hct) , (2) hemBHct represents the baseline value, whereas AHct represents the value post-MLD):All blood parameters assessed in this study were corrected to plasma volume changes according to (2) the equation according to Van Beaumont , as Hct p-values < 0.05 were considered as statistically significant. All statistical analyses were performed using IBM SPSS  software. GraphPad Prism  was used to generate the figures. Figures display mean values ± standard error of the mean (SEM).Data are presented as mean ± standard deviation (SD). Analysis of variances (two-way) ANOVA with repeated measurements was conducted in order to determine effects over three weeks of lymphedema therapy and due to manual lymphatic drainage averaged over three weeks. Correlation was tested using Pearson’s correlation with Fisher z-transformation to calculate a mean correlation over all time-points. In total, thirteen patients  with stage II primary or secondary lymphedema were included in this study. Out of these patients, 10 were diagnosed with bilateral lower limb lymphedema, whereas 3 were diagnosed with unilateral lymphedema. All patients completed the study protocol. = 31; p < 0.001; ε = 0.74). This is a total loss of 0.392 L or 3.1% ((17) = 4.98; p = 0.0001). No changes in leg volume were seen pre vs. post MLD  = 0.42; p = 0.527).As expected, leg volume decreased over three weeks of therapy from 12.840 ± 1.899 L to 12.448 ± 1.977 L  = 2.60; p = 0.041). The highest reduction was seen between day 1 (3.068 ± 0.640 L) and day 2  = 3.03; p = 0.006) as well as day 7  = 2.75; p = 0.011) of CDT  = 1.04; p = 0.321).Extracellular limb volume (limbECF) reduced over three weeks of CDT (F) of CDT B. LimbEC = 4.11; p = 0.005) and increased post-MLD  = 6.85; p = 0.017). LimbICF was significantly different between day 2 (1.920 ± 0.453 L) and day 14 (1.811 ± 0.430 L) of therapy (t(19) = 3.43; p = 0.003). MLD led to an average increase in limbICF from 1.837 ± 0.037 L pre-MLD to 1.879 ± 0.056 L post-MLD, which reflects an increase of 2.3% (Intracellular fluid in the limbs (limbICF) decreased over three weeks  = 2.77; p = 0.057; ε = 0.79) and did not change due to MLD  = 3.05; p = 0.097) (LimbTBW tended to reduce over three weeks of CDT from 4.987 ± 0.910 L to 4.824 ± 0.926 L (F= 0.097) A. = 4.49; p = 0.003), but did not change due to MLD  = 0.28; p = 0.601). Post-test showed a major reduction between day 1 and day 2 of CDT (p = 0.003)  significantly reduced over three weeks of CDT (F= 0.003) .r = 0.635).Perometry and limbTBW moderately correlated over all measurement time points  = 7.04; p = 0.001) and increased post-MLD  = 11.84; p = 0.011). The major reduction of %TBW occurred between the first and second day of CDT (t(13) = 3.09; p = 0.009), with a reduction from 50% to 46.5%. Overall reduction over three weeks of CDT was from 50% to 47% total body water. The average increase in %TBW due to MLD was from 46% pre-MLD to 48% post-MLD  reduced over three weeks of therapy  = 4.15; p = 0.007), with the highest decrease between day 1 and day 2 (t(13) = 2.48; p = 0.029). Overall reduction was 0.8% (from 20.7% to 19.9%) in whole body extracellular fluid. MLD did not lead to changes in %ECF  = 2.17; p = 0.172) (Percentage of extracellular fluid (%ECF) decreased over three weeks of CDT (F= 0.172) B. = 15; p = 0.003). This represents an increase of 1 percentage point. No changes in %ICF were seen over three weeks of CDT  = 4.04; p = 0.063) (Percentage of intracellular fluid (%ICF) increased post-MLD (F= 0.063) C. = 8.97; p = 0.013). No changes occurred due to three weeks of CDT  = 0.56, p = 0.521) (Ratio of ECF/ICF significantly changed due to MLD over three weeks of CDT (F= 0.521) D. = 0.88, p = 0.489)  . = 23.71; p = 0.005 and F = 6.63; p = 0.049, respectively) from 4.33 ± 0.09 to 4.47 ± 0.03 mg/L and 1.61 ± 0.06 to 1.67 ± 0.04 mg/L, which is an increase of 3.3% and 4.1%, respectively  = 5.04; p = 0.066). Total protein concentrations did not change over three weeks of therapy  = 0.96; p = 0.445) , increased after MLD  A. = 1.01; p = 0.440; ε = 0.48 and F = 1.02; p = 0.366; ε = 0.31, respectively) or due to MLD  = 2.14; p = 0.239 and F = 