Niger's President Mohamed Bazoum smiles before a working lunch with French President Emmanuel Macron Niger — Niger's mutinous soldiers say they will prosecute deposed President Mohamed Bazoum for "high treason" and undermining state security hours after they said they were open to dialogue with West African nations to resolve the mounting regional crisis The announcement on state television on Sunday night said the military regime had "gathered the necessary evidence to prosecute before competent national and international authorities the ousted president and his local and foreign accomplices for high treason and for undermining the internal and external security of Niger." was ousted by members of his presidential guard on July 26 and has since been under house arrest with his wife and son in the presidential compound in the capital People close to the president as well as those in his ruling party say their electricity and water have been cut off and they're running out of food The junta dismissed these reports Sunday night and accused West African politicians and international partners of fueling a disinformation campaign to discredit the junta International pressure is growing on the junta to release and reinstate Bazoum the West African regional bloc ECOWAS gave the regime seven days to return him to power or threatened military force but that deadline came and went with no action from either side ECOWAS ordered the deployment of a "standby" force but it's still unclear when or if it would enter the country Become an NPR sponsor Samuel Eto’o’s expression reflected emotion and consternation as he extended his condolences to the people of Bazou who lost their traditional chief on 6 December 2024 in Switzerland following an illness Samuel Eto’o arrived in Bazou on 22 January 2025 to pay his respects and was warmly welcomed by the local authorities of the commune The first-degree traditional chief of the Bazou people passed away in Switzerland on 6 December 2024 Widely known as a true guardian of the traditions of this region of Cameroon had been mayor of Nkongsamba when it was Cameroon’s third largest city His reign was marked by powerful acts that make his stay unforgettable he received the mortal remains of Fo’o Kemayou Paul Bernard which he carefully preserved for 25 years despite the opposition of interim chief Kezembou Marcel Aka Tayo Marce Amid this time of mourning and lament, Samuel Eto’o showed his solidarity to the people and the traditional Bazou community. Eto’o’s trip to the West came a day after Cavaye Yeguié Djibril, President of Cameroon’s National Assembly, elevated him to the rank of Grand Notable of the Tokombere chiefdom. les actualités camerounaises et la revue de presse Lebledparle.com à ne pas manquer Δdocument.getElementById("ak_js_1").setAttribute("value",(new Date()).getTime()) Actualité du Cameroun ce matinSamuel Éto'o Lebledparle.com is a site dedicated to news in Cameroon news from Cameroon and all useful information analyzes and all the news in real time and continuously Deliver pizza to live the Quebecois dream in Mon Bazou If you also like weird games that are oddly specific and a clear labor of love which in Quebecois French translates roughly to "my piece of shit car," is a game about making money doing odd jobs in order to soup up the titular Bazou into a real race car around "the hot scene of tuned cars and wanna-be streets racers." build a sugar shack to harvest maple syrup scavenge the junkyard to get good parts for your rides Just make sure to make enough money to keep gas in the tank live the rural Canadian dream of making your own poutine and growing weed in the woods There are lots of upgrades to be done on the titular car It's a rural and pedestrian game with a strong sense of humor It's about being happily engrossed in one's hobbies You can find Mon Bazou on Steam. Keep up to date with the most important stories and the best deals, as picked by the PC Gamer team. ContributorJon Bolding is a games writer and critic with an extensive background in strategy games. When he's not on his PC, he can be found playing every tabletop game under the sun. Volume 10 - 2022 | https://doi.org/10.3389/fphy.2022.920687 This article is part of the Research TopicNew Trends in Acoustofluidics: Modeling, Experiments, and ApplicationsView all 6 articles This study presents a proof of concept to demonstrate the ability of ultrasounds to perform acoustophoretic processes in hybrid millifluidic resonators that include channels laterally embedded in extremely soft media with physical properties close to those of liquids particles are driven by acoustic radiation forces toward hydrodynamic/acoustic equilibrium positions in a similar way to that produced in conventional microfluidic resonators with solid structures; 20 um-sized polystyrene beads immersed in deionized water flow channelized throughout an aqueous-based gel between an inlet and outlet in a resonant chamber while being exposed to ultrasounds at a frequency of 1.54 MHz The liquid channel formed presents irregular walls and variable geometry defined by the sample injection pressure Particles collect rapidly along a central line equidistant from the walls regardless of whether they are parallel or not as observed for different channel geometries and cross-sectional dimensions the particles collect in acoustic pressure nodes established with the 2D spatial distribution These results break the paradigm of solid structures as essential physical elements to support acoustophoresis demonstrating the ability to produce these processes in media without a consolidated structure It opens a door to bioprinting applications It can also be applied in environmental and agri-food applications for early detection and sorting of unwanted microelements in water and waste Acoustophoresis in microfluidics/milifluidics can be performed based on bulk acoustic waves (BAW) or surface acoustic waves (SAW), but recent technological developments show that PAW (plate acoustic wave) devices are also efficient in cell separation [45] BAW approaches base their performance on resonances that are established inside the treatment channel which requires the establishment of a liquid-phase standing wave between the channel walls requiring strict parallelism of the walls and rigid chip materials to achieve high reflectivity (silicon or glass are typically used for the chip structure in BAW devices) These devices have a high manufacturing cost and have little versatility in their performance for collecting cells at fixed positions defined by the relationship between the wavelength and the cross-sectional dimensions of the channel Devices using SAW can operate as standing or nonstationary waves and are more versatile than BAW as they have no restriction specifications for channel width or stiffness allowing variable positions for particle collection defined by selecting the electrical displacements of their interdigital transducers (IDTs) they are severely restricted at low frequencies due to physical gap requirements between the electrodes of their TDIs as well as their widths which are in the order of half a wavelength The configuration of IDTs to operate at frequencies below 10 MHz requires very large dimensions of IDTs and much larger thicknesses of the piezoelectric substrate to avoid Lamb waves The SAW isolator literature refers to operating frequencies above 9 MHz They have a high manufacturing cost and require clear rooms for fabrication However, the basic mechanisms of acoustophoresis can be induced on other microfluidic platforms with 2D and 3D ultrasonic resonances of the entire chip structure containing the treatment channel. The authors of the current study have demonstrated the feasibility of polymeric materials as alternative materials [46, 53, 54] in efficient BAW ultrasonic separators that base their actuation on 3D resonances of the whole chip structure The ability to perform acoustophoresis on vibrating plate structures, PAW, a unique case of BAW models restricted to 2D structural plate-like vibrations set on very thin polymeric chips with a strategic surface-to-volume ratio, has also been demonstrated [46] These flexible ultrasonic separators present advantages of each of BAW and SAW type devices but discard some of their respective disadvantages PAW devices are a real alternative to conventional acoustophoretic devices as they feature simple geometric designs and allow structural materials with a wide range of physical and chemical properties to suit a specific application including biocompatibility for bioapplications they have economic advantages associated with their low manufacturing cost and are easy to fabricate and integrate with other on-chip components for future assembly configurations Previous literature studies report the handling of acoustic particles and cells in soft matter, such as droplets and gels [55–60] there are no studies reporting acoustophoresis within channels made in these extremely soft media—in particular liquid samples flowing through gels and exposed to ultrasound we wanted to discover the limits of acoustophoretic device requirements beyond the boundary of solid chips to test the feasibility of manipulating particles in aqueous suspensions flowing in channeled paths through an irregular and extremely soft aqueous gel The principle of operation of acoustophoresis is based on the concept of radiation force, induced by a nonlinear interaction of the incident wave and waves scattered by particles. This hydrodynamic force acoustically induced acts on every single particle with a size much smaller than the acoustic wavelength λ. In a one-dimensional standing wave, this axial force FRAx can be expressed as follows [61] (Gor’kov 1962): where Vp is the particle volume; ϕ=5ρp−2ρ02ρp+ρ0−βpβ0 is the acoustic contrast factor; ρ0 and ρp and β0 and βp are the density and compressibility of the fluid and particle respectively; and “x” is the distance from the particle to the nearest node of pressure in the standing wave particles collect in locations separated by a half wavelength distance (λ/2) Sign ϕ indicates the motion of particles either toward nodes (ϕ > 0) or to antinodes in the standing wave (ϕ < 0) “Eckart” large-scale acoustic streaming is due to energy absorption in the fluid phase and drives cells out of desired position platforms they are negligible in short pathlength trap chambers but the boundary conditions must be well established for the generation of the standing wave in the case of the BAW acoustic waveform performance that requires the establishment of pressure nodes within the channel In devices driven by surface acoustic waves no requirement is necessary on the boundary conditions for the establishment of pressure nodes; only a suitable shift between the faced IDTs promotes the generation of pressure nodes at different locations inside the channel without the establishment of standing waves A recent study was performed by Vargas et al. in 2021 to address it [63]. It reported a theoretical prediction for the acoustic behavior of multiple particles in 3D standing waves in resonant chambers with both rigid and free walls has been recently performed. The authors assumed free walls along one direction (x-axis) and rigid walls in the other two transverse directions (along y- and z-axes) (Figure 1) 3D FR acting on a spherical particle within a rectangular chamber of dimensions lx and lz with a standing pressure field px (x) (A) Chamber with all rigid walls; (B) rectangular chamber with free walls in x derived an expression for 3D acoustic potential Uac (x z) established in this chamber actuated by a one-dimensional plane wave assuming hybrid boundary conditions with freedom along the x-axis: A 3D acoustic radiation force is derived from this acoustic potential: FR=−∇UAc: In a short pathlength chamber with z-direction ≈λ/4, this field reduces to a 2D standing wave with a spatial distribution of pressure nodes and antinodes limited to the x–y plane, simplifying Eqs 2,3 with term reduction we present a microfluidic hybrid resonant system combining rigid materials for the top and bottom of a circular chamber (quartz glass and aluminum respectively) containing a coupling water-based gel crossed by a channel with flowing liquid samples the channel acquires extremely soft lateral walls made up entirely of gel with a variable geometry defined by the pressure of injection of samples we investigate and demonstrate the feasibility of acoustophoretic manipulation of particles in straight and tortuous channels with irregular and extremely soft walls within unstructured media Particle collection effects achieved in the experiments at a frequency of f = 1.54 MHz are similar to those achieved in conventional microfluidic platforms with solid structures actuated by ultrasounds The ‘‘sample-containing’’ active area (inner diameter) of the cylindrical steel body had a diameter of 18 mm The signal was supplied by a function generator (Agilent 33220A The thicknesses of different layers were selected to give a highly resonant system (A) Schematic diagram of the setup used to perform the acoustophoretic manipulation; (B) channelized path formed by the suspension flowing between the chamber inlet and outlet Aqueous-based suspension of polystyrene micron-sized beads; (A) flowing through a gel embedded in the resonating chamber; (B) channel imprint remaining as a hollow channel embedded in the gel volume after removing the suspension; (C) and (D) polystyrene-aqueous suspensions flowing at two different flow rates between the chamber inlet and outlet to draw “liquid channelizations” with different geometry and width throughout the gel matrix which depends on the flow velocity of the sample Samples consist of aqueous-based suspensions containing polystyrene 20 µm-sized beads in a concentration of approximately Cv∼0.01% in deionized distilled water These spherical particles present positive acoustic contrast factor ϕ to collect at the pressure nodes of the acoustic wave established in the area of the chamber occupied by the liquid phase The channel shape and width are defined by hydrodynamic parameters associated with the injection pressure and flow rate of the sample, which are in turn influenced by the particle concentration and physical properties of both, particles and liquid, as well as the texture of the surrounding gel (viscosity). According to them, it acquires paths with different geometry and tortuosity (Figures 3C,D) which provide higher channel tortuosity due to