Increased accumulation of liposomes (LSs) conjugated to antibodies against αvβ3 was observed in TCM networks compared to non-conditioned networks, indicating αvβ3 may be a potential target for the delivery of drugs specifically to tumour vasculature. Furthermore, the use of microbubbles (MBs) and ultrasound (US) to further enhance the delivery of LSs to TCM-conditioned vasculature was investigated. Quantification of fluorescent LS accumulation post-perfusion of the vascular network showed 3-fold increased accumulation with the use of MBs and US, suggesting that targeted LS delivery could be further improved with the use of locally administered MBs and US.The vascular system is the primary route for the delivery of therapeutic drugs throughout the body and is an important barrier at the region of disease interest, such as a solid tumour. The development of complex 3D tumour cultures has progressed significantly in recent years however, the generation of perfusable vascularised tumour models still presents many challenges. This study presents a microfluidic-based vasculature system that can be induced to display properties of tumour-associated blood vessels without direct incorporation of tumour cells. Conditioning healthy endothelial–fibroblast cell vasculature co-cultures with media taken from tumour cell cultures was found to result in the formation of disorganised, tortuous networks which display characteristics consistent with those of tumour-associated vasculature. Integrin α Increased accumulation of targeted liposomes was observed in cultures mimicking tumour-associated vasculature, when compared to healthy vasculature cultures. Microbubble-mediated liposome delivery was found to further increase liposome accumulation. One area of particular interest is the development of microfluidic models that recapitulate the tumour vasculature and allow for the evaluation of anti-cancer therapeutics. Systems incorporating multiple healthy cell types alongside cancer cells have been developed and used to investigate a range of cancer-related processes, such as cancer cell response to chemical gradients and invasion into healthy tissues.1–8 Whilst endothelial cells have been incorporated into many tumour models, these are typically used in basic monolayer or pre-patterned configurations which do not accurately recreate the structure and function of tumour-associated vasculature.9–11 The endothelium presents the first barrier for therapeutics delivered intravenously and, as such, the absence of authentic vasculature components in tumour models often limits the validity of the test outcomes.The successful development of increasingly complex microfluidic cell culture or organ-on-chip models has demonstrated the ability of these systems to effectively recreate the structure and function of many 12–14 Endothelial cells and fibroblasts are seeded in a fibrin matrix into a microfluidic device, and then supplied with the appropriate growth factors to induce angiogenesis – the formation of new blood vessels. The co-culture of endothelial cells and fibroblasts recreates the process of vasculogenesis and angiogenesis that occurs at the site of wounds undergoing tissue repair and recapitulates the process of healthy network formation. Critically, the use of microfluidics overlaid in this process allows cells to be exposed to the physiological shear stresses and pressures required to induce vessel formation, whilst also allowing access to the vessel network by using connected side channels that mimic larger blood vessels. These vascularised organ-on-a-chip microfluidic systems present a promising platform for the investigation of many physiological processes and have recently been used for large-scale drug screening.15 However, without the influence of tumour cells, the vasculature produced in these systems will not display the necessary tumour-associated characteristics. The addition of tumour cells to vasculature cultures has allowed for the production of systems that recreate the in vivo responses of many anti-cancer therapeutics.16 However, the incorporation of tumour cells with healthy vasculature presents many challenges to ensure that the cultures progress in a similar fashion to tumours growing in vivo. Optimisation of seeding densities and timing is required to prevent tumour cells from overwhelming the healthy vasculature. Conversely, if cells are seeded too sparsely, then tumour development and influence over the vasculature will be limited.Recently, it has been discovered that vasculature networks can be produced by inducing the self-assembly of endothelial cells and fibroblasts into vessel structures in the presence of a continuous