0.08; p = 0.789, respectively).Plasma osmolality and oncotic pressure did not change over three weeks of CDT  = 4.35; p = 0.014; Chloride: F = 4.35; p = 0.014; Potassium: F = 4.62; p = 0.011). Sodium, chloride and potassium levels reduced post-MLD on day one, but increased on all other measurement days compared to pre-MLD. Electrolyte levels did not change over three weeks of CDT  = 0.86; p = 0.509; Chloride: F = 1.10; p = 0.389; Potassium: F = 0.92; p = 0.474). Finally, no changes were seen due to MLD  = 1.00; p = 0.374; Chloride: F = 0.35; p = 0.585; Potassium: F = 0.06; p = 0.824).Plasma electrolyte levels showed an interaction between therapy days and MLD (p = 0.057). Intracellular limb fluid increased due to manual lymphatic drainage. A moderate correlation was observed between total leg volume (assessed via perometry) and total leg fluid . Whole-body fluid assessment showed a decrease in total body—and extracellular—fluid over three weeks of therapy and an increase after manual lymphatic drainage. Our results suggest that a major fluid change occurred between the first and second day of lymphedema therapy for limb extracellular fluid (p = 0.006), whole-body total fluid (p = 0.009) and extracellular fluid (p = 0.002). We further observed an increase in plasma volume due to manual lymphatic drainage of, on average, 1.5%. Albumin concentrations as well as albumin-to-globulin ratio increased post lymphatic drainage.We observed the expected decrease in limb volume over three weeks of complete decongestive therapy. Extracellular—and intracellular—fluid in the limbs decreased over time, whereas total leg fluid only tended to reduce due to three weeks of lymphedema therapy (p < 0.001). Cohen and colleagues (2011) reported a reduction of 368 mL post-therapy in a case study. The therapy protocol used in their study included eight sessions over three months [Our results show an overall reduction of leg volume of 392 mL (3.1%) over three weeks of complete decongestive therapy (e months . Comparee months . A recene months . We obsep = 0.527). This is a novel finding of our study, as in the previous studies, perometry was assessed before and after several weeks of physical therapy, but not directly before and after manual lymphatic drainage [Leg volume, however, did not change due to manual lymphatic drainage p = 0.52. This isdrainage ,30,31. Adrainage . This isp = 0.057). Different studies have previously investigated extracellular and intracellular fluid shifts during lymphedema therapy via bioelectrical impedance spectroscopy [Over three weeks of complete decongestive therapy, extracellular—as well as intracellular—fluid reduced in the lower limbs. This is also reflected in a reduction in the inter-leg ratio of extracellular—to intracellular—fluid (expressed as the ratio of Ri to R0) over three weeks of physical therapy. We also observed a tendency towards a reduced total limb fluid (limbTBW) after three weeks of physical therapy  observed a volume reduction of 55% from day 1 to day 2 of therapy, a volume reduction of 11% and 1% between day 2 to 3 and day 4 to day 6, respectively . Our resp = 0.017). This is in accordance with the results of a study performed by Maher and colleagues (2012). They did also not observe changes in limbECF after manual lymphatic drainage in arm lymphedema patients [We tested the hypothesis that extracellular fluid in the limbs (limbECF) decreases after manual lymphatic drainage (as lymphatic drainage should enhance lymphatic outflow), and that the limb intracellular fluid will not change. Our data disprove this hypothesis, as manual lymphatic drainage in the limbs did not lead to extracellular fluid changes. In accordance with this observation, no changes were seen in limbECF/limbICF ratio due to MLD. Only intracellular limb fluid increased  between perometry (assessing leg volume) and bioelectrical impedance spectroscopy analysis (assessing leg fluid) at all measurement timepoints. Previously, Jain and colleagues (2010) reported that bioelectrical impedance spectroscopy produces valid and reliable data as compared to the gold standard assessment, which is perometry . However(13) = 3.09; p = 0.009 and %ECF: t(13) = 2.48; p = 0.029). Our results indicate that in terms of fluid mobilization, one week of therapy may be enough, as no changes occur afterwards.Another hypothesis that we tested was that whole-body total and extracellular fluid decreases following three weeks of lymphedema therapy. We observed decreases in total body water (%TBW) and extracellular fluid (%ECF) after three weeks of therapy, which is in accordance with our proposed hypothesis. Similar to what we observed in the segmental analysis, the greatest fluid reduction in the whole-body assessment occurred between day 1 and day 2 of therapy  changes over the three weeks of therapy. Pereira de Godoy and colleagues (2019) reported increases in intracellular fluid after 7 days of intensive therapy. In contrast to that, our results show a reduction of intracellular fluid over three weeks of therapy, but an increase of intracellular fluid due to manual lymphatic drainage. The differences in our findings could be attributed to the methodology used: they assessed only the trunk and upper abdomen , whereasFurthermore, whole-body ratio of extracellular to intracellular fluid (ECF/ICF) decreases due to manual lymphatic drainage, averaged over three weeks of physical therapy. This is in contrast with what has been reported. For instance, a recent review by Thompson and colleagues (2020) reported that in further stages of lymphedema (stage II–III) manual lymphatic drainage may not provide an additional benefit in combination with complex decongestive therapy . While tManual lymphatic drainage has been suggested to enhance lymphatic activity in healthy patients  and to iIn contrast to that we expected, albumin levels increased after manual lymphatic drainage, which is also reflected in changes in the albumin-to-globulin ratio. Plasma volume changes have previously been reported to affect plasma protein concentrations ,55. HoweDue to the increased plasma albumin levels that we observed post-therapy, we also expected accompanying changes in oncotic pressure. However, we did not observe changes in the oncotic pressure. Furthermore, concentrations of electrolytes such as sodium, chloride and potassium did not change over three weeks of therapy. A possible reason could be the relatively small increase in plasma volume (approximately 1.5%). In accordance with these observations, osmolality did not show any changes as well. Our results further showed that on the first day of therapy, osmolality and electrolyte levels decrease post-MLD, whereas they increase on all other measurement time-points. Therefore, significance was not achieved comparing pre- vs. post-MLD. This could be because the greatest changes in fluid mobilization occurred between the first and second day of CDT.Overall, our results suggest that the equation according to Van Beaumont is a suitable tool to assess minor plasma volume changes, as they occur due to fluid shifts during lymphedema therapy. Although our results indicate that fluid entering the blood stream has a higher protein content than plasma, it seems as if manual lymphatic drainage does not lead to major changes in plasma components  in our patients. Future studies should investigate if other plasma components such as hormones and blood cells are affected by physical therapy and if plasma volume and plasma component analysis could provide a promising and cost-effective method to assess lymphatic (out-)flow as well as lymphatic fluid concentration in different lymphedema patient groups  . n = 3). We do not, however, believe that this is a limitation, as exclusion of the non-affected legs did not influence our results.A possible limitation of this study could be that data sets of all lower limbs were pooled. Therefore, we did not calculate ratios between affected and non-affected lower extremities. The reason for this is that the majority of the patients included in this study were diagnosed with bilateral lower leg lymphedema and the number of non-affected legs available for analysis was limited , affected body part (upper and/or lower limbs) and different therapeutical approaches.