higher resistance within the gel This is associated with the viscous resistance of the gel to the opening of a channel by the suspension during its flow across it between the chamber inlet and outlet the weaker the force exerted by the liquid to flow through the gel tracing a more tortuous and less straight channel higher flow rates generate thinner and straighter gel–liquid interfaces for z = λ/8) with the buoyancy-corrected gravitational force The pressure was demonstrated to be linearly proportional to the voltage applied to the transducer at low supplied voltages pressure amplitudes close to 0.05 MPa were obtained within the chamber at the frequency of f = 1.54 MHz and voltages around 5 VP-P approximately 70% of the chamber is filled with the water-based gel It means pressure amplitudes lower than 0.05 MPa were obtained in the chamber full of water heating effects of the ultrasound actuation on the sample temperature have been discarded in current experiments carried out for the very low supplied voltages and short times of actuation of few seconds They considered excessive heating due to vibration damping and other system losses potentially compromise the biocompatibility of the SAW technique with motile Chlamydomonas reinhardtii algae cells In the current work presented in this study weak temperature variations below 1°C are not relevant for successful acoustophoresis results because it refers to experiments performed with polystyrene particles our samples were flowing at high velocities varying from 2 mm/s up to 20 mm/s while being exposed to the ultrasonic actuation The exposure of liquid samples to a flow motion prevents heating effects following the same cooling principle typically applied with smart devices developed for outer space applications A microscopy imaging setup provided images with a spatial resolution of 10 µm per pixel polystyrene 20-µm-diameter particles used in the experiments occupied 2 pixels ±1 pixel ∼20 ± 10 µm in filmed images An estimate of the particle flow velocity before and during the ultrasonic actuation was achieved by using the PIV code of MATLAB and Image J freeware from consecutive filmed frames in each movie resulting in vp¯ = 7 pixels/frame = 21 mm/s Each particle crosses the entire channel length of 18 mm in somewhat less than 1 s in the case of straight channelization but it requires longer time for channels with certain path tortuosity Once the acoustic field is applied in the chamber flowing particles rapidly collect along a line within the liquid phase as if a pressure node had been established on such a line centered between the liquid–gel interfaces The application of ultrasounds at the frequency of 1.54 MHz generates a 2D resonance in the chamber including the gel and liquid sample within the channel A radiation force is acoustically induced to act on particles which were rapidly driven toward pressure nodes established in the standing wave where they collect according to their positive acoustic contrast factor Figure 4A shows polystyrene beads filmed during their flow motion in deionized water before the ultrasound actuation, Figure 4B shows particles flowing while exposed to ultrasounds aligned along a central line in the channel, and Figure 4C shows particles collected in 2D pressure nodes when the sample is in rest and exposed to the ultrasonic wave (A) Polystyrene beads of size 20 µm flowing in an aqueous suspension within a channel embedded in a gel without ultrasound exposure; (B) flowing at the same velocity and exposed to the acoustic wave at f = 1.54 MHz The particles collect along a central line equidistant to the lateral gel–liquid interfaces; (C) collection of the particles exposed to ultrasounds in quiescent samples forming aggregates without flow motion; (D) particles flowing homogeneously distributed within the channel path before their exposure to ultrasounds; (E) particle flow collected along a line once exposed to the ultrasonic wave Acoustic drift motion and collection effects were not expected on particles in the channel of our device because these acoustophoretic motions arise from the radiation force induced by ultrasounds and exerted on every single particle, directly related to the establishment of standing waves inside the channel. The impedance of the liquid medium in the channel and that of the gel are really similar (Table 1) and no wave reflection is expected at the interface between both media required for the establishment of a standing wave the experimental results have been different and show the feasibility of ultrasounds to collect particles along a central line in the channel regardless of the impedance relationship Properties of water and coupling aquasonic gel at room temperature of 20°C Both media differ mainly in their respective viscosities (this parameter defines a fluid’s resistance to flow or an induced motion) with much higher magnitudes in gels than in water viscosity is not a parameter involved in the acoustic impedance definition Z = ρc (defined by density and sound speed) which defines the reflectivity of acoustic waves on the interface between the two media (channel walls) the relevance of this experimental study that demonstrates the feasibility of acoustophoresis mainly based on a single parameter A certain difference in viscosity between the flowing liquid sample and the gel in the liquid–gel interface provides a certain consistency to channel walls and promotes a wave reflection to allow the particle collection inside the channel It must be remarked that this is a proof of concept, without quantitative analyses of the influence of flow rates or acoustic frequency on the effects of particle collection observed in the experiments. We also observed repeatedly that particles collected in a central line within the channel always in the midline between the walls, regardless of whether they were parallel, divergent, or convergent (Figures 4D,E) Despite the fact that the lateral limits of the channel are not exactly flat or parallel to each other and despite the shape of the channel the acoustic collection of particles occurs in a few seconds once the acoustic field is applied These successful results obtained in more than 20 experiments performed at different flow velocities (varying from quiescent samples without moving up to 21 mm/s) show the need for a revision in the theoretical study of the radiation force in resonant chambers and Our findings point to the need for a revision in the theoretical definition of the axial and lateral expressions of the radiation force FR for resonant chambers with extremely soft but not free walls which probably should include in its expression added terms referred to as the viscosities of both walls and liquid phase The study presented in this work is a proof of concept to analyze the limits in the physical property requirements of soft matter-based resonant devices for the manipulation of particles by acoustophoresis we experimentally demonstrate the ability of ultrasound to collect particles in microfluidic channels created in extremely soft media without a solid structure the concept of acoustophoresis can be extended to extremely soft media with a liquid phase flowing through aqueous-based gels with close density but different viscosity the parallelism between channel walls is not a key factor for the particle collection the slight difference in viscosity and density between the gel and liquid phase allows to keep confined the liquid phase between lateral gel edges particles in flowing samples rapidly collect along a single central line established between lateral channel edges where they continue their flow motion chained in the central line between the gel–liquid interfaces that act as pseudo-walls The flow motion combined with strong spatial gradients of acoustic pressure amplitudes drives the particles toward a central position in the liquid phase This is an unexpected behavior repeatedly observed in our 20 experiments We must emphasize that although the gel is a medium with an extremely smooth texture and properties very close to those of the liquid suspension the particles’ drift motion has been found similar to that of conventional microfluidic resonators with a solid structure surrounding the liquid phase flowing in the channel These results break the paradigm of solid structures as essential physical elements to support acoustophoresis in channels based on the BAW actuation This study confirms the ability of ultrasounds to handle particles in irregular channels embedded in extremely soft and unstructured media It opens a door to future biomedical applications involving bio-printing procedures mimicking soft organic matter to handle flowing cells or particles in irrigation channelizations The results of this study open a door to a new technique that could be of interest in upcoming biomedical applications involving bioprinting procedures to handle flowing cells or particles in bioprinted mimicking samples that present channelization The original contributions presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author JL and PC have done equal contributions as first authors to the experimental research MC has contributed to the work by participating in the experiments DB has provided the trap device and knowledge from previous studies This research was supported and funded by the National Research Project DPI2017-90147-R of Spain and by the National Research Council of Spain CSIC through project i-COOPA20348 The trap device belonging to Bazou was used for the experiments The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher Med Biol Eng Comput (2010) 48:999–1014 PubMed Abstract | CrossRef Full Text | Google Scholar Microfluidic device based on a micro-hydrocyclone for particle–liquid separation PubMed Abstract | CrossRef Full Text | Google Scholar Blood plasma separation in elevated dimension T-shaped microchannel Biomed Microdevices (2013) 15(3):415–25 PubMed Abstract | CrossRef Full Text | Google Scholar Particle separation and sorting in microfluidic devices: A review Microfluid Nanofluid (2014) 17:1–52 CrossRef Full Text | Google Scholar Low-cost blood plasma separation method using salt functionalized paper PubMed Abstract | CrossRef Full Text | Google Scholar Passive blood plasma separation at the microscale: A review of design principles and microdevices J Micromech Microeng (2015) 25:083001–0820024 CrossRef Full Text | Google Scholar Continuous-flow electrokinetic-assisted plasmapheresis by using three-dimensional microelectrodes featuring sidewall undercuts PubMed Abstract | CrossRef Full Text | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar Microfluidic self-sorting of mammalian cells to achieve cell cycle synchrony by hydrophoresis PubMed Abstract | CrossRef Full Text | Google Scholar Efficient microfluidic particle separation arrays CrossRef Full Text | Google Scholar Microdevice for the isolation and enumeration of cancer cells from blood Biomed Microdevices (2009) 11:883–92 PubMed Abstract | CrossRef Full Text | Google Scholar High-throughput size-based rare cell enrichment using microscale vortices Biomicrofluidics (2011) 5(2):0222061–10 PubMed Abstract | CrossRef Full Text | Google Scholar Recent advances and prospects in the isolation by size of epithelial tumor cells (ISET) methodology Technol Cancer Res Treat (2013) 12(4):295–309 PubMed Abstract | CrossRef Full Text | Google Scholar Size-selective collection of circulating tumor cells using Vortex technology PubMed Abstract | CrossRef Full Text | Google Scholar release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip PubMed Abstract | CrossRef Full Text | Google Scholar Label-free cell separation and sorting in microfluidic systems Anal Bioanal Chem (2010) 397:3249–67 PubMed Abstract | CrossRef Full Text | Google Scholar Isolation and retrieval of circulating tumor cells using centrifugal forces PubMed Abstract | CrossRef Full Text | Google Scholar Slanted spiral microfluidics for the ultra-fast label-free isolation of circulating tumor cells PubMed Abstract | CrossRef Full Text | Google Scholar Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells PubMed Abstract | CrossRef Full Text | Google Scholar Isolation of circulating tumor cells using a microvortex-generating herringbone-chip Proc Natl Acad Sci U S A (2010) 107:18392–7 PubMed Abstract | CrossRef Full Text | Google Scholar Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology PubMed Abstract | CrossRef Full Text | Google Scholar Versatile label free biochip for the detection of circulating tumor cells from peripheral blood in cancer patients Biosens Bioelectron X (2010) 26:1701–5 PubMed Abstract | CrossRef Full Text | Google Scholar Isolation of rare circulating tumour cells in cancer patients by microchip technology PubMed Abstract | CrossRef Full Text | Google Scholar Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation PubMed Abstract | CrossRef Full Text | Google Scholar physical capture of circulating tumor cell clusters PubMed Abstract | CrossRef Full Text | Google Scholar Continuous sorting and washing of cancer cells from blood cells by hydrophoresis CrossRef Full Text | Google Scholar Membrane microfilter device for selective capture electrolysis and genomic analysis of human circulating tumor cells J Chromatogr A (2007) 1162(2):154–61 PubMed Abstract | CrossRef Full Text | Google Scholar Plasma preparation from whole blood using ultrasound Ultrasound Med Biol (2000) 26:881–8 PubMed Abstract | CrossRef Full Text | Google Scholar combining ultrasound standing waves and laminar flow Sensors Actuators B: Chem (2001) 75:213–22 CrossRef Full Text | Google Scholar Carrier medium exchange through ultrasonic particle switching in microfluidic channels PubMed Abstract | CrossRef Full Text | Google Scholar Manipulation of cells using an ultrasonic pressure field Ultrasound Med Biol (2005) 31:857–64 PubMed Abstract | CrossRef Full Text | Google Scholar A micro-particle positioning technique combining an ultrasonic manipulator and a microgripper J Micromech Microeng (2006) 16:1562–70 CrossRef Full Text | Google Scholar Continuous particle size separation and size sorting using ultrasound in a microchannel CrossRef Full Text | Google Scholar Ultrasonic standing wave manipulation technology integrated into a dielectrophoretic chip PubMed Abstract | CrossRef Full Text | Google Scholar Chip integrated strategies for acoustic separation and manipulation of cells and particles PubMed Abstract | CrossRef Full Text | Google Scholar Fractionation