fluid flow.et al. used TCM taken from HT1080 cells to condition human umbilical vein endothelial cells (HUVECs) grown on Matrigel and found increased proliferation and angiogenic tube formation compared to vascular endothelial growth factor (VEGF) stimulated HUVECs. Analysis of protein expression found most differentially expressed proteins were downregulated (88%) with TCM, compared to an upregulation of 59% when VEGF was used to stimulate angiogenesis – revealing that the induction of tumour angiogenesis does not rely on VEGF alone.17 This highlighted the importance of using the appropriate tumour-derived angiogenic stimuli when seeking to recapitulate tumour angiogenesis. Hida et al. also investigated the effects of conditioning HUVECs with TCM and found that endothelial cells acquire drug-resistant characteristics when cultured in TCM. It was discovered that these characteristics are reversed when cells are re-cultured in TCM-free media.18 The presence of high levels of VEGF and other various inflammatory cytokines  have been reported in media taken from patient-derived colorectal tumours and have been proposed as the main drivers of endothelial cell conditioning.19 Sawa-Wejksza et al. showed that media taken from several colorectal cell lines  could be used to induce THP-1 monocytes to differentiate into a non-adherent population of M1 and M2 immunosuppressive macrophages, consistent with tumour-associated macrophages found in vivo.20 These observations, along with various other studies,21,22 demonstrate that the cytokines and soluble factors tumour cells secrete, influence tumour-associated cells in a paracrine fashion, and can be used to condition cells to display similar properties to in vivo tumour-associated cells.The indirect influence of tumour cells has also been previously investigated as a means of inducing healthy cells to display tumour-associated characteristics. Katanasaka 2,23 Further details regarding the choice of design features and experimental setup can be found throughout the materials and methods section.This study combines the use of TCM conditioning and perfusable microfluidic vasculature models to develop a method of producing vasculature-on-chip networks that display tumour-associated properties. vβ3 – an integrin found to be upregulated in tumour vasculature. Integrin αvβ3, an ECM-binding receptor found primarily on endothelial cells, has also been of recent interest due to its upregulation in tumour tissues. Integrin αvβ3 binds to various matrix proteins found around newly forming blood vessels, such as fibrinogen, vitronectin and fibronectin.24 As a result, integrin αvβ3 has been found to be crucial for angiogenesis and blocking of the signalling pathway has been found to reduce neovascularisation in tumour tissues.25–27 The constant pro-angiogenic signalling induced by tumour cells results in αvβ3 being upregulated in tumour vasculature, presenting it as a potential target for drug-loaded carriers.28,29 Furthermore, integrin αvβ3 upregulation in mammary fibroblasts conditioned with pro-inflammatory breast cancer factors has been previously reported, indicating that soluble factors secreted by tumour cells affect integrin expression.30By conditioning networks with media taken from tumour cell cultures, the soluble factors secreted by tumours induce vasculature to form similarly as if it were associated directly with tumour cells. Network functionality was demonstrated by using this system to evaluate the efficacy of targeting model drug carrier particles to integrin α31 As a result, MBs have found use as US contrast agents in clinical imaging and have been used to facilitate the imaging of tumour vascular structures.32–34 MB also offer a potential means of enhancing drug delivery to cells through a mechanism known as sonoporation. Low acoustic pressure US waves can be used to induce MB to undergo stable cavitation, in which bubbles oscillate with small amplitudes at the US driving frequency. The rapid expansion and contraction of MBs exert forces on nearby cell membranes, creating pores (sonoporation) and increasing membrane permeability.35–37 Higher acoustic pressures can also be used to induce MBs to expand too much greater diameters then collapse and fragment into smaller particles. Significantly higher pressures and temperatures are generated when MBs collapse resulting in the generation of shockwaves. This process referred to as inertial cavitation has been associated with increased cellular uptake and increased irreversible sonoporation – where the pores created in cell membranes fail to reseal and result in cell death.38,39 Multiple in vitro studies have demonstrated the ability of