Osteosarcoma arising from cortical surface is classified into parosteal, periosteal and high-grade surface osteosarcoma. Along the spectrum, parosteal osteosarcoma occupies the well-differentiated end. It is a relatively rare disease entity, comprised only 4% of all osteosarcomas and barely reported in the literature. The objective of this study is to describe cases of parosteal osteosarcoma as well as a variety of treatment options amenable to such entity. Six cases of parosteal osteosarcoma were identified based on histopathological reports in a tertiary referral hospital in Jakarta, Indonesia between January 2001 and December 2019. The mean age was 29.8 years old; four of them (66.7%) were male. Distal end of femur was the most commonly involved bone . The patients were treated with wide excision followed by several different reconstruction methods: replacement with endoprosthesis, extracorporeal irradiation, knee arthrodesis, or prophylactic fixation. One of our patients presented with dedifferentiated component, and therefore was treated by limb ablation. While two cases died of pulmonary metastasis, other patients reported fair to excellent functional outcome. Osteosarcoma is a primary bone malignancy characterized by malignant cells of mesenchymal origin depositing immature osteoid matrix. It is one of the most common neoplasms of the musculoskeletal system, accounting for approximately one-fifth of all primary malignant bone tumours. Osteosarcoma encompasses various types of bony lesions, with the typical manifestation which is a high-grade sarcoma arise intramedullary. A few of the disease entities arise on the outer cortical surface; such lesions are termed surface osteosarcomas which are further divided into parosteal osteosarcoma, periosteal osteosarcoma, and high-grade surface osteosarcoma , 2. ParoParosteal osteosarcoma has been rarely reported in the literature. To date, there are no data regarding this tumour in our region. The objective of this study is to describe cases of parosteal osteosarcoma as well as a variety of treatment options amenable to such entity.We searched through the clinicopathological reports and histopathological archives of patients diagnosed with parosteal osteosarcoma who visited Cipto Mangunkusumo Hospital, Jakarta, Indonesia, between the period of January 1999 and August 2018. Data regarding age, sex, tumour site, duration of symptoms, neoadjuvant chemotherapy, surgical treatment, and presence of local recurrence or metastasis were recorded. The patients were further followed up and asked to visit our Musculoskeletal Oncology outpatient clinic where the musculoskeletal tumour society score (MSTS) was evaluated. All patients agreed to participate in the study and provided written consent prior to the publication.Six cases of parosteal osteosarcoma were identified . We follWe put an emphasis for case no. 6, who was a 37-year-old female who was diagnosed as osteochondroma elsewhere and twice underwent simple excision of the thought-to-be-benign tumour. Upon admission to our center, the tumour grew at an alarming speed. Without any histopathological report or slide from previous hospital, the tumour was biopsied and demonstrated appearance of irregular bony trabeculae with cellular foci of high-grade tumour Figures , indicatWhile all patients received a wide margin of tumour resection, this series showed that parosteal osteosarcoma was amenable to a variety of reconstruction methods. Case no. 1 was treated with extracorporeal irradiation for the tumour on the distal humerus. Unfortunately, at 30-month follow-up, the patient died from lung metastasis that was unrecognized at the time of diagnosis.Moreover, our case nos. 2 and 3 underwent joint replacement with endoprosthesis . HoweverOut of the six patients, only case no. 4 was administered a regimen of neoadjuvant chemotherapy because of the patient manifested lung and intramedullary metastasis at the time of diagnosis. After tumour excision, he underwent knee arthrodesis using Küntscher intramedullary nail, plate, and bone cement . He diedFurthermore, case no. 5 was a 14-year-old boy who was brought to medical attention early when the tumour was relatively small in size. The patient underwent hemicortical excision and prophylactic fixation using a plate . CurrentLastly, our case no. 6, who demonstrated a dedifferentiated pattern of parosteal osteosarcoma, was performed high transfemoral amputation and augmentation using the patient's ipsilateral ablated tibia . At the Parosteal osteosarcoma is a relatively rare bone tumour. It is reported