of polydisperse colloid with acousto-optically generated potential energy landscapes PubMed Abstract | CrossRef Full Text | Google Scholar Performance of a quarter-wavelength particle concentrator PubMed Abstract | CrossRef Full Text | Google Scholar Strategies for single particle manipulation using acoustic and flow fields PubMed Abstract | CrossRef Full Text | Google Scholar Manipulation of microparticles using phase-controllable ultrasonic standing waves J Acoust Soc Am (2010) 128:EL195–9 PubMed Abstract | CrossRef Full Text | Google Scholar Surface acoustic wave actuated cell sorting (SAWACS) PubMed Abstract | CrossRef Full Text | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar Controlled cell aggregation in a pulsed acoustic field PubMed Abstract | CrossRef Full Text | Google Scholar On-chip manipulation of single microparticles and organisms using surface acoustic waves Proc Nat Acad Sci USA (2012) 109:11105–9 PubMed Abstract | CrossRef Full Text | Google Scholar Acoustofluidics 23: Acoustic manipulation combined with other force fields PubMed Abstract | CrossRef Full Text | Google Scholar A polymeric chip for micromanipulation and particle sorting by ultrasounds based on a multilayer configuration Sensors Actuators B: Chem (2010) 144:310–7 CrossRef Full Text | Google Scholar Optimizing polymer lab-on-chip platforms for ultrasonic manipulation: Influence of the substrate Proc 6th Int Conf Microtechnologies Med Biol (2011) 6:60–1 CrossRef Full Text | Google Scholar A lateral mode flow-through PMMA ultrasonic separator Int J Appl Biomed Eng (2012) 5(8):20–7 Google Scholar Continuous acoustic separation in a thermoplastic microchannel CrossRef Full Text | Google Scholar Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip Ultrasound Med Biol (2007) 33(1):145–51 PubMed Abstract | CrossRef Full Text | Google Scholar Acoustic whole blood plasmapheresis chip for prostate specific antigen microarray diagnostics PubMed Abstract | CrossRef Full Text | Google Scholar Ultrasonic enrichment of flowing blood cells in capillars: Influence of the flow rate CrossRef Full Text | Google Scholar label-free enrichment of prostate cancer cells in blood based on acoustophoresis PubMed Abstract | CrossRef Full Text | Google Scholar Introduction to the special issue on the theory and applications of acoustofluidics CrossRef Full Text | Google Scholar Ultrasound-induced acoustophoretic motion of microparticles in three dimensions PubMed Abstract | CrossRef Full Text | Google Scholar Acoustic radiation- and streaming-induced microparticle velocities determined by microparticle image velocimetry in an ultrasound symmetry plane PubMed Abstract | CrossRef Full Text | Google Scholar Motile cells as probes for characterizing acoustofluidic devices PubMed Abstract | CrossRef Full Text | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar Particle separation inside a sessile droplet with variable contact angle using surface acoustic waves PubMed Abstract | CrossRef Full Text | Google Scholar Ultrasound patterning technologies for studying vascular morphogenesis in 3D PubMed Abstract | CrossRef Full Text | Google Scholar A new immobilisation method to arrange particles in a gel matrix by ultrasound standing waves Ultrasound Med Biol (2005) 31(2):261–72 PubMed Abstract | CrossRef Full Text | Google Scholar Google Scholar 3D modeling of acoustofluidics in a liquid-filled cavity including streaming CrossRef Full Text | Google Scholar Ultrasound Med Biol (2022) 48(7):1202–14 PubMed Abstract | CrossRef Full Text Proc Ultrason Int (2004) 42(1):319–24 CrossRef Full Text | Google Scholar J Colloid Interf Sci (2010) 351(2):407–14 PubMed Abstract | CrossRef Full Text | Google Scholar On the forces acting on a small particle in an acoustical field in an ideal fluid CrossRef Full Text | Google Scholar Microstreaming effects on particle concentration in an ultrasonic standing wave CrossRef Full Text | Google Scholar Physical enviroment of 2-D animal cell aggregates formed in a short pathlength ultrasound standing wave trap Ultrasound Med Biol (2005) 31(3):423–30 PubMed Abstract | CrossRef Full Text | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar Relative transmissivity of ultrasound coupling agents commonly used by therapists in the UK Ultrasound Med Biol (2007) 33:120–8 PubMed Abstract | CrossRef Full Text | Google Scholar Temperature-controlled MPa-pressure ultrasonic cell manipulation in a microfluidic chip PubMed Abstract | CrossRef Full Text | Google Scholar Thermal considerations for microswimmer trap-and-release using standing surface acoustic waves PubMed Abstract | CrossRef Full Text | Google Scholar Numerical simulation of 3D boundary-driven acoustic streaming in microfluidic devices PubMed Abstract | CrossRef Full Text | Google Scholar Bazou D and González I (2022) Acoustophoretic particle manipulation in hybrid solid/gel resonators Received: 14 April 2022; Accepted: 28 July 2022;Published: 01 September 2022 Copyright © 2022 Luzuriaga, Carreras, Candil, Bazou and González. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Itziar González, aWNpYXIuZ29uemFsZXpAY3NpYy5lcw== Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Two recently appointed Conway Fellows, Dr Despina Bazou and Dr Aisling Coughlan will receive funding awards under the SFI-IRC Pathway programme announced by Minister for Further and Higher Education Patrick O’Donovan TD on 11 July 2024 Pictured (L-R): Dr Despina Bazou & Dr Aisling Coughlan The SFI-IRC Pathway programme is a collaborative initiative between Science Foundation Ireland (SFI) and the Irish Research Council (IRC) to support early-career research across all disciplines and to encourage a cohesive research ecosystem in Ireland It is a €14.6 million investment in 25 projects over a four-year period that will also provide additional support to each awardee to recruit a postgraduate student who will be primarily supervised by them Dr Despina Bazou and Dr Aisling Coughlan are two of seven awardees based in University College Dublin Both researchers focus on the area of multiple myeloma - a cancer of the plasma cells which are a type of white blood cell made in the bone marrow.  Dr Bazou from UCD School of Biomolecular & Biomedical Science is specifically interested in precisely how malignant plasma cells migrate outside of the bone marrow as the disease progresses known as extramedullary disease (EMD).  no official guidelines regarding EMD treatment are available globally Clinicians treat it as the primary disease even though it is already resistant to first-line therapy There is urgent need to understand EMD at a molecular level to identify therapeutic targets and improve patient outcomes Dr Bazou will use advanced techniques to compare the molecular signatures of the disease before and after it progresses beyond the bone marrow and understand the interaction with other cells involved in the progression of the disease.  “I am grateful to SFI and the IRC for this opportunity to improve our understanding of the tumour microenvironment in multiple myeloma The results originating from my project will help to optimise precision treatment for multiple myeloma patients.”  Dr Coughlan from UCD School of Medicine is also interested in improving therapeutic options in multiple myeloma She will use advanced techniques to investigate the changes that impact gene activity in this disease.  Dr Coughlan wants to more fully understanding at a molecular level what happens in therapy resistance – where drugs that initially work for a patient become less effective over time “Epigenetic cancer therapies are promising particularly for patients that cannot handle aggressive treatment regimes such as chemotherapy I am delighted to receive this funding to explore the epigenomic differences that underlie drug sensitivity in multiple myeloma.” Minister O’Donovan said: “This Pathway funding will help facilitate a defined journey from post-doctoral research to independent researcher Bridging this gap is a challenging but critical milestone and the research selected for investment will address key challenges and opportunities ranging from new therapies for Parkinsons to the development of sustainable supercapacitors.” Science for Society at Science Foundation Ireland said: “We are delighted to work in partnership with our colleagues in the IRC to deliver the SFI-IRC Pathway programme It provides targeted support to early-career researchers who will use the funding to pursue independent research at the frontiers of knowledge Investment in these projects will generate novel discoveries and insights across diverse research topics from environmental sustainability to disease treatment and prevention UCD Conway Institute of Biomolecular and Biomedical Research T: +353 1 716 6700E: conway@ucd.ie NEW BEDFORD — Central African royalty visited the city Friday receiving a warm welcome and a flag raising ceremony that symbolized the forming of a bond that officials hope will result in lasting partnerships of Cameroon's Bazou Kingdom and Mayor Jon Mitchell exchanged gifts and talked of connecting youth program efforts and trade across the continents at a flag raising ceremony on the steps of city hall Friday afternoon "This is a great pleasure for Cameroon and the Cameroonian population to have our flag raised and we thank the Mayor for giving us this opportunity," King Kemajou said through a translator at the ceremony The Cameroonian king said he "spent two days roaming around the city" and made stops at various places including the Chamber of Commerce and the Custom House He spoke of hopes that the talks would lead to "fruitful" partnerships in the future "It's been a pleasure to welcome King Vincent here today," Mitchell said New Bedford is a place where French and French Canadian immigrants came over many decades and worked in the city and the textile mills."  The tour of the city also included a meeting with members of Youth Build New Bedford and the Cameroonian king said he shared a vision for improving the lives of youth with members of the city's youth organizations we met with his majesty earlier and I think he sees Youth Build as a model to bring back to Cameroon," said Lisa Mello-Frost program manager for Youth Build New Bedford Mello-Frost added that she hopes to plan Skype conversations with youth groups in Cameroon and share ideas about agriculture Metrics details Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs) with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow which are both elevated in the tumor microenvironment it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1 its activity and its export from the nucleus we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression Our results suggest that HDAC1 modulates angiogenesis in response to flow providing a new target for modulating vascularization in the clinic little is known about their role in sprouting angiogenesis in response to transwall flow stimuli that are prevalent in hyperpermeable vessels during inflammation or tumor growth we provide new insight into the role of post-translational modifications induced by mechanical forces in vascular endothelial cell morphogenesis as it is a poorly understood modulator of vessel morphogenesis We show that angiogenic morphogenesis is HDAC1-dependent and that interstitial flow increases the phosphorylation of HDAC1 a protease involved in angiogenic sprouting is regulated by HDAC1 and that inhibition of HDAC1 nuclear export downregulates MMP14 protein expression Our results suggest the existence of flow-dependent and -independent pathways involving VEGF and HDAC1 that contribute to vascular morphogenesis Angiogenic sprouting in a microfluidic device (a) Schematic of the PDMS microfluidic device HUVECs are seeded into two channels (red) separated by the polymerized collagen gel (purple) Transendothelial interstitial flow (arrow) is applied across the collagen gel Each HUVEC channel has independent input and outlet ports allowing strict flow control in both channels Close - up view of the boxed area shows three apertures that allow invasion and sprouting of the lumenized vessel segments (red) into the central collagen gel (purple) (b) Immunofluorescence staining for Notch-1 (white) and Dll4 (red) expression in GFP transduced HUVECs sprouting in 3D collagen (c) Angiogenic sprouting and invasion into the 3-D collagen matrix under interstitial flow (dashed arrow indicates flow direction) and a VEGF gradient is significantly inhibited in response to 1000 nM of sunitinib Control devices treated with DMSO proceeded with extensive sprouting (Scale bar (d) Schematic of the transwell macroscale device Collagen and fibronectin were loaded into a 6-well transwell chamber and HUVECs (106/ml) in EGM media were allowed to spread and form a confluent monolayer for 24 h prior to flow initiation Interstitial flow (0.5 μm/sec) across the HUVEC monolayer was driven using a programmable syringe pump (Harvard Apparatus) Boxed area shows a close-up schematic of interstitial flow imposed across the 6-well transwell collagen gel substrate and the transwell ports analogous to an apical/lumen - basal flow which allowed stimulation of larger numbers of endothelial cells and collection of sufficient protein samples The most dramatic phosphorylation event detected in this array was in HDAC1 prompting us to further investigate the role of HDAC1 in flow-induced angiogenic morphogenesis HDAC1 enables angiogenic invasion under flow Blocking HDAC1 with the selective HDAC1 inhibitor DHOB (1000 nM) for 24 h (a) under static conditions (without a VEGF gradient) has no effect on angiogenic invasion while (b) in the presence of interstitial flow and a VEGF gradient sprouting is significantly reduced The dashed arrow indicates the flow direction Control devices (treated with DMSO) showed significant invasion under static and flow conditions (c) Quantification of invasion (displayed as the % of spout area) after one day of continuous DHOB treatment under static and flow conditions (d) DHOB at 1000 nM did not promote EC apoptosis after 1 and 24 h of continuous perfusion as determined by western blot analysis for cleaved caspase 3 (e) Knockdown of HDAC1 was achieved by siRNA transfection (f) Silencing HDAC1 with siRNA under static (no VEGF gradient) conditions (top row) has a small effect on angiogenic sprouting while in the presence of interstitial flow and a VEGF gradient (bottom row) sprouting is significantly reduced Exogenous VEGF (50 ng/ml) was also included in the media