sonoporation to increase drug delivery directly to tumour cells and endothelial cell-MB studies have been used to show increased permeability due to sonoporation, indicating that increased passage of therapeutics would occur.40 However, many of these investigations are carried out on 2D cell monolayers or in pre-patterned 3D configurations.11,36,41,42 Each of which neglect to recreate the authentic structure of vasculature which primarily consists of endothelial cells supported by a basement membrane layer and surrounding stromal cells.The use of microbubbles (MBs) to further facilitate the uptake of targeted therapeutics was tested to evaluate a combined localised delivery approach. MBs are micron-sized gas particles typically stabilised by a lipid shell. Due to the compressibility of their gas core, MBs are highly responsive to ultrasound (US) which causes bubbles to oscillate and scatter US waves.43,44 Loading of anti-cancer therapeutics into LS has long been of interest and has been found to decrease systemic toxicity, allowing for higher drug doses to be tolerated.45–47 The conjugation of drug-loaded LSs to MBs to create therapeutic/drug-loaded MBs has been suggested as a potential means of delivering anti-cancer therapeutics locally to the tumour site, using US as a local release mechanism.48 If shown to be reliable and effective, this method of localised drug delivery to the tumour would overcome one of the major challenges anti-cancer treatments face – damage to healthy tissues from systemic drug exposures. The attachment of targeted antibodies to MB has also been explored as a method of increasing MB accumulation in tumour tissues – further increasing local drug-loaded LSs accumulation. Antibodies targeting VEGF pathway proteins have been of interest due to the upregulation of VEGF found in tumour-induced angiogenesis.49,50 A recent study by Ingram et al. observed the increased efficacy of cytotoxic drugs loaded into liposomes and attached to VEGFR2-targeted MBs. The US-triggered delivery was found to enhance localised tumour drug accumulation whilst simultaneously limiting the bioavailability of drugs in normal tissues.51 Integrin αvβ3 targeting has also recently been tested and a study using αvβ3-targeted MBs found that some sonoporation events induced endothelial cells to retract from neighbouring cells, resulting in the formation of a large gap in the endothelium.36,39,50 However, this investigation was conducted using a basic endothelial cell monolayer model – thus limiting the validity of these conclusions. The use of integrin αvβ3-targeted MBs has been investigated as a means of imaging tumours and tumour angiogenesis using US.52–56 Anderson et al. observed a 5-fold increase in targeted MBs US intensity compared to controls and found significantly decreased intensity when mouse tumours were pre-treated with an antibody to block integrin αvβ3.57 It is evident that αvβ3-targeted MBs offer a promising means of targeting tumour vasculature, however further research into the impact of targeting on MB-mediated drug delivery is still required.The use of sonoporation as a means of facilitating the delivery of drug-loaded LS (LSs) is of particular interest for the treatment of cancer.vβ3-targeted LS and MBs to increase LS accumulation in tumour tissues. Networks mimicking tumour vasculature were created by growing vasculature in TCM taken from HCT116 colorectal cell cultures. Integrin αvβ3 was observed to be upregulated in HUVECs and normal human lung fibroblasts (NHLFs) grown in TCM and was found to directly correlate with the concentration of TCM used. Networks conditioned with TCM were observed to undergo increased rates of angiogenesis, forming a disorganised network with larger diameter vessels. Targeted-LS were perfused through the networks and their accumulation in healthy and TCM-conditioned networks were first compared to evaluate the impact of αvβ3 upregulation. Antibody targeting concentrations of 2 and 4 μg mL−1 were tested and compared to isotype controls of the same concentrations. Targeted LS were then conjugated to MBs which were perfused through the networks before being exposed to US. The efficacy of MB-mediated delivery of αvβ3-targeted LS in TCM-conditioned networks was then evaluated and compared to those in healthy networks. This study reports a simple approach to condition on-chip vasculature to display disease-like characteristics and highlights the importance of producing accurate organ-on-chip models for the successful development of new therapeutics.This study uses microfluidic vasculature cultures to investigate the efficacy of using α5 HUVEC cells were plated in a T75 flask and incubated in