that, in our national referral hospital, the incidence of osteosarcoma was 16.8 cases per year . From thCharacteristics of patients in the current study such as age, sex, and tumour location were also in accordance with the big picture from literature. Parosteal osteosarcoma has slight female predominance and more frequently encountered in the third decade of life . PosteriParosteal osteosarcoma is a slow-growing tumour arising from the outer layer of the periosteum. It represents well-differentiated end of the spectrum of surface osteosarcomas. The tumour is relatively distinguishable from other types of osteosarcoma by a combination of radiographic and histopathological characteristics. Radiographically, it appears as a sessile, ossified, lobulated mass which attaches to the underlying bone via a broad pedicle but does not penetrate the cortex to involve the medulla. In addition, there was little or no periosteal new bone formation. Histologically, parosteal osteosarcoma typically presents with irregular bony trabeculae and bland-appearing spindle cells within the fibrous stromal tissue. Markedly, atypical cells and atypical mitoses are not present .Those radiographical and histological characteristics can therefore be mistaken for benign lesions such as osteochondroma. They have similar predilections in age and tumour sites. Moreover, both tumours could be a sessile mass covered by a thin cartilage cap , 2. One The cases of dedifferentiation that were discovered at the time of repeated excision after local recurrence have been reported elsewhere , 9. On tAfter tumour resection, the remaining bone defect could be managed by a variety of reconstructive surgeries. Lesions at the distal portion of a long bone, especially if the adjacent joint is involved, are best treated with endoprosthesis. However, the availability of the implants is relatively precarious in developing countries. If endoprosthesis is unavailable, extracorporeal irradiation followed by reimplantation (case no. 1) or resection arthrodesis using metallic plus bone cement (case no. 4) could be the alternative methods of surgical reconstruction. Both methods have been found to result in a reasonable functional outcome even in later stages of the disease .Patients with earlier stages of parosteal osteosarcoma are amenable to cortical resection with or without prophylactic fixation. Our patient no. 5 with relatively localized tumour, minimal cortical involvement, and little soft tissue involvement was treated by hemicortical excision and prophylactic plating. On the contrary, patients in a very late stage of local tumour with local recurrence after several excisions (such as our case no. 6) should be treated with limb ablation. When the tumour is large and circumferential with neurovascular involvement, contaminated from previous biopsy or procedure, or could not be excised with a clear wide margin, ablation should be considered [Regarding systemic treatment, we administered neoadjuvant chemotherapy only for case no. 4 who had lung metastasis at the time of diagnosis. Current chemotherapeutic agents offer little or no benefit parosteal osteosarcoma, with degree of tumour necrosis and disease survival after chemotherapy being unclear , 13, 14.Parosteal osteosarcoma carries much better prognosis than the classical, high-grade osteosarcoma. Four of our patients lived with no evidence of disease for a median follow-up of 41 months. The five-year disease-free survival of parosteal osteosarcoma is approximately 90% –19. In tPresented as a benign-looking tumour, parosteal osteosarcoma could be mistaken for severalmusculoskeletal lesions such as osteochondroma. Consequently, the diagnosis of parostealosteosarcoma should be established carefully on the basis of clinical, radiological, andhistopathological findings. Our series further emphasized that inadequate diagnosis or unplanned excision could lead to local recurrence, which was associated with the development of the more aggressive dedifferentiated pattern. In addition to wide-margin excision, the tumour was amenable to a wide range of reconstruction modality with fair to excellent outcome.