control devices with cells transfected with non-targeting siRNA proceeded with significant invasion under static and flow conditions (g) Quantification of the % of invasion area after one day of under static and flow conditions for HDAC1 and non-targeting siRNA treated endothelial cells To further validate our results with the DHOB inhibitor, we selectively knocked down HDAC1 using siRNA (Fig. 2e). As in the pharmacological blocking experiments, invasion was significantly reduced under flow when HDAC1 was genetically suppressed (Fig. 2f,g) Under static conditions there was a smaller inhibition of invasion by the HDAC1 knockdown cells indicating that HDAC1 may also be involved in static sprouting or that there was some residual convective flow in the microdevices our results strongly suggest that HDAC1 mediates EC morphogenesis in response to mechanical flow stimuli Interstitial flow-induced HDAC1 phosphorylation and nuclear export in endothelial cells (e) Immunofluorescence staining showing increased p-HDAC1 outside the nuclear (DAPI positive) area under flow (middle row) in relation to static (top row) LMB (20 nM) treatment blocked the increase (bottom row) (Scale bar (f) Quantification of the area fraction of cytoplasmic t- and p-HDAC1 under static Inhibition of p-HDAC1 nuclear export inhibits angiogenic invasion (a) Angiogenic invasion into the 3-D collagen matrix under static conditions (no VEGF gradient) (top row) is not inhibited by a 24 h LMB (20 nM) treatment Under flow and a VEGF gradient (bottom row) LMB treatment results in reduced sprouting and invasion Dashed arrow indicates the direction of flow Control (DMSO) devices (both static and flow) proceeded with extensive sprouting (b) Quantification of the % of invasion area under static and flow in response to 24 h of continuous leptomycin B (LBM) treatment MMP14 expression is regulated by HDAC1 (a) Representative western blot showing that HDAC1 blockade with DHOB (1000 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1 (b) Representative western blot showing that inhibition of HDAC1 nuclear export with LMB (20 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions (c) MMP14 selective blockade (100 μg/ml anti-MMP14) resulted in significant reduction of invasion under static (no VEGF gradient) and (d) flow (dashed arrow indicates the direction of flow) and VEGF gradient (bottom row) conditions Control (CTL-100 μg/ml IgG) devices (both static and flow) proceeded with extensive sprouting (e) Quantification of the % area of invasion further supports the microscopic observations Potential role of HDAC1 in flow-induced angiogenesis Interstitial (Transwall) flow and VEGF result in increased HDAC1 phosphorylation in the nucleus as well as HDAC1 nuclear export Nuclear export (dashed blue line) can be reduced with LMB treatment while HDAC1 activity potentially through its role as a transcriptional regulator VEGFR2 phosphorylation is integral for both static and flow-induced angiogenic sprouting as revealed by the blocking experiments with sunitinib HDAC1 appears to complement and amplify the VEGF pathway when flow signals are present It remains to be determined whether the various members of the HDAC family perform different functions during EC morphogenesis or how they coordinate the responses to mechanical stimuli we found that inhibition of HDAC1 export with LMB reduces the increase in MMP14 protein expression in response to flow This suggests that HDAC1 regulation of MMP14 may not be limited to its nuclear activity but may also be related to its cytoplasmic accumulation Given the short time frame of the experiments performed (lysates were collected 1 h after DHOB or LMB inhibition in the presence or absence of flow) it is possible that the regulation of MMP14 expression in response to flow lies beyond gene transcription and translation further supporting the potential involvement of the cytoplasmic pool of t-HDAC1 and p-HDAC1 It will be interesting for future studies to elucidate if and how HDAC1 regulates MMP14 protein expression in response to flow to effect EC morphogenesis It is currently unknown whether any of these HDAC1 associated proteins is involved in flow-induced angiogenesis As elevated interstitial flow and abnormal angiogenesis are extensive in pathologies such as cancer understanding how ECs sense and respond to flow via HDAC1 will not only advance our knowledge of the role of mechanotranduction and chromatin remodeling in angiogenesis but also open up the possibilities of designing novel anti-angiogenic drugs and regimens to advance cancer therapies As HDAC1 activity and phosphorylation also regulate many other cellular processes such as cell proliferation the understanding of mechanical regulation of HDAC1 activity will also provide mechanistic insights into a range of cell functions beyond pathological angiogenesis Human umbilical vein endothelial cells (HUVECs) were acquired from the Center for Excellence in Vascular Biology HUVECs (passage number 1–5) were used non-labelled or labelled with the Cell Tracker Green CMFDA probe (5 μM) (Invitrogen each device consisted of two layers of 12:1 base to curing agent poly(dimethylsiloxane) (PDMS The top layer was constructed using soft lithography to form the negative relief features ~50 μm in height The top layer with the features was irreversibly sealed against a planar PDMS layer (~2 μm thick) via treatment with plasma oxygen (Harrick heated to 60 °C for 30 min and then sterilized by exposure to UV light for ~30 min The system recreates two quiescent blood vessels separated by 300 μm with multiple regions for potential endothelial sprouting Fluid flow was controlled with a programmable syringe pump (Harvard Apparatus) The flow medium was the same as the growth medium (EGM) Before initiation of flow-based experiments clear polypropylene barbed elbow fittings (1/16 inch Cole-Parmer) connected to silicone tubing (Saint-Gobain) were inserted into the 1.5-mm diameter inlet/outlet ports of the HUVEC channels The opposite inlet/outlet ports of the HUVEC channels were connected to luer adapters (Cole-Parmer) which served as fluid reservoirs To subject endothelial cells solely to interstitial flow (transverse convection) we pulled media through the collagen matrix from the reservoirs connected to the other endothelial-lined channel while exposing both channels to negligible levels of tangential shear A pressure gradient across the collagen gel is also generated convects through the collagen and intravasates into the other channel The flow rate was 7.3 μl/h corresponding to a flow velocity of 60 μm/sec Devices without imposed flow served as controls (“static”) To expose a larger number of cells to trans-wall flow for protein or RNA analyses, we constructed the device shown in Fig. 1d 500 μl of collagen (3 mg ml−1) and fibronectin (10 □g ml−1) were loaded into a 6-well cell culture insert with 0.4 μm pore-size membrane (Corning The gel was allowed to polymerize at 37 °C for 2 h 2 ml of HUVECs (non-labeled) at a concentration of 106/ml in EGM media were then added slowly on top of the collagen gel to prevent gel disruption Cells were allowed to spread and form a confluent monolayer for 24 h prior to flow initiation Cells in the macroscale device were subjected to flow for 1 h using a programmable syringe pump (Harvard Apparatus) (Fig. 1d) The flow rate for both configurations was 0.7 ml/h corresponding to a bulk flow velocity of 0.5 μm/sec The flow medium was the same as the growth medium (i.e Exogenous VEGF (50 ng/ml) was added to the media as per experimental requirements Gels not exposed to flow served as control (“static”) To investigate the role of HDAC-1 in endothelial morphogenesis cells were treated with the HDAC-1-selective inhibitor 4-(dimethylamino)-N-[6-(hydroxyamino)-6-oxohexyl]-benzamide (Santa Cruz Biotechnology DHOB was continuously introduced into the HUVEC-lined channels of the microfluidic device by flow (60 μm/sec) for 24 h at concentrations of 30 In control devices without flow (“static”) DHOB was added to the luer reservoirs connected to the four ports of the device and allowed to passively diffuse into the HUVEC channels Nuclear export of HDAC1 was blocked with leptomycin B (LMB) (20 nM) (Santa Cruz Biotechnology a pharmacological inhibitor of CRM1 (exportin 1) – dependent transport VEGFR2-Tyr951 inhibition was performed with the tyrosine receptor kinase inhibitor Sunitinib (LC Laboratories MMP14 activity was inhibited with a specific anti-MMP14 antibody (Dyax The experimental protocols for Sunitinib and anti-MMP antibody were as described for DHOB above All treatments described here were performed in EGM supplemented with 50 ng/ml of exogenous VEGF Phosphorylation profiling was performed using the Phospho Explorer Antibody Array (ELISA-based) from Full Moon Biosystems (CA This array consists of 1318 antibodies from over 30 signaling pathways relevant to our study; it assays for serine 2 mg of protein collected from cells exposed to static or flow conditions in the transwell device were sent to Full Moon Biosystems for automated analysis Accell SMARTpool siRNA against HDAC1 (E-003493-00) was purchased from Dharmacon (CO HUVECs were seeded in 25 cm2 flasks and then transfected with siRNA HDAC1 or with non-targeting siRNA SMARTpool (Dharmacon D-001950-01) according to the manufacturer’s instructions 24 h post-transfection cells were seeded on one channel of the device and allowed to spread and form a confluent monolayer for 24 h prior to flow initiation HUVEC monolayers (static and flow) in the macroscale flow device were harvested in lysis buffer and clarified by centrifugation The protein concentrations in the lysates were determined using the BioRad protein assay 40 μg of whole lysate was applied to SDS-PAGE and transferred to a Hybond PVDF membrane (GE Health) which was then incubated with antibodies to HDAC1 (1:1000 α-tubulin or GAPDH were used as loading controls The bound primary antibodies were detected using a horseradish peroxidase (HRP)-conjugated secondary antibody and the ECL detection system (GE Health) Band density was quantified using the Image J software (NIH HDAC1 activity assay was performed with the Immunoprecipitation and activity assay from Biovision (CA HDAC1 was immunoprecipitated from 100 μg of HUVEC cell lysate with rabbit anti-HDAC1 antibody The beads with antibody-HDAC1 complex were washed three times with PBS centrifuged for 10 s at 14,000 g before assaying for HDAC1 activity EC monolayers exposed for 1 h to interstitial flow in the macroscale device were initially washed with Phosphate Buffered Saline (PBS) to remove any cell media washed three times with PBS and subsequently permeabilized with 0.5% Triton in PBS for 10 min at 4 °C Co-cultures were then rinsed three time with PBS-Glycine followed by serum blockade (5% donkey serum/0.1% Triton in PBS for 60 min) Monolayers were labeled with anti-HDAC1 (1:200 Rockland Immunochemicals Inc.) and anti-pSer421-HDAC1 (1:200 Appropriate fluorescently labelled secondary antibody was applied for 60 min and washed three times with saline Cell nuclei were stained with DAPI nuclear stain (Invitrogen 1:200) and washed again three times with saline prior to confocal microscopy ECs in the microfluidic device were stained for Notch 1 (1:100 R&D Systems) and Alexa Fluor 488- Phalloidin (2 U/ml) according to the aforementioned protocol Phase contrast and corresponding fluorescence images were acquired with an Olympus IX81 microscope (20× air lens with 2 μm slice thickness) equipped with an automated stage and the OpenLab software The area of cell invasion into the collagen gel was quantified using the ImageJ analysis software the area of invasion from each aperture of the microfluidic device was measured at day 1 and then normalized relative to the invasion at day 0 with the exception of the leptomycin B experiments where non-normalized data are illustrated Projections of confocal images were produced using ImageJ For the analysis of the t- and p-HDAC1 cytoplasmic staining we identified the nuclear area (defined as ‘DAPI-positive’ area) and created a binary image where the value 0 was assigned to the nuclear area we multiplied the DAPI binary image with the t- and p-HDAC1 binary images eliminating the nuclear region from our analysis The area fraction of t- and p-HDAC1 outside the nucleus per field of view was quantified and normalized to the total number of cells in each field of view Analysis of means was performed with a one-tailed t-test or one-way analysis of variance (ANOVA) (GraphPad Prism software Differences were considered significant at P values less than 0.05 Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting Branching morphogenesis and antiangiogenesis candidates: tip cells lead the way VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia Flow-mediated endothelial mechanotransduction Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology Intussusceptive microvascular growth in a human colon adenocarcinoma xenograft: a novel mechanism of tumor angiogenesis VEGF receptor signalling - in control of vascular function Mechanosensitive mechanisms in transcriptional regulation Activation of integrin alpha5 mediated by flow requires its translocation to membrane lipid rafts in vascular endothelial cells Proceedings of the National Academy of Sciences of the United States of America 113 Shear stress-initiated signaling and its regulation of endothelial function Role of integrins in endothelial mechanosensing of shear stress Interstitial fluid flow intensity modulates endothelial sprouting in restricted Src-activated cell clusters during capillary morphogenesis Interstitial flow through the internal elastic lamina affects shear stress on arterial smooth muscle cells Anastomosis of endothelial sprouts forms new vessels in a tissue analogue of angiogenesis Integrative biology: quantitative biosciences from nano to macro 4 RhoA mediates flow-induced endothelial sprouting in a 3-D tissue analogue of angiogenesis Fluid forces control endothelial sprouting Proceedings of the National Academy of Sciences of the United States of America 108 Role of histone deacetylases in vascular cell homeostasis and arteriosclerosis Epigenetic histone acetylation modifiers in vascular remodelling: new targets for therapy in cardiovascular disease Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors The physiological roles of histone deacetylase (HDAC) 1 and 2: complex co-stars with multiple leading parts Protein kinase CK2 regulates the dimerization of