virus-containing media for 16 hours. Fluorescent assisted cell sorting (FACS) was then used to sort transduced cells based on pre-determined gates based on the level of EGFP expression. Positive cells were reseeded and grown for a minimum of 48 hours to ensure sufficient viability. GFP-HUVECs were expanded to p3 before being frozen. Normal human lung fibroblasts (NHLF) were purchased from CellSystems and grown using FibroLife S2 (LifeLine Cell Technology) media. NHLFs were expanded to p3 and frozen. Both cell types were defrosted and grown for 2 days at p4, then used for the experimental setup.Human umbilical vein endothelial cells (HUVEC) were purchased from TCS Cellworks (ZHC-2102) and grown using endothelial growth media-2 . HUVECs were labelled at passage 1 with an enhanced green fluorescent protein (EGFP). Lentivirus production for the transduction was carried out following Tronolab protocols using HEK 293 T cells. 5 × 10Polydimethylsiloxane (PDMS) microfluidic devices were fabricated using standard photolithography and soft lithography techniques. SU8-2075 photoresist was spin-coated onto a 4 in. silicon wafer at 2000 rpm to produce a 100 μm thick layer. The photoresist was baked for 60 min then a direct-write 375 nm UV laser  was used to pattern the device design by selectively exposing the photoresist. The wafer was then baked for a further 60 min then the excess photoresist was removed using Microposit EC solvent . PDMS  was mixed in a 10 : 1 base : curing agent ratio poured onto the wafer and desiccated for 40 min to remove any bubbles. PDMS was poured to produce 1 mm thick devices which minimised US attenuation through the PDMS. PDMS was then cured at 80 °C for 1 h. Devices were then cut, hole-punched and bonded to PDMS-coated coverslips using oxygen plasma.Fabricated devices were then sterilised in an autoclave at 120 °C for 40 min. Reservoirs were fabricated from 16 mm diameter polycarbonate (Engineering and Design Plastics) cut into 32 mm long pieces. Reservoirs were fabricated to contain a 2 mm wide spout protruding from the bottom of each container. This allowed for simple interfacing with 2 mm hole-punched inlets and outlets in the microfluidic chips. Thicker pieces of PDMS were then bonded over inlet/outlet locations to secure reservoir interfacing with the chip. Reservoir lids were fabricated by gluing 0.22 μm PTFE filters (Cole-Parmer) onto the top of Delrin (Par-group) rings, which were then slotted over the top of the reservoirs. 0.22 μm filters prevented bacterial contamination of the media. Reservoirs were autoclaved at 120 °C for 40 min and the underside of the reservoir lids were sterilised under UV light for 30 min prior to experimental use.−1 solution. The solution was then separated into 20 μL aliquots and frozen at −20 °C. Single aliquots of both fibrinogen and thrombin were defrosted prior to experimental use.Fibrinogen  was prepared by dissolving 100 mg of power into 10 mL of sterile distilled water containing 0.9% NaCl and warmed to 37°. The solution was gently agitated until a cloudy solution was formed. The solution was then separated into 200 μL aliquots and frozen at −20 °C. Thrombin powder  was dissolved in 2 mL of PDMS containing 0.1% w/v bovine serum albumin  to give a 50 U mL7 cells per mL and 5 × 106 cells per mL, respectively. 5 μL of the fibrinogen-cell mixture was then rapidly mixed with 0.5 μL of thrombin and pipetted into the centre chamber inlet of the microfluidic chip. Chips were then placed on a hotplate at 37 °C for 20 min to allow for fibrin polymerisation. Basement Membrane Matrix  was then mixed with laminin  and pipetted through the reservoirs, into the side channels. Chips were placed on the hotplate at 37 °C for a further 15 min to allow the BME mixture to partially polymerise. EGM-2 was then manually pipetted through each side channel to remove the majority of the BME mixture, leaving a coating remaining along the channel walls. Reservoirs were then filled with EGM-2 with 15 mmH2O and 5 mmH2O being added to inlet and outlet reservoirs, respectively. Flow direction was reversed daily to encourage complete anastomosis and media in each reservoir was releveled to ensure continuous flow and pressure differences. Fresh media was added every 48 hours to ensure consistent growth factor concentrations. EGM-2 complete with all growth factors was used for the first 24 hours before being replaced with growth factor reduced EGM-2 without VEGF or fibroblast growth factor (FGF), which was used throughout the rest of the experiment.Both HUVEC and NHLF cells were detached from their culture flasks and resuspended together in fibrinogen at a concentration of 1 × 1058 is a software developed