Urinary tract infections (UTI) represent a substantial burden to the healthcare system. In the early 2000s annual UTI admissions numbered 100,000, and these infections resulted in over 1 million emergency department (ED) visits. While only a fraction of total UTI volume, the estimated cost of complicated (cUTI) to the healthcare system exceeded &3.5 billion. We set out to evaluate the contemporary burden of cUTI in the US in terms of ED visits annually.We conducted a retrospective multicenter cohort study within the National Emergency Department (NEDS) database, a 20-percent stratified sample of all US hospital-based EDs, from 2012-2018, to explore characteristics of patients discharged with a cUTI diagnosis. We applied a previously published algorithm to identify cUTI using administrative coding. We applied survey methods to develop national estimates. Among 3,010,997 ED visits with cUTI, 43.3% were female, and 59.0% were age 65 years or older. Commensurately, Medicare was the primary payor in 62.8% of the visits. The majority of the patients (59.1%) presented to metropolitan teaching hospitals, and plurality were in the Southern US (39.6%). There was a narrow range in the visits’ seasonal variation, from 6.4% occurring in February to 7.9% in October. cUTI was the principal diagnosis in 48.5% of all cUTI visits. In the remaining 51.5%, sepsis was the most common principal diagnosis (33.9%), but severe sepsis and septic shock codes each appeared in 4.9%. Of all cUTI ED visits, 21.4% had catheter-associated UTI. While only 19.8% had a code for pyelonephritis, 2,050,548 (68.1%) were admitted to the hospital. Mortality in the ED was 0.02%.During the seven-year span, there were over 3 million ED visits for cUTI. Although fewer than 1 in 10 patients met criteria for severe sepsis/septic shock, approximately 2/3rds of cUTI patients presenting to the ED were subsequently hospitalized.Marya Zilberberg, MD, MPH, Cleveland Clinic (Consultant)J&J (Shareholder)Lungpacer Merck (Grant/Research Support)scPharma (Consultant)Sedana Spero (Grant/Research Support) Brian Nathanson, PhD, Lungpacer (Grant/Research Support)Merck (Grant/Research Support)Spero (Grant/Research Support) Kate Sulham, MPH, Spero Therapeutics (Consultant) Andrew F. Shorr, MD, MPH, MBA, Merck (Consultant)


Drosophila melanogaster has become an excellent model organism to explore the genetic mechanisms underlying tumour progression. Here, by using well‐established Drosophila tumour models, we identified Toll‐7 as a novel regulator of tumour growth and invasion.Transgenic flies and genetic epistasis analysis were used. All flies were raised on a standard cornmeal and agar medium at 25°C unless otherwise indicated. Immunostaining and RT‐qPCR were performed by standard procedures. Images were taken by OLYMPUS BX51 microscope and Zeiss LSM 880 confocal microscope. Adobe Photoshop 2020 and Zeiss Zen were used to analyse the images. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0.Toll‐7 suppresses RasV12/lgl−/−‐induced tumour growth and invasion, as well as cell polarity disruption‐induced invasive cell migration, whereas expression of a constitutively active allele of Toll‐7 is sufficient to promote tumorous growth and cell migration. In addition, the Egr‐JNK signalling is necessary and sufficient for Toll‐7‐induced invasive cell migration. Mechanistically, Toll‐7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Moreover, Toll‐7 activates the EGFR‐Ras signalling, which cooperates with the Egr‐JNK signalling to promote Yki‐mediated cell proliferation and tissue overgrowth. Finally, Toll‐7 is necessary and sufficient for the proper maintenance of EGFR protein level.Loss of Drosophila, which shed light on the pro‐tumour function of mammalian Toll‐like receptors (TLRs).Our findings characterized Toll‐7 as a proto‐oncogene that promotes tumour growth and invasion in  In this study, we identified Toll‐7 as a proto‐oncogene that promotes tumour growth and invasion through Egr‐JNK and EGFR‐Ras signalling. Mechanistically, Toll‐7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Toll‐7 promotes EGFR expression post‐transcriptionally to activate ERK. Our results provide in‐vivo evidence and underlying genetic mechanism for the role of Toll‐7 in promoting tumorigenesis. While the roles of Toll in embryonic patterning and innate immunity have been well‐documented, recent studies suggest Toll signalling is also involved in other biological processes including cell death, wound healing and cell competition.The Toll signal pathway is highly conserved from insect to mammal, with the mammalian counterparts of Toll being named Toll‐like receptors (TLRs).In this study, we identified Toll‐7 as a proto‐oncogene that promotes tumour growth and invasion by activating both Egr‐JNK and EGFR‐Ras signalling. The Egr‐JNK signalling is necessary and sufficient to trigger invasive cell migration, while the EGFR‐Ras signalling cooperates with the Egr‐JNK signalling to promote Yki‐mediated cell proliferation and tissue