histone deacetylase 1 (HDAC1) and HDAC2 during mitosis Histone deacetylase (HDAC) inhibitors down-regulate endothelial lineage commitment of umbilical cord blood derived endothelial progenitor cells Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells Role of histone deacetylases in transcription factor regulation and cell cycle modulation in endothelial cells in response to disturbed flow Proceedings of the National Academy of Sciences of the United States of America 109 Control of endothelial cell proliferation and migration by VEGF signaling to histone deacetylase 7 Proceedings of the National Academy of Sciences of the United States of America 105 Fluid shear stress stimulates phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and eNOS Histone deacetylases modulate vascular smooth muscle cell migration induced by cyclic mechanical strain doi: 10.1016/j.jbiomech.2009.01.012 (2009) Histone deacetylase 3 is critical in endothelial survival and atherosclerosis development in response to disturbed flow doi: 10.1161/CIRCULATIONAHA.109.890491 (2010) HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells Visualization of endothelial actin cytoskeleton in the mouse retina Filopodia are dispensable for endothelial tip cell guidance Cancer cell glycocalyx mediates mechanotransduction and flow-regulated invasion Integrative biology: quantitative biosciences from nano to macro 5 Interstitial flow induces MMP-1 expression and vascular SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism Histone deacetylase 1 phosphorylation promotes enzymatic activity and complex formation HDAC1 nuclear export induced by pathological conditions is essential for the onset of axonal damage Epigenetic regulation of Smad2 and Smad3 by profilin-2 promotes lung cancer growth and metastasis The protease MT1-MMP drives a combinatorial proteolytic program in activated endothelial cells FASEB journal: official publication of the Federation of American Societies for Experimental Biology 26 Crosstalk between neovessels and mural cells directs the site-specific expression of MT1-MMP to endothelial tip cells Dynamic distribution of HDAC1 and HDAC2 during mitosis: association with F-actin Histone deacetylase (HDAC) activity is critical for embryonic kidney gene expression gene regulation and functional analysis in mouse models Inhibition of histone deacetylase attenuates hypoxia-induced migration and invasion of cancer cells via the restoration of RECK expression histone modifications and regulator recruitment in human matrix metalloproteinase 9 gene transcription doi: 10.1128/MCB.24.12.5496-5509.2004 (2004) 6 and 8 are critical for invasion in breast cancer Regulation of tissue-specific and extracellular matrix-related genes by a class I histone deacetylase doi: 10.1002/(SICI)1097-4652(200007)184:1&lt;1::AID-JCP1&gt;3.0.CO;2-7 (2000) Identification of a Dual Inhibitor of SRPK1 and CK2 That Attenuates Pathological Angiogenesis of Macular Degeneration in Mice Role for casein kinase 2 in the regulation of HIF-1 activity Retinal pigment epithelium cells produce VEGF in response to oxidized phospholipids through mechanisms involving ATF4 and protein kinase CK2 A passive pumping method for microfluidic devices Download references The authors would like to acknowledge funding from the National Institutes of Health (R01HL106584) Bazou Despina and Ng Mei Rosa contributed equally to this work Massachusetts General Hospital and Harvard Medical School Department of Mechanical and Aerospace Engineering performed most experiments and wrote the manuscript performed experiments and provided expertise with the microfluidic devices directed the research and wrote the manuscript The authors declare no competing financial interests Download citation Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Speculation about Rigobert Song’s trip to the Central African Republic was put to rest on Monday. The former Indomitable Lions has been officially appointed as coach of CAR’s Fauves du Bazou Bangui His appointment was confirmed in the afternoon of 13 January 2025 by the Minister for Youth Joining him on the coaching team are Eloge Yamissi Enza Rigobert Song Bahana boarded a flight to the Central African Republic in the early morning of 13th January accompanied by influencer Steve Fah Various sources had already hinted at the significance of the trip particularly as Faustin-Archange Touadéra’s country needed a new coach for its national team After a meeting between the Minister for Youth the President of the Republic and Rigobert Song former coach of Cameroon’s Indomitable Lions has finally secured a a new role after being without a team for almost two years He takes charge of the Wildcats of Bazou Bangui in the wake of their disqualification from the 2025 African Cup of Nations in Morocco His primary challenge as new coach will be to lead his team in the quest for a spot in the 2026 World Cup with qualifying matches set to resume next March Actualité du Cameroun ce matinRigobert Song Metrics details Evidence supports the advantages of inhalation over other drug-administration routes in the treatment of lung diseases Although data obtained from animal models and conventional in vitro cultures are informative testing the efficacy of inhaled chemotherapeutic agents requires human-relevant preclinical tools we developed and characterized in vitro models for the efficacy testing of inhaled chemotherapeutic agents against non-small-cell lung cancer (NSCLC) These models recapitulated key elements of both the lung epithelium and the tumour tissue namely the direct contact with the gas phase and the three-dimensional (3D) architecture Our in vitro models were formed by growing human adenocarcinoma (A549) cells as multilayered mono-cultures at the Air-Liquid Interface (ALI) The in vitro models were tested for their response to four benchmarking chemotherapeutics demonstrating an increased resistance to these drugs as compared to sub-confluent monolayered 2D cell cultures Chemoresistance was comparable to that detected in 3D hypoxic tumour spheroids the multilayered monocultures demonstrated to be compatible with testing drugs administered as a liquid aerosol by a clinical nebulizer offering an advantage over 3D tumour spheroids we demonstrated that our in vitro models provide new human-relevant tools allowing for the efficacy screening of inhaled anti-cancer drugs The current methods used to administer chemotherapeutics for lung cancer treatment (namely intravenous injection or oral ingestion) are a constituent component of the problem inhalation is a needle-free non-invasive administration method which increases the patients’ acceptance of treatment regimens The clinical translation of inhaled chemotherapeutics is however impaired by the complete lack of preclinical models capable of predicting the behaviour and action of such compounds in humans The aim of this study is to facilitate such translation by developing novel in vitro models of non-small-cell lung cancer (NSCLC) with increased predictive capability of the efficacy of inhaled anti-cancer agents as a large proportion of the dose adheres to the rodent’s hair is then ingested by the animal and contributes to inaccurate pharmacokinetics conclusions Three-dimensional (3D) cultures can satisfy this need These in vitro platforms are in fact better models of the biological and biochemical characteristics of human tissues than conventional 2D cell cultures 3D spheroids do not mimic the direct contact of the lung epithelium with the gas phase a key feature of the respiratory tract structure and function these cultures are not suitable models for testing the efficacy of aerosolized drugs we speculated that they could also be cultured at the Air-Liquid Interface (ALI) ALI MCCs of human adenocarcinoma (A549) cells our MCCs also enabled the testing of four anti-cancer drugs delivered by a clinical nebulizer in the form of a liquid aerosol ALI MCCs structure: (A) Schematics of the TranswellTM supports used to form the MCCs (B) Schematics of the ALI multilayered mono-cultures developed Representative LSCM images are also reported showing the organization of the F-actin (in red) and of cell nuclei (in blue) in these cultures The Ki67 protein expression throughout the layers is also shown (in green) clearly demonstrating the 3D architecture of the model developed were reconstructed with ImageJ software to obtain the side view shown Properties of ALI MCCs: Representative LSCM images of the (A) F-actin organization (in red) and (B) Ki67 protein expression (in green) in ALI multilayered mono-cultures at different time-points Full datasets for all time-points are reported in the Supporting Information (A) Cell nuclei were also stained with Hoechst 33342 (in blue) clearly demonstrate the multilayered structure of the in vitro models developed (B) Z-stack images of the apical side of the cultures were reconstructed and are shown as three-dimensional projections By quantifying the ATP levels and the percentage of live cells in the cultures, it was found that ALI MCCs models were viable for up to 14 d (Fig. 3A and Supporting Figure S2). Time-dependent phenotype modifications in ALI MCCs: (A) Time-dependent changes in: ATP levels % of LY passage and Papp values in ALI multilayered mono-cultures grown up to 14 d Data are shown as average ± standard error of the mean (nreplicates = 2; ntests = 3) The symbols (**) and (***) indicate statistically significant changes as compared to the values measured at 24 h (p < 0.01 and 0.001 respectively) (two-way ANOVA and Bonferroni post-test) (B) Western blot analysis of E-cadherin (epithelial marker) vimentin and fibronectin (mesenchymal markers) in A549 cells forming ALI multilayered mono-cultures and cultured up to 14 d Abbreviations “n1” and “n2” indicate different experimental replicates β-actin expression is also reported as proteins loading control Cell culture architecture influences the response to anti-cancer drugs administered by direct inoculation: (A) Details of the four anti-cancer drugs tested in this study Their half-maximal inhibitory concentration (IC50) is listed as reported in the GDSC database for the A549 cell model (in the text referred to as “nominal IC50”) (B) Changes in cell viability of sub-confluent mono-cultures of A549 cells grown on plastic substrates and exposed to the four anti-cancer drugs at their nominal IC50 concentration for 72 h GDSC database experimental conditions were reproduced in our assay shown as average ± standard error of the mean (nreplicates = 3; ntests = 3) are normalized to the cell viability of the untreated control (NT) The symbol (***) indicates statistically significant differences from NT (p < 0.001) (one-way ANOVA followed by Dunnett post-test) (C,D) Percentage (%) of live A549 cells (C) and % cytotoxicity (D) detected in MCCs cultured for 14 d (from left to right) either on TranswellTM supports (in ALI or in submerged conditions) or on plastic substrates (in submerged conditions) and then exposed to four anti-cancer drugs at their nominal IC50 concentration for 72 h Data are reported as average ± standard error of the mean (nreplicates = 2; ntests = 3) (**) and (***) represent significant differences from the corresponding NT (p values < 0.05 (E) Histograms of the LDH activity in the experimental controls: untreated mono-cultures (NT) and positive controls (PT) A significant LDH activity was detected in supernatants harvested from PT Data are reported as average ± standard error of the mean (nreplicates = 3; ntests = 3) p < 0.01 and p < 0.001 indicate a significant difference from NT (t-test) our results proved that multilayered mono-cultures were more chemoresistant than the sub-confluent cell model formed by the same cell line ALI multilayered mono-cultures show chemoresistance: (A,B) Histograms showing the (A) units of caspases 1–10 activity and (B) the levels of cytochrome c released from the mitochondria into the cell cytoplasm as detected in ALI multilayered mono-cultures grown for 14 d and then exposed to the four anti-cancer drugs at their nominal IC50 for 72 h Cell cultures were exposed to drugs by direct inoculation Untreated cultures were also tested as negative control (NT) Dotted lines indicate the levels of caspases activity and cytochrome c release by NT Data are presented as average ± standard error of the mean (nreplicates = 2; ntests = 3) p < 0.05 indicates significant differences from NT (one-way ANOVA with Dunnett post-test) (C) Western blot analysis of the expression of phospho-p53 (p-p53) PARP and its cleaved form (cleaved PARP) in A549 cells cultured as ALI multilayered mono-cultures for 14 d and then exposed to docetaxel (Doc) cytarabine (Cyt) or methotrexate (Met) at their nominal IC50 for 72 h Untreated cultures (NT) were also analysed “n2” and “n3” indicate different biological replicates β-actin expression is also reported as proteins’ loading control Efficacy of anti-cancer drugs delivered as a liquid aerosol by nebulization in ALI MCCs Percentage (%) of live A549 cells (top histogram) and cytotoxicity (bottom histogram) detected in ALI multilayered mono-cultures In vitro models were exposed to four anti-cancer drugs (docetaxel vinblastine and methotrexate) at their nominal IC50 concentration for 72 h by direct inoculation (on the left) or nebulization (on the right) p values indicate significant differences (two-way ANOVA and Bonferroni post-test) Comparison of the chemoresistance detected in ALI multilayered mono-cultures and 3D tumour spheroids: (A,B) Percentage (%) of live A549 cells (A) and ATP levels (B) detected in (from left to right) ALI multilayered mono-cultures and 3D tumour spheroids Both in vitro models were grown for 14 d and then exposed to four anti-cancer drugs (docetaxel (**) and (***) indicate significant differences (p values < 0.05 (C) Changes in cell viability in 3D tumour spheroids cultured for 4 7 and 14 d and analysed for their percentage of live cells (in black) and total ATP levels (in grey) The symbol (***) indicates a significant difference from values at t = 4 d (one-way ANOVA and Dunnett post-test) (D) Representative LSCM images of the F-actin organization (in red) in 3D tumour spheroids at different time-points Cell nuclei were also stained with Hoechst 33342 (in blue) clearly demonstrate the growing thickness of the spheroids overtime (E) Live 3D tumour spheroids stained for cell nuclei (in blue) and hypoxia (in green) at different time-points Z-stack images of the cultures were reconstructed and are shown as three-dimensional projections MDR mechanism in ALI multilayered mono-cultures: (A) Schematics of MDR in cancer cells triggered by overexpression of MRP1/ABCC1 and MDR1/ABCB1 drug efflux pumps (B) Western blot analysis of the expression of MRP1/ABCC1 and MDR1/ABCB1 drug efflux pumps in A549 cells forming ALI multilayered mono-cultures grown for 14 d and then exposed to docetaxel (Doc) cytarabine (Cyt) or methotrexate (Met) at their nominal IC50 concentration for 72 h The expression of these pumps in untreated cultures (NT) is also reported for comparison “n2” and “n3” indicate different experimental replicates β-actin expression is reported as proteins loading control (C) Schematics of the mechanism of action of reversan (D) Histogram of the LDH activity in the experimental controls: untreated ALI multilayered mono-cultures (NT) ALI multilayered mono-cultures exposed to reversan (10 μM) for 72 h No significant LDH activity was detected following reversan treatment p < 0.01 indicates a significant difference from NT (one-way ANOVA with Dunnett post-test) (F) Percentage (%) cytotoxicity detected by LDH cytotoxicity assay in ALI multilayered mono-cultures grown for 14 d and exposed to 10 concentrations of docetaxel for 72 h in the presence or absence of reversan (10 μM) Values for untreated cultures (NT) and positive control (LDH PT) are also shown Differences were not significant (two-way ANOVA with Bonferroni post-test) For the efficacy testing of inhaled anti-cancer drugs it is important to mimic the direct contact of the lung epithelium with the gas phase ALI cultures are the only in vitro model available that can reproduce this feature these in vitro models allow aerosols to directly deposit onto semi-dry apical cell surfaces drug deposition and dissolution occur in a small volume of cell lining fluid and mimic closely the delivery of liquid drug aerosol on the lung surface of human patients These properties make ALI in vitro models ideal candidates for testing inhaled drugs our study aimed at creating novel in vitro models that incorporated both the ALI culturing conditions and the 3D architecture of the tumour tissue which is generally mimicked in in vitro cancer research experiments through the adoption of 3D tumour spheroids we developed ALI multilayered mono-cultures of A549 cells and we investigated how the features of such in vitro models would affect the cellular response to four different benchmark anti-cancer drugs delivered by direct inoculation or as a liquid aerosol by means of a clinical nebulizer This might be reflected by our results showing that the mesenchymal features (i.e fibronectin up-regulation overtime) were acquired by A549 cells forming ALI MCCs even without the loss of E-cadherin expression we believe that in our experiments the multilayered structure of ALI MCCs acted as a physical barrier to drugs’ penetration hindering them to reach their subcellular targets in the lower layers of the cultures and cell cultures’ thickening could be responsible for hindering nutrition diffusion gradients with the downstream effect of triggering the cells population to shift into the G0 phase Inhibition of MRP1/ABCC1 with reversan confirmed that the high MRP1/ABCC1 expression in ALI multilayered mono-cultures triggered chemoresistance to all four drugs tested we included in the study a valuable comparison of the chemoresistance of A549 cells forming ALI multilayered mono-cultures to that found in 3D tumour spheroids of the same cell line We demonstrated that chemoresistance was generally comparable among the two in vitro models with the main advantage of ALI MCCs over 3D tumour spheroids of being able to reproduce the direct contact of the lung epithelium with the gas phase we suggest that the decreased efficacy detected in ALI MCCs exposed by nebulization could be due to the poor water solubility of the drugs The latter is one of the main serious limitations associated with the pulmonary delivery of chemotherapeutics be inhaled in their traditional form and require a special drug delivery system (e.g carrier) to be deposited directly onto the lung epithelium our data showed that nebulized drugs were less effective in inducing cytotoxicity in ALI MCCs Therapeutic efficacy and testing of novel inhaled chemotherapeutics must ultimately be demonstrated in preclinical animal models prior to their clinical evaluation like the development and characterization of three-dimensional MCCs cultures of NSCLC provides the in vitro tools capable of guiding the rational selection of inhaled anti-cancer candidates for animal testing thus minimizing the number of animals used per study (principle of Reduction the tissue-mimetic model developed herein has demonstrated to increase the prediction efficiency of the current in vitro approaches by: (i) incorporating the necessary levels of biological complexity (3D architecture) (ii) achieving a “closer relevancy to the patient model” by reproducing MDR mechanisms observed in human NSCLC patients and (iii) integrating culturing conditions at the Air-Liquid Interface that are compatible with aerosol administration methods the global valence of the presented preclinical model is its applicability to other sectors of the pharmaceutical and chemicals industries (e.g. as a valid alternative to animal-based inhalation studies A549 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco Ireland) supplemented with glucose (1,000 mg/l) gentamicin (5 μg/ml) and 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich cells were detached from cell culture flasks’ substrate with TryplE™ (Gibco counted using a Countess™ Automated Cell Counter (Invitrogen Ireland) and diluted in the supplemented culture medium at concentrations appropriate for each experiment The seeding concentration of A549 cells was kept constant among all cell models (1.5 × 105 cells/ml) with the exception of sub-confluent monolayered mono-cultures grown on plastic substrate and 3D spheroids which were seeded at lower concentrations (5 × 103 cells/ml and 1.25 × 105 cells/ml A549 cells were seeded in 96-well plates (Nunc Ireland) in supplemented DMEM medium (final volume/well: 200 μl; seeding concentration: 3.1 × 103 cells/cm2) Cells were cultured for 24 h at 37 °C and 5% CO2 obtaining sub-confluent monolayered cell cultures (15% cell confluence c.a. A549 cells were seeded on 24-well plates (Costar Ireland) (final volume/well: 500 μl) or on the apical side of Transwell™ Permeable Supports (Costar The cell concentration per cm2 was equal to 1.5 × 105 cells/cm2 in both in vitro models The Transwell™ supports were formed by polyethylene terephthalate (PET) membrane inserts of 6.5 mm of diameter (growth area: 0.33 cm2) and pore size of 0.4 μm attached onto hanging supports fitting 24-well plates In multi-layered mono-cultures grown on PET 700 μl supplemented DMEM medium was also added to the basolateral chamber Cultures were grown for 14 d and both apical and basolateral media changed every 3 d 700 μl supplemented DMEM medium was added to the wells of 24-well plates and Transwell™ Permeable Supports were inserted into the wells A549 cells were added to the apical compartment of the Transwell™ supports (final volume/support: 200 μl; cell concentration: 1.5 × 105 cells/cm2) and incubated for 24 h at 37 °C and 5% CO2 to allow cell attachment to the membrane the media in the apical compartment was removed leaving A549 cells in direct contact with the gas phase The ALI multilayered mono-cultures were cultured for up to 14 d and medium in the basolateral chamber was changed every 3 d Spheroids of A549 cells were formed by means of the GravityPLUSTM Hanging Drop System (InSphero Europe GmbH A549 cells were suspended in supplemented DMEM medium and 40 μl of cell suspension was added to each well of the GravityPLUSTM plate cell medium was changed by aspirating 20 μl from each well and delivering 20 μl of fresh supplemented DMEM medium with a pipette 3D spheroid formation was assessed by inverted microscopy 3D spheroids were transferred to the raster plate provided in the GravityPLUSTM Hanging Drop System by slowly adding 70 μl of fresh medium to each well Successful transfer was validated through microscopic inspection of the wells using an inverted microscope 3D spheroids were then cultured up to 14 d 100 µl of cell suspension was analysed for each sample Measurements for each sample were carried out in duplicate to ensure data reliability Two replicates of the same sample were included in each test (nreplicates = 2) and experiments repeated three times (ntests = 3) Results are presented as average ± standard error of the mean Quantitative results were confirmed by Laser Scanning Confocal Microscopy (LSCM) inspection of the live specimens stained with Hoechst 33342 and ethidium homodimer-1 (Eth-1) (Invitrogen Ireland) was added to the cultures and incubated for 30 min on a plate shaker (ambient temperature) Supernatants were then transferred to 96-well clear bottom black microplates (Corning Ireland) and luminescence read by an FLx800 plate reader (BioTek Ireland) (integration time = 0.5 s/well; gain = 135) Each time-/end-point was tested in duplicate (nreplicates = 2) and experiments were repeated three times (ntests = 3) Data are presented as average ± standard error of the mean where CB is LY concentration in the basolateral compartment as determined experimentally VB the volume in the basolateral compartment (700 μl) C0 the initial LY concentration in the apical compartment (400 μg/ml) and VA the volume in the apical compartment (200 μl) Finally, the apparent permeability coefficient (Papp) was determined based on Equation (4) t refers to time (equal to 1 h in our experimental design) while A corresponds to the surface area of the filter (0.3 cm2) Cell cultures were exposed to four chemotherapeutic drugs: anhydrous docetaxel cytarabine and methotrexate (Sigma-Aldrich Selection criterion was the efficacy in inducing A549 cells death based on the GDSC database while methotrexate was the less effective in inducing cancer cells death Drugs were purchased as in the form specified by the European Pharmacopoeia In vitro models were exposed to drugs for 72 h in duplicate (nreplicates = 2) Experiments were repeated three times (ntests = 3) To validate the sensitivity to chemotherapy of our A549 cells batch sub-confluent monolayer mono-cultures were exposed to the four anti-cancer drugs at a concentration equal to the nominal half-maximal inhibitory concentration (IC50) reported for these anti-cancer agents by the GDSC database for A549 cells Drugs were diluted in supplemented DMEM medium and added to the cultures with a pipette (direct inoculation) following supernatants removal Cells viability was measured after 72 h exposure using a fluorescence-based assay cells were first fixed with 3.6% FA for 30 min and then stained with the fluorescent DNA stain Hoechst 33342 (1 µg/ml) for 1 h The fluorescent signal intensity was quantified by an area scan for each well by means of FLx800 plate reader (λexcitation = 360/40 nm; λemission = 460/40 nm) Cell cultures exposed to supplemented DMEM medium were used as negative controls (NT) Fluorescence intensity data were normalized on the negative control (NT) read-outs 3D tumour spheroids and submerged multilayered mono-cultures were exposed to the four drugs diluted in supplemented DMEM medium Supernatants were first removed from these cultures and drug-containing medium (70 μl in 3D spheroids and 200 μl in submerged multilayered cultures) was then added to the in vitro models via pipetting to ensure the direct contact of the epithelium with the gas phase Cell cultures exposed to saline or supplemented DMEM medium were included as negative controls (NT) in the experimental design The high ionic strength of the physiological saline used as drug vehicle ensured optimal flow output This was equal to their nominal IC50 concentration following drug exposure were quantified by flow cytometry and LDH cytotoxicity assay where ΔOD is the rate of increase in optical density (OD) for each sample or for the blank (i.e. Levels of cytochrome C in the cell cytoplasm of A549 cells forming ALI multilayered mono-cultures were quantified by Enzyme ImmunoSorbent Assay (ELISA) (Cytochrome c ELISA Kit ALI MCCs were exposed for 72 h to docetaxel Drugs were tested at their nominal IC50 concentration and were added to the cultures by direct inoculation Detection of cytochrome c released from the mitochondria to the cytosol was achieved by selective lysis of the cell membrane using a Cell Extraction Buffer (Invitrogen supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF) (both from Santa Cruz Biotechnology Inc. the optical density of each well at λ = 450 nm was determined using an Epoch microplate reader ALI multilayered mono-cultures were exposed by direct inoculation to nine concentrations of docetaxel (Table 1) (ten-fold dilution series over a 108-fold concentration range) in the absence and presence of the inhibitor reversan (10 μM) (Santa Cruz Biotechnology Reversan was dispersed in drug-containing hypertonic saline at the desired concentration LSCM was used to assess F-actin organization Ki67 protein expression and hypoxia detection LSCM imaging was carried out by means of a ZEISS 510 Meta confocal microscope equipped with a Zeiss Zen software (Carl Zeiss Series of z-stack images were acquired and then analysed by ImageJ software A549 cells were fixed with 3.6% FA for 10 min at ambient temperature permeabilized with 0.25% Triton X-100 in PBS for 10 min and incubated with 5% BSA in PBS for 1 h (blocking step) Specimens were then stained with Hoechst 33342 (1 µg/ml) for nuclei rhodamine phalloidin (1:50) for F-actin or Mouse anti-human Ki-67 (FITC) (1 µg/ml) for Ki67 expression (all supplied by Invitrogen The staining solutions were prepared in 1% BSA in PBS For cultures grown on TranswellTM supports solutions were added in both apical and basolateral compartment The specimens were incubated at ambient temperature for 3 h in the dark and rinsed with PBS When monitoring hypoxia in 3D tumour spheroids live specimens were stained with Image-iT® Hypoxia Reagent (1:100 in fresh medium) (Invitrogen Ireland) and Hoechst 33342 (1 µg/ml) for 30 min in the dark Specimens were then immediately imaged by LSCM fixed specimens were mounted on glass slides in transparent mounting medium (VECTASHIELD PET membranes were detached from the plastic support with a scalpel blade Fixed 3D tumour spheroids were carefully transferred to glass slides by means