for the quantitative analysis of vasculature network morphologies from microscope images. Contrast and brightness enhancement was first applied to vasculature images to prevent misidentification of vessel structures. Multiple vessel diameters were selected for identification (10–150 μm in 5 μm increments) to ensure all vessels were correctly identified. The small particle removal maximum was set to 200 μm and the fill holes value set to 1200 μm, to ensure imaging artifacts and the intervascular space was correctly removed.AngioTool−1). On-chip flow conditions were setup as described above and the calcein solution added to the high-pressure side channel, whilst PBS was added to the low-pressure channel. Videos were captured of fluorescent calcein diffusion across the fibrin gels then FIJI (ImageJ) was used to analyse the velocity of the diffusion. Velocity values were expressed in μm s−1 and compared to optimal anastomosis interstitial flow values reported throughout literature. Fibrinogen concentrations which gave optimal interstitial flow values were then chosen for on-chip experiments.The interstitial flow velocity through fibrin hydrogels was measured by perfusing 2 μM Calcein  through blank fibrin gels produced using a range of fibrinogen concentrations . Beads were recorded flowing down the channels and their linear velocities were determined using Mosaic ImageJ particle tracking plugin (https://imagej.net/plugins/mosaicsuite). Linear velocities for reservoir height differences ranging between 2–20 mm were determined and compared to their theoretical values.Flow rates down each side channel, induced by the hydrostatic pressures from reservoir height differences, were measured by using 2 μm green, fluorescent beads  through the networks using the same on-chip flow conditions described previously. Perfusion was repeatedly imaged at multiple locations across the chip in 5 minute intervals and the leakage of the fluorescent dextran observed. The commonly implemented permeability model developed by Yuan et al. and further simplified by Lee et al. was then used to calculate vessel permeability using the known initial, final, and background fluorescent intensities.59,60The permeability of the vascular endothelium was determined by flowing Texas Red-Dextran  to the desired TCM : EGM ratio . EGM-2 supplementary growth factors were then added to ensure the overall concentration remained constant.HCT116 (ECACC 91091005) were obtained from ECACC (UK) and grown using DMEM 10% FBS , with 1% Glutamax , in an incubator at 37 °C, 5% CO4 NHLF cells were first seeded in Fibrolife S2 media in each well of a 24-well plate then allowed to grow to form a confluent monolayer for 96 hours. HUVEC cells (1 × 107 cells per mL) were then suspended in fibrinogen (7.5 mg mL−1) and mixed with thrombin (50 U mL−1) to create the same fibrin-cell suspension used on-chip. 10 μL of this suspension was then seeded directly onto the NHLF monolayer. These cultures were grown in EGM : TCM media ratios of [1 : 0], [2 : 1], [1 : 1], and [1 : 2] for a further 96 hours at which point immunostaining and flow cytometry was used to determine αvβ3 -expression in each of the culture conditions.HUVEC and NHLF cells were seeded off-chip in a similar configuration as on-chip vasculature cultures. 1 × 10−1) for 60 minutes. Cells were then detached, washed, and resuspended in a solution of PBS (10% FBS) and anti- αvβ3 antibody  diluted 1 : 2000 with PBS (3% BSA). Cells were incubated for 30 minutes then centrifuged at 300 g for 5 minutes and washed 3 times with PBS. Cells were then resuspended in Alexa Fluor 647 secondary antibody  diluted 1:2000 with PBS (3% BSA) and incubated in the dark for a further 30 minutes. Cells were again centrifuged at 300 g for 5 minutes and washed in PBS 3 × then resuspended to a final concentration of 1 × 106 cells per mL in PBS (10% FBS). Flow cytometry was performed by Dr Ruth Hughes and Dr Sally Boxall in the Bioimaging and FACS facility within the faculty of Biological Sciences at the University of Leeds. A CytoFLEX S (Beckman Coulter) cytometer was used to excite GFP-HUVECs and the Alexa Fluor 647 secondary antibody with 488 nm and 638 nm lasers, respectively. A minimum of 10 000 valid events were captured for each sample to allow for robust data analysis. Fluorescence minus one compensation controls were first performed using αvβ3-stained NHLFs and GFP-HUVECs as red-only and green-only controls, respectively. Unstained NHLFs were also used as double-negative controls. Flow cytometry data was gated with respect to these controls to determine the number of αvβ3-positive cells. Immunofluorescent intensities were plotted as histograms and a