overgrowth. Mechanistically, Toll‐7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. In addition, Toll‐7 promotes EGFR expression post‐transcriptionally. Therefore, these data provide in‐vivo evidence and underlying genetic mechanism for the role of Toll‐7 in promoting tumour growth and invasion, which shed light on the pro‐tumour function of mammalian TLRs.22.1yw, ey‐FLP1; tub‐Gal80, FRT40A; act>y+>Gal4, UAS‐GFP (40A tester), lgl4, FRT40A, UAS‐RasV12. Additional fly stocks used in this study: ptc‐Gal4, GMR‐Gal4s, GMR‐Gal4, UAS‐GFP, UAS‐GFP‐IR, hh‐Gal4, UAS‐LacZ, UAS‐BskDN, UAS‐Puc, UAS‐EgrW, UAS‐EgrRegg, act‐Gal4, tub‐Gal80ts, UAS‐scrib‐RNAi, diap1‐LacZ, wg‐LacZ, ex‐LacZ, ban‐LacZ were previously described.UAS‐Myc‐Egr‐HA was obtained from Xue lab. UAS‐Toll‐7CY was a kind gift from Dr. A. Hidalgo, aos‐LacZ (BL2513) was a kind gift from Dr. Jian Zhu. UAS‐Toll‐7‐RNAi (30488), UAS‐Rab5‐GFP (43336), UAS‐EGFR‐RNAi (25781), UAS‐EGFR (5368) were obtained from the Bloomington stock center, UAS‐Toll‐7‐RNAi (39176), UAS‐yki‐RNAi (40497), UAS‐egr‐RNAi (45253) were obtained from the VDRC stock center.All flies were raised on a standard cornmeal and agar medium at 25°C unless otherwise indicated. Fluorescently labelled invasive tumours were dissected in 3rd instar larval eye discs using the following strains: 2.2Antibody staining of imaginal discs was performed by standard procedures. Primary antibodies included mouse anti‐MMP1 , rabbit anti‐Cleaved Dcp‐1 , Phalloidin 555 , rat anti‐DE‐cadherin , mouse anti‐β‐integrin , rabbit anti‐phospho‐Histone H3 , rabbit anti‐phospho‐JNK , mouse anti‐DLG , rabbit anti‐Egr , rabbit anti‐Rab5 , mouse anti‐Myc‐Tag (1:100 CST 2276), mouse anti‐β‐Gal , mouse anti‐Wg  and mouse anti‐EGFR . Second antibodies included goat anti‐rabbit‐Cy3 , goat anti‐mouse‐Cy3 , goat anti‐mouse‐Cy5  and goat anti‐rat‐Cy3 .2.3Toll‐7 RNAi lines, act‐Gal4; tub‐Gal80ts driver was used. Animals were raised at 25°C for 2 day, and then shifted to 29°C for 3 day before dissecting the larvae.To assess the knockdown efficiencies of Primers used were as follows:rp49‐FP: TACAGGCCCAAGATCGTGAA.rp49‐RP: TCTCCTTGCGCTTCTTGGA.Toll‐7‐FP: ATCCATCGCAACCCAGTGG.Toll‐7‐RP: GCTGTCGCTCAATGAGACG.2.4T‐test were used to compute p‐values, p‐value < 0.05 was considered as significant. n.s means not significant. *, **, **** and **** represent p‐value less than 0.05, 0.01, 0.001 and 0.0001, respectively.Adobe Photoshop 2020 was used to measure the tumour size and quantified as relative volume to that of wild‐type. Adobe Photoshop 2020 was also used to count the migrating cell number. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0. A combination of One‐way ANOVA with Bonferroni's multiple comparison test and the 33.1V12) and lgl mutation (lgl−/−) in Drosophila eye‐antennal discs could induce massive tumour‐like overgrowth and invasive metastasis to the ventral nerve cord (VNC).Toll‐7 by two independent RNAi lines significantly inhibited RasV12/lgl−/−‐triggered tumour growth in the eye‐antennal discs  staining, respectively. Knockdown of Toll‐7 significantly suppressed RasV12/lgl−/−‐triggered cell proliferation  assay was performed to check the knockdown efficiencies of the Toll‐7 RNAi lines .Toll‐7 suppressed scrib depletion‐induced MMP1 expression, while expression of Puc served as a positive control , the fly orthologue of JNK. Blocking Egr‐JNK signalling significantly impeded Toll‐7CY‐induced overgrowth and migration phenotypes  other than Egr‐JNK. The Hippo‐Yki signalling is a well‐known pathway that regulates cell proliferation and tissue growth.Toll‐7CY . To furtexpanded  and bant  expression was significantly reduced upon Toll‐7 depletion (Figure c‐Jun N‐terminal kinase signalling executes either pro‐ or anti‐tumour function in different cellular contexts, depending on EGFR‐Ras signalling‐mediated switch of Yki activity.n Figure . It remaMalignant tumour development requires multiple factors and signal transduction pathways, which have not been fully unveiled. Identifying novel regulators of tumour progression provides potential drug targets for cancer prevention and therapy. Toll/TLR signal pathway, which has conserved functions in innate immunity, is also considered to play a role in cancer inflammation.The authors declare no competing interest.X.D., Z.L. W.L. and L.X. conceived and designed the experiments. X.D., Z.L. and W.L. performed the experiments and analysed the data, G. L. and L.X. supervised the study, X.D., Z.L. and L.X. wrote the manuscript. All authors approved the final manuscript.Figure S1‐S8Click here for additional data file.