of a pipette; whereas live specimens were imaged in their growing environment Cell cultures were washed with ice-cold PBS RIPA buffer (Santa Cruz Biotechnology Inc. Ireland) supplemented with sodium orthovanadate (Santa Cruz Biotechnology Inc. protease inhibitor cocktail and PMSF was used as lysis buffer Supplemented RIPA buffer was added to the apical compartment of the Transwell™ supports and A549 cells scraped to favour cell lysis 3D spheroids were transferred to Eppendorf tubes containing supplemented RIPA buffer and placed on ice vigorous pipetting was used to ensure complete cell lysis Following sonication for 10 min in a sonic bath to favour cell lysis all lysates were centrifuged for 15 min at 15,000 rpm at 4 °C The protein content of each lysate obtained was quantified using the Pierce BCA Protein Assay Kit (Product no 23225; Thermo Scientific β-actin or α-tubulin protein bands were used as loading controls The membranes were then washed and incubated with their respective HRP-linked secondary antibodies (anti-rabbit IgG HRP-linked antibody or anti-mouse IgG HRP-linked antibody HRP-linked Antibody (Cell Signalling Technology Inc Ireland) was used to stain the protein ladder incubated with HRP substrate (LuminataTM Forte Western HRP Substrate Ireland) and protein bands visualised by chemiluminescent detection on CL-XPosure Film (Thermo Scientific Relative proteins’ expression levels were quantified by ImageJ software USA) was used to carry out the statistical analysis A p value < 0.05 was considered statistically significant The statistical tests used for each dataset are specified in the corresponding figure caption All data generated or analysed during this study are included in this article The raw datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Ferlay, J. et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. International journal of cancer 127, 2893–2917, https://doi.org/10.1002/ijc.25516 (2010) Tsvetkova, E. & Goss, G. D. Drug resistance and its significance for treatment decisions in non-small-cell lung cancer. Current oncology 19, S45–51, https://doi.org/10.3747/co.19.1113 (2012) Ahmad, J. et al. Nanotechnology-based inhalation treatments for lung cancer: state of the art. Nanotechnology, science and applications 8, 55–66, https://doi.org/10.2147/NSA.S49052 (2015) Labiris, N. R. & Dolovich, M. B. Pulmonary drug delivery. Part I: physiological factors affecting therapeutic effectiveness of aerosolized medications. British journal of clinical pharmacology 56, 588–599, https://doi.org/10.1046/j.1365-2125.2003.01892.x (2003) Zhou, Q. T., Tang, P., Leung, S. S., Chan, J. G. & Chan, H. K. Emerging inhalation aerosol devices and strategies: where are we headed? Advanced drug delivery reviews 75, 3–17, https://doi.org/10.1016/j.addr.2014.03.006 (2014) Sung, J. C., Pulliam, B. L. & Edwards, D. A. Nanoparticles for drug delivery to the lungs. Trends in biotechnology 25, 563–570, https://doi.org/10.1016/j.tibtech.2007.09.005 (2007) Perspective on Nanoparticle Technology for Biomedical Use Fernandes, C. A. & Vanbever, R. Preclinical models for pulmonary drug delivery. Expert opinion on drug delivery 6, 1231–1245, https://doi.org/10.1517/17425240903241788 (2009) Schlesinger, R. B. Comparative deposition of inhaled aerosols in experimental animals and humans: a review. Journal of toxicology and environmental health 15, 197–214, https://doi.org/10.1080/15287398509530647 (1985) Cryan, S. A., Sivadas, N. & Garcia-Contreras, L. In vivo animal models for drug delivery across the lung mucosal barrier. Advanced drug delivery reviews 59, 1133–1151, https://doi.org/10.1016/j.addr.2007.08.023 (2007) Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations Toxicological sciences: an official journal of the Society of Toxicology 55 Hartung, T. & Sabbioni, E. Alternative in vitro assays in nanomaterial toxicology. Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology 3, 545–573, https://doi.org/10.1002/wnan.153 (2011) Benam, K. H. et al. Engineered In vivo disease models. Annu Rev Pathol 10, 195–262, https://doi.org/10.1146/annurev-pathol-012414-040418 (2015) Establishment of a tumour-stroma airway model (OncoCilAir) to accelerate the development of human therapies against lung cancer Alternatives to laboratory animals: ATLA 44 Agu, R. U. & Ugwoke, M. I. In vitro and in vivo testing methods for respiratory drug delivery. Expert opinion on drug delivery 8, 57–69, https://doi.org/10.1517/17425247.2011.543896 (2011) Sant, S. & Johnston, P. A. The production of 3D tumor spheroids for cancer drug discovery. Drug discovery today. Technologies 23, 27–36, https://doi.org/10.1016/j.ddtec.2017.03.002 (2017) Production and characterization of multiple-layered populations of animal cells Postconfluency MDCK monolayers as an in vitro model of solid tumor chemosensitivity Comparative chemosensitivity of exponential- versus plateau-phase cells in both in vitro model systems Differential killing efficacy of twenty antitumor drugs on proliferating and nonproliferating human tumor cells Plateau-phase cultures: an experimental model for identifying drugs which are bioactivated within the microenvironment of solid tumours Ribatti, D. A revisited concept: Contact inhibition of growth. From cell biology to malignancy. Experimental cell research, https://doi.org/10.1016/j.yexcr.2017.06.012 (2017) Minchinton, A. I. & Tannock, I. F. Drug penetration in solid tumours. Nature reviews. Cancer 6, 583–592, https://doi.org/10.1038/nrc1893 (2006) Multicellular membranes as an in vitro model for extravascular diffusion in tumours Limited penetration of anticancer drugs through tumor tissue: a potential cause of resistance of solid tumors to chemotherapy Clinical cancer research: an official journal of the American Association for Cancer Research 8 Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours Penetration of anticancer drugs through solid tissue: a factor that limits the effectiveness of chemotherapy for solid tumors Clinical cancer research: an official journal of the American Association for Cancer Research 5 Kyle, A. H., Huxham, L. A., Chiam, A. S., Sim, D. H. & Minchinton, A. I. Direct assessment of drug penetration into tissue using a novel application of three-dimensional cell culture. Cancer research 64, 6304–6309, https://doi.org/10.1158/0008-5472.CAN-04-1099 (2004) Grantab, R. H. & Tannock, I. F. Penetration of anticancer drugs through tumour tissue as a function of cellular packing density and interstitial fluid pressure and its modification by bortezomib. BMC Cancer 12, 214, https://doi.org/10.1186/1471-2407-12-214 (2012) Choi, M. S., Kim, S. H. & Kuh, H. J. Penetration of paclitaxel and 5-fluorouracil in multicellular layers of human colorectal cancer cells. Oncology reports 25, 863–870, https://doi.org/10.3892/or.2011.1138 (2011) Lee, J. H., Na, K., Song, S. C., Lee, J. & Kuh, H. J. The distribution and retention of paclitaxel and doxorubicin in multicellular layer cultures. Oncology reports 27, 995–1002, https://doi.org/10.3892/or.2012.1650 (2012) Al-Abd, A. M., Lee, J. H., Kim, S. Y., Kun, N. & Kuh, H. J. Novel application of multicellular layers culture for in situ evaluation of cytotoxicity and penetration of paclitaxel. Cancer science 99, 423–431, https://doi.org/10.1111/j.1349-7006.2007.00700.x (2008) Extravascular diffusion of tirapazamine: effect of metabolic consumption assessed using the multicellular layer model International journal of radiation oncology Extravascular transport of the DNA intercalator and topoisomerase poison N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA): diffusion and metabolism in multicellular layers of tumor cells The Journal of pharmacology and experimental therapeutics 297 Multicellular resistance to tirapazamine is due to restricted extravascular transport: a pharmacokinetic/pharmacodynamic study in HT29 multicellular layer cultures Miura, S., Suzuki, H. & Bae, Y. H. A Multilayered Cell Culture Model for Transport Study in Solid Tumors: Evaluation of Tissue Penetration of Polyethyleneimine Based Cationic Micelles. Nano today 9, 695–704, https://doi.org/10.1016/j.nantod.2014.10.003 (2014) The multilayered postconfluent cell culture as a model for drug screening Critical reviews in oncology/hematology 36 In vitro chemosensitivity testing of multilayered microcultures Postconfluent multilayered cell line cultures for selective screening of gemcitabine Cytotoxic effects of anticancer agents on subconfluent and multilayered postconfluent cultures Cichon, M. A., Gainullin, V. G., Zhang, Y. & Radisky, D. C. Growth of lung cancer cells in three-dimensional microenvironments reveals key features of tumor malignancy. Integrative biology: quantitative biosciences from nano to macro 4, 440–448, https://doi.org/10.1039/c1ib00090j (2012) Barthes, J. et al. Priming cells for their final destination: microenvironment controlled cell culture by a modular ECM-mimicking feeder film. Biomaterials science 3, 1302–1311, https://doi.org/10.1039/c5bm00172b (2015) Shen, Y. I. et al. Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting. Biomaterials science 2, 655–665, https://doi.org/10.1039/C3BM60274E (2014) Strehmel, C., Zhang, Z. F., Strehmel, N. & Lensen, M. C. Cell phenotypic changes of mouse connective tissue fibroblasts (L-929) to poly(ethylene glycol)-based gels. Biomaterials science 1, 850–859, https://doi.org/10.1039/c3bm60055f (2013) Sunami, H., Yokota, I. & Igarashi, Y. Influence of the pattern size of micropatterned scaffolds on cell morphology, proliferation, migration and F-actin expression. Biomaterials science 2, 399–409, https://doi.org/10.1039/c3bm60237k (2014) Movia, D. et al. The curious case of how mimicking physiological complexity in in vitro models of the human respiratory system influences the inflammatory responses. A preliminary study focused on gold nanoparticles. Journal of Interdisciplinary Nanomedicine 2, 110–130, https://doi.org/10.1002/jin2.25 (2017) Cooper, J. R. et al. Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype. PLoS One 11, e0164438, https://doi.org/10.1371/journal.pone.0164438 (2016) Sekine, W., Haraguchi, Y., Shimizu, T., Umezawa, A. & Okano, T. Thickness limitation and cell viability of multi-layered cell sheets and overcoming the diffusion limit by a porous-membrane culture insert. J Biochip Tissue chip S1, https://doi.org/10.4172/2153-0777.S2-001 (2011) Ren, H., Birch, N. P. & Suresh, V. An Optimised Human Cell Culture Model for Alveolar Epithelial Transport. PLoS One 11, e0165225, https://doi.org/10.1371/journal.pone.0165225 (2016) Monolayers of human alveolar epithelial cells in primary culture for pulmonary absorption and transport studies Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability Clinical and experimental allergy: journal of the British Society for Allergy and Clinical Immunology 28 Thiery, J. P. Epithelial-mesenchymal transitions in tumour progression. Nature reviews. Cancer 2, 442–454, https://doi.org/10.1038/nrc822 (2002) Ye, Z. et al. Prognostic Values of Vimentin Expression and Its Clinicopathological Significance in Non-Small Cell Lung Cancer: A Meta-Analysis of Observational Studies with 4118 Cases. PLoS One 11, e0163162, https://doi.org/10.1371/journal.pone.0163162 (2016) Dauphin, M. et al. Vimentin expression predicts the occurrence of metastases in non small cell lung carcinomas. Lung cancer (Amsterdam, Netherlands) 81, 117–122, https://doi.org/10.1016/j.lungcan.2013.03.011 (2013) Yang, W. et al. Genomics of Drug Sensitivity in Cancer (GDSC): a resource for therapeutic biomarker discovery in cancer cells. Nucleic Acids Res 41, D955–961, https://doi.org/10.1093/nar/gks1111 (2013) Aram, L., Yacobi-Sharon, K. & Arama, E. CDPs: caspase-dependent non-lethal cellular processes. Cell Death Differ 24, 1307–1310, https://doi.org/10.1038/cdd.2017.111 (2017) Kumar, M. et al. NF-kappaB regulates mesenchymal transition for the induction of non-small cell lung cancer initiating cells. PLoS One 8, e68597, https://doi.org/10.1371/journal.pone.0068597 (2013) Riffle, S. & Hegde, R. S. Modeling tumor cell adaptations to hypoxia in multicellular tumor spheroids. J Exp Clin Cancer Res 36, 102, https://doi.org/10.1186/s13046-017-0570-9 (2017) The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport Wangari-Talbot, J. & Hopper-Borge, E. Drug Resistance Mechanisms in Non-Small Cell Lung Carcinoma. J Can Res Updates 2, 265–282, https://doi.org/10.6000/1929-2279.2013.02.04.5 (2013) Sharom, F. J. ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics 9, 105–127, https://doi.org/10.2217/14622416.9.1.105 (2008) Burkhart, C. A. et al. Small-molecule multidrug resistance-associated protein 1 inhibitor reversan increases the therapeutic index of chemotherapy in mouse models of neuroblastoma. Cancer research 69, 6573–6580, https://doi.org/10.1158/0008-5472.CAN-09-1075 (2009) Singer, B. B. et al. Deregulation of the CEACAM expression pattern causes undifferentiated cell growth in human lung adenocarcinoma cells. PLoS One 5, e8747, https://doi.org/10.1371/journal.pone.0008747 (2010) Identification and characterization of different subpopulations in a human lung adenocarcinoma cell line (A549) Clonal variation of cadmium response in human tumor cell lines Watanabe, N. et al. A549 subclones demonstrate heterogeneity in toxicological sensitivity and antioxidant profile. American journal of physiology. Lung cellular and molecular physiology 283, L726–736, https://doi.org/10.1152/ajplung.00025.2002 (2002) Roman, J., Ritzenthaler, J. D., Roser-Page, S., Sun, X. & Han, S. alpha5beta1-integrin expression is essential for tumor progression in experimental lung cancer. Am J Respir Cell Mol Biol 43, 684–691, https://doi.org/10.1165/rcmb.2009-0375OC (2010) Jia, D. et al. Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion. BMC Cancer 10, 364, https://doi.org/10.1186/1471-2407-10-364 (2010) Cytoplasmic redistribution of E-cadherin-catenin adhesion complex is associated with down-regulated tyrosine phosphorylation of E-cadherin in human bronchopulmonary carcinomas Lim, S. C., Jang, I. G., Kim, Y. C. & Park, K. O. The role of E-cadherin expression in non-small cell lung cancer. Journal of Korean medical science 15, 501–506, https://doi.org/10.3346/jkms.2000.15.5.501 (2000) Influence of E-cadherin dysfunction upon local invasion and metastasis in non-small cell lung cancer Yang, Y. L., Chen, M. W. & Xian, L. Prognostic and clinicopathological significance of downregulated E-cadherin expression in patients with non-small cell lung cancer (NSCLC): a meta-analysis. PLoS One 9, e99763, https://doi.org/10.1371/journal.pone.0099763 (2014) alpha-catenin and gamma-catenin but not beta-catenin in human lung cancer The expression of beta-catenin in non-small-cell lung cancer: a clinicopathological study Liu, D. et al. E-cadherin expression associated with differentiation and prognosis in patients with non-small cell lung cancer. Ann Thorac Surg 71, 949–954; discussion 954–945, https://doi.org/10.1016/S0003-4975(00)02545-5 (2001) In vitro bioassays for anticancer drug screening: effects of cell concentration and other assay parameters on growth inhibitory activity The Ki-67 protein: from the known and the unknown doi:10.1002/(SICI)1097-4652(200003)182:3<311::AID-JCP1>3.0.CO;2-9 (2000) Fulda, S. & Debatin, K. M. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798–4811, https://doi.org/10.1038/sj.onc.1209608 (2006) Degterev, A., Boyce, M. & Yuan, J. A decade of caspases. Oncogene 22, 8543–8567, https://doi.org/10.1038/sj.onc.1207107 (2003) Pop, C. & Salvesen, G. S. Human caspases: activation, specificity, and regulation. J Biol Chem 284, 21777–21781, https://doi.org/10.1074/jbc.R800084200 (2009) Apaf-1 oligomerizes into biologically active approximately 700-kDa and inactive approximately 1.4-MDa apoptosome complexes Okouoyo, S. et al. Rescue of death receptor and mitochondrial apoptosis signaling in resistant human NSCLC In vivo. International journal of cancer 108, 580–587, https://doi.org/10.1002/ijc.11585 (2004) Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy Saelens, X. et al. Toxic proteins released from mitochondria in cell death. Oncogene 23, 2861–2874, https://doi.org/10.1038/sj.onc.1207523 (2004) Moll, U. M., Wolff, S., Speidel, D. & Deppert, W. Transcription-independent pro-apoptotic functions of p53. Current opinion in cell biology 17, 631–636, https://doi.org/10.1016/j.ceb.2005.09.007 (2005) Gogvadze, V., Orrenius, S. & Zhivotovsky, B. Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochimica et biophysica acta 1757, 639–647, https://doi.org/10.1016/j.bbabio.2006.03.016 (2006) Immunohistochemical detection of multidrug resistance protein in human lung cancer and normal lung Clinical cancer research: an official journal of the American Association for Cancer Research 4 Li, J. et al. Expression of MRP1, BCRP, LRP, and ERCC1 in advanced non-small-cell lung cancer: correlation with response to chemotherapy and survival. Clinical lung cancer 10, 414–421, https://doi.org/10.3816/CLC.2009.n.078 (2009) LRP and ERCC1 as prognostic factors in non-small cell lung cancer patients receiving postoperative cisplatin-based chemotherapy The International journal of biological markers 24 and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels Clinical cancer research: an official journal of the American Association for Cancer Research 7 Yamada, K. M. & Cukierman, E. Modeling tissue morphogenesis and cancer in 3D. Cell 130, 601–610, https://doi.org/10.1016/j.cell.2007.08.006 (2007) Hirschhaeuser, F. et al. Multicellular tumor spheroids: an underestimated tool is catching up again. Journal of biotechnology 148, 3–15, https://doi.org/10.1016/j.jbiotec.2010.01.012 (2010) Zanoni, M. et al. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Scientific Reports 6, 19103, https://doi.org/10.1038/srep19103 (2016) Spheroids in radiobiology and photodynamic therapy In Unraveling the Safety Profile of Nanoscale Particles and Materials Blais, C. M. et al. Methacholine Challenge: Comparison of Airway Responsiveness Produced by a Vibrating Mesh Nebulizer Versus a Jet Nebulizer. Journal of aerosol medicine and pulmonary drug delivery, https://doi.org/10.1089/jamp.2017.1392 (2017) Huber, J. M. et al. Evaluation of assays for drug efficacy in a three-dimensional model of the lung. J Cancer Res Clin Oncol 142, 1955–1966, https://doi.org/10.1007/s00432-016-2198-0 (2016) Dekali, S. et al. Assessment of an in vitro model of pulmonary barrier to study the translocation of nanoparticles. Toxicology Reports 1, 157–171, https://doi.org/10.1016/j.toxrep.2014.03.003 (2014) Download references by the Irish Research Council under the Government of Ireland Postdoctoral Fellowship scheme to DM and Science Foundation of Ireland through the Advanced Materials and BioEngineering Research (AMBER) project (Grant #SFI/12/RC/2278) The authors would like to thank Dr Luisana Di Cristo for technical assistance and useful discussions on western blotting techniques Department of Clinical Medicine/Trinity Translational Medicine Institute (TTMI) First Moscow State Sechenov Medical University designed and performed the biological experiments The authors declare no competing interests Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-018-31332-6 Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research UCD awardee Dr Sujit Jung Karki with Dr Ruth Freeman at UCD Rosemount Environmental Research Station Credit: Carl Gibney Eight early career researchers at University College Dublin are among more than two dozen receiving €14.6 million investment under the (opens in a new window)SFI-IRC Pathway programme.The 25 research projects have been awarded financial support covering a four-year period that will see their postdoctoral awardees transition into independent research leaders. The funding will also provide additional support for a postgraduate student who will be primarily supervised by the awardee.The eight UCD projects funded are: Patrick O’Donovan TD said the SFI-IRC Pathway programme provided a “defined journey from post-doctoral research to independent researcher”. “Bridging this gap is a challenging but critical milestone ranging from new therapies for Parkinsons to the development of sustainable supercapacitors.”Commenting on the awards added: “[The SFI-IRC Pathway programme] provides targeted support to early-career researchers who will use the funding to pursue independent research at the frontiers of knowledge. “Investment in these projects will generate novel discoveries and insights across diverse research topics to wireless network security.”The SFI-IRC Pathway programme is a collaborative initiative between Science Foundation Ireland (SFI) and the Irish Research Council (IRC) to support early-career research across all disciplines and to encourage a cohesive research ecosystem in Ireland By:  David Kearns UCD University Relations (with materials from Emma Loughney To contact the UCD News & Content Team Sie haben erfolgreich Ihre Einwilligung in die Nutzung von Transfermarkt mit Tracking und Cookies widerrufen Sie können sich jetzt zwischen dem Contentpass-Abo und der Nutzung mit personalisierter Werbung (Business in Cameroon) - A private investor (not named so far) in partnership with the Cameroonian ministry of economy which oversees agropole projects in the country has just launched an agropole of freshwater fish production in Bazou The project which has been officially launched on January 12 cost CFA1.6 billion and CFA500 million was disbursed by the government for its realization. The sponsor revealed that thanks to this investment 241 tons of freshwater fishes and 1.3 million fries will be produced every year.  It should be reminded that agropole projects are aimed at creating income-generating activities in rural areas through agro pastoral projects which will increase the national production and at the same time reduce the massive imports of foodstuffs Kribi Bitumen Plant Set to Start Construction in 2025 with Government Backing CEMAC Bond Market Hits CFA 8.45 Trillion in March 2025, Interest Rates Drop Cameroon’s Timber Output Projected to Rise in 2025 Despite Higher Export Taxes Central Africa Stock Exchange Sees 98% Drop in Trading Value in Q1 2025 Every week the economy and investment news from Cameroon Mboa Paris Trains 30 Young Cameroonians to Boost Tech and Entrepreneurship Cameroon Audit Targets Former Officials for Mismanagement in Agricultural Project Camwater Seeks Global Bids to Launch Bottled Water Lines in Five Cities Bafoussam Workshop Highlights Benefits of Cameroon-EU Trade Agreement Cameroon Could Reach 350,100 Tons of Cotton in 2025 (Beac) Paul Biya Appoints Johnny Razack as Chair of Cameroon’s National Investment Company Cameroon Refuses Work Visa Renewal for Casino and Super U Boss Over Toxic Workplace Claims Cameroon Joins Global Charter to Fight Illegal Fishing Less than 24 hours after the Cameroonian was appointed to lead the Wildcats of Bazou Bangui the executive committee of the Central African Football Federation has expressed its opposition urges the country’s Ministry of Sport to avoid any possible conflict between the FCF and the Ministry This call is particularly crucial given that CAR is in the process of restoring peace Rigobert Song is currently in a delicate position as his appointment on Monday does not meet with the unanimous approval of Central Africans He finds himself in the same position as Marc Brys his successor as coach of the Indomitable Lions The Central African Football Federation has learned with dismay and surprise via social media of the decree N°002/MP/SEC/DIR-CAB.2025 of 13 January 2025 appointing the coaches of the senior national team The Executive Committee of the Central African Football Federation meeting in extraordinary session on 14 January 2025 informs the national and international opinion that it was neither involved in nor consulted on this decision it declares that it does not recognise itself in this unilateral decision which contradicts the provisions in force: “Decisions concerning the recruitment of members of management structures must remain among the rights and competences reserved solely for the Executive Committee of the Federations” the Central African Football Federation has decided to nationalise the post of Fauves A coach the interim position has been entrusted to an all-Central African technical staff led by Eloge Enza Yamissi Following the Central African Republic’s latest qualification result for the African Nations Championship (CHAN) and at a time when the President of the Republic and Head of State is stepping up his efforts to consolidate peace between the country’s sons and daughters would like to avoid any action aimed at creating an unnecessary crisis between the Ministry of Sport and the body responsible for managing Central African football DUBA NAN: Danna “See First” karkashin karkashin "Following “ don samun labaran Legit.ng a shafinka na Facebook akai-akai Niger – An cire Mohammed Bazoum daga kan karagar mulki a Nijar ta hanyar wani danyen juyin mulki da aka gudanar a ranar Larabar nan Rahoton da mu ka samu daga Reuters ya tabbatar da cewa wasu sojoji sun yi wa gwamnati tawage sun kifar da Shugaba Mohammed Bazoum A wata sanarwa da su ka fitar ta bakin Kanal Amadou Abdramane sojojin sun sanar da cewa sun kawo karshen gwamnati mai-ci a kasar Nijar A daren yau Amadou Abdramane ya karanto sanarwar tare da manyan sojoji tara a gefensa LURA: Shin kana son bamu labari da tattaunawa da marubutanmu Kakakin sojojin ya ce tabarbarewar rashin tsaro da rashin kyawun shugabanci da ake fama da shi ya jawo su ka yi juyin mulkin France 24 ta kuma tabbatar da sojojin sun rufe duka wasu iyakokin jamhuriyyar sannan an kakaba dokar kulle tare da dakatar da duk ayyuka su ka nuna ba za su yarda wata kasar tayi masu katsalandan ba tare da yin alkawarin tsare lafiyar Bazoum Zuwa yanzu ba a san halin da Mai girma Bazoum mai shekaru 63 yake ciki ba sai dai da-dama daga mutanen kasar su na goyon bayan gwamnatin shi Manyan kasashen Duniya su na bukatar Jamhuriyyar Nijar domin su iya yakar ‘yan ta’addan da ke tada zaune tsaye a kasashen Mali da Burkina Faso Amurka wanda ta kashe Dala miliyan 500 wajen tsamar da tsaro a Nijar daga 2012 zuwa yau tayi kira ga sojojin da su yi gaggawar sakin shugaban Aljazeera ta ce Sakataren gwamnatin Amurka Antony Blinken ya shaidawa manema labarai a New Zealand cewa ba su goyon bayan juyin mulki Rahoton ya ce wannan shi ne juyin mulki na bakwai da aka yi a nahiyar Afrikar a cikin shekaru uku A tarihin kasar mai makwabtaka da Najeriya Bazoum ne shugaban farko da ya gaji mulkin farar hula bayan shekaru fiye da 60 da samun’yancin kai Twitch streamer Jeremy "Jerma985" had a hilarious moment occur during a recent livestream while playing the game Mon Bazou Jerma noticed a rather peculiar object in the in-game world and was taken aback The Twitch streamer did not know what a log splitter looked like in real life and was shocked to see the tool in-game Expressing his confusion regarding the log splitter debate he was engaging in with his Twitch chat Jerma hosted a recent broadcast on April 21 and focused on playing the sandbox and simulation game Mon Bazou Mon Bazou is a single-player, choose your own adventure game where the player is required to build a car from scratch by installing various important parts by themselves Players are required to make money by performing various actions in-game One such action that Jerma was required to do was to cut wood in the game. However, the Twitch streamer seemed pretty clueless about what a log splitter looked like and mistook it for a trailer His Twitch chat guided him and mentioned that there was a log splitter located behind his in-game house Jerma failed to understand how the tool worked He hilariously called the log splitter a trailer and continued to express his confusion The 36-year-old streamer said the following as he struggled to cut a piece of wood in-game: He then sat inside the pickup truck in-game and read a few messages posted by his viewers Many fans in the Twitch chat exclaimed that the tool behind his house was indeed a log splitter He opened up Google Images on his second monitor to have a look at what a log splitter looks like As soon as he realized that his viewers were correct He still seemed confused regarding the log splitter and the trailer situation and stated: The streamer's hilarious realization came to an end a few minutes later and he continued to play Mon Bazou for the rest of his livestream Audiences on Reddit were amused to see how clueless the streamer was. Many fans made fun of the streamer in a reaction thread on the r/LivestreamFail subreddit A discussion surrounding the log splitter also popped up in the comment section of the thread Jerma985 is a well-known content creator who kicked off his streaming career in November 2016 He currently has more than 900k followers on his Twitch channel and garners around 14k concurrent viewers per stream Are you stuck on today's Wordle? Our Wordle Solver will help you find the answer Your perspective matters!Start the conversation