Lorentzian peak fitting function used to find the modal peak intensity for each sample. Immunostaining of microfluidic vasculature cultures was performed by perfusing the networks with PBS (+Ca and +Mg) for 30 minutes Comment using the same on-chip growth flow conditions. Cultures were then fixed with 4% paraformaldehyde for 90 minutes which was followed by a 60 minute PBS wash. Cultures were permeabilised by flowing 0.3% Triton-X100 for 30 minutes then a blocking solution of PBS  was perfused for 60 minutes. The primary antibody  was diluted 1 : 1000 in PBS (3% BSA) and perfused through the network overnight at 4 °C. A 60 minute PBS wash was then performed then the secondary antibody  diluted 1 : 1000 in PBS (3% BSA) was perfused for 3 hours. A final 90 minute PBS wash was performed then the cultures were imaged using a Leica DMi8/SP8 confocal microscope.Cell-containing fibrin hydrogel clots were first digested using nattokinase . LS concentration was determined using a NanoSight NS300  and was typically found to be between 2–4 × 1012 LS mL−1. The average LS diameter was determined to be 335 ± 65 nm, found using a Zetasizer . Biotin – avidin binding was used to conjugate αvβ3 – antibodies to the surface of the LS. Biotin αvβ3 antibody  was mixed with LS (1011 mL−1) to give a final concentration of 2 or 4 μg mL−1. Neutravidin  was then added, the solution mixed, and the antibodies allowed to bind for 20 minutes before perfusion. The same protocol was used for biotinylated isotype antibody controls . 3 kDa cascade blue dextran  was also added to the LS solution to allow for perfusion through the networks to be imaged alongside LS perfusion.LS were prepared from a mixture of DSPC , Cholesterol , DSPE-PEG2000 Biotin  and DOPE-647 N (Sigma-Aldrich 42247) in a 55 : 40 : 4.6 : 0.4 M ratio with a total lipid concentration of 12 mg mL−1. Lipids were dissolved in a 1 : 1 mixture of chloroform and methanol and dried under nitrogen to remove the solvent. Lipids were then resuspended in PBS solution containing 1% glycerol and tip sonicated  at 4 °C for 40 min. The lipid solution was transferred to a 1.75 mL glass vial  C4F10 gas  was then bubbled through the lipid solution for 2 minutes using a pressure of 200 mbar. Immediately after bubbling, the polypropylene cap was replaced, secured with parafilm, then placed in a shaker  for 45 seconds. After MB production, size and concentration distribution were measured optically using bright-field microscopy and analysed using a custom MATLAB  script. MBs were typically produced at a concentration between 3–7 × 109 MB mL−1 with an average diameter between 1 and 2 μm. MBs were mixed with targeted-LS for a final MB : LS ratio of 109 : 1011 and allowed to bind for 20 minutes on ice prior to perfusion. This previous ratio of MB : LS : Neutravidin has been previously shown to produce MB–LS conjugates using fluorescent microscopy to observe the association of fluorescent LSs to the MB surface40 and has also been used to produce targeted-LS–MB complexes in related literature.51MBs were prepared from a mixture of DPPC and DPSE-Biotin-PEG2000 in a 95 : 5 M ratio and a total lipid concentration of 2 mg mLz-stacks using a 1.5 μm slice thickness.The inlet reservoir for perfusion was first filled with 2.2 mL of media and the corresponding outlet filled with 0.5 mL. The inlet and outlet reservoirs in the adjacent media channel were filled with 0.3 mL and 0.1 mL, respectively. This configuration maximised the interstitial pressure across the vasculature chamber, maximising the intramural flow through the network. Once the flow was initiated, 30 μL of LSs, MBs, or LS–MBs were deposited into the bottom of the inlet reservoir and allowed to perfuse through the network for 1 hour. Perfusion through the network was imaged using a Leica DMi8/SP8 confocal microscope, with the GFP and atto647N being excited with 488 nm and 638 nm diode lasers, respectively. The mark and find feature was used to periodically image the network in 5 minutes intervals at multiple  locations to observe LS, MB, and dextran perfusion across the network. Microfluidic devices were insonated once MBs or LS–MBs were observed to have populated the entire network. 1 hour after perfusion began, fresh media was added to the inlet reservoir and allowed to perfuse through the network for an additional hour. This rinsed all remaining LS solution from the network, ensuring that all of the remaining LS were those which had accumulated within the vasculature. Post-perfusion images were then taken using a 100× objective, which captured confocal via a 2 cm thick gel standoff pad  and coupling gel to ensure only far-field, uniform US waves reached the device. These US parameters have been previously used in on-chip MB-related experiments and have been shown to burst approximately 99.6% of MBs contained within a PDMS device with a 1 mm wall thickness.40 Characterisation of US attenuation through 1 mm of PDMS has been shown to reduce peak negative pressure to 0.81 ± 0.04 MPa, a 16% reduction from the 0.94 ± 0.01 MPa pressure measured through degassed ultrapure water. This resulted in a mechanical index (M.I) value of 0.54 ± 0.03.Microfluidic devices were insonated using a 2.25 MHz centre frequency unfocussed transducer with a 6 mm element diameter . Ultrasound pulses were controlled by a function generator  and consisted of a duty cycle of 1%, a pulse repetition frequency of 1 kHz for a total duration of 2 seconds. The free field transducer output was previously calibrated as described here and found to produce a peak negative pressure of 0.81 ± 0.04 MPa when driven by a +546 dB RF power amplifier . This produced a mechanical index (MI) of 0.54 ± 0.03. The transducer was coupled to the top of the microfluidic device at a 180° angle z-stack images of LS fluorescence were used to quantify the amount of accumulation within the vasculature networks. ImageJ particle analysis tool was used to identify LS and quantify their fluorescent intensity. Contrast enhancement was first performed on captured images to reduce any background fluorescence and ensure that LSs were correctly identified. Z-Stacks were duplicated, and the duplicates were binarised using the threshold tool. Thresholding values were determined using images of vasculature which had not been perfused with LSs. Measurements were set to the original stack, using the binarized stack as a mask for the original stack. The analyse particles tool was then used to quantify LS fluorescence. The values from each stack of images were averaged and plotted as a single data point on a boxplot graph. Imaging locations were kept relatively consistent across each microfluidic chip with the choice of locations spanning the area of vasculature network. Between 5–8 z-stacks were taken for each microfluidic chip used, depending on time constraints. Each set of accumulation data was capture from 3–7 separate microfluidic chips used across a minimum of 3 separate experiments. Accumulation data was then subject to a Mann–Whitney non-parametric statistical test which ranks data by value then determines the P value by the number of times a point from one dataset is higher than a point from another.Post-perfusion Rhyd is dictated by channel length, L, and the overall flow rate is dictated by the height difference, ΔH. Serpentine side channels were therefore used to increase the hydraulic resistance down the channel and decrease flow rates to physiological levels. Further information on the equations governing hydrostatic flow can be found in the supplementary material. The pressure distribution along each channel, shown in −1 depending on the relative difference in reservoir fluid levels which could range up to ΔH = 22 mmH2O. The asymmetric positioning of serpentines allowed for a pressure differential to be created across the centre chamber whilst maintaining equal rates of flow down each channel. Side channels were designed to produce flow rates ranging from 100–800 μm s−1, which is well within the range of flow rates reported to be optimal in inducing angiogenesis and anastomosis (0.1–6 μm s−1).61 From these observations, it was decided that a final fibrinogen working concentration of 7.5 mg mL−1 would be used to form the fibrin gels. This provided a sufficiently dense scaffold for cells to interact with whilst allowing for sufficient interstitial flow and nutrient exchange.The effect of this pressure differential was observed by measuring the interstitial flow across the channel by imaging the diffusion of Calcein across a blank fibrin gel. −1 fibrinogen) then perfusing and tracking the trajectory of 2 μm green, fluorescent beads down a side channel. 13, agreeing closely with the theoretical value of 2.6 × 1013. Theoretical flow rates were initially plotted using a capillary pressure, Pcap = 0, which fixes the intercept to the origin. However, the intercept from measured flow rates allowed Pcap to be calculated and found to be 23 ± 5 Pa. Theoretical flow rates were replotted using this value to demonstrate the agreement with measured flow rates. Overall, it was shown that physiological rates of flow could be provided using hydrostatic pressure and reservoirs could provide reasonable flow rates for up to 24 hours, at which point fluids were re-levelled and the initial flow rate restored.Flow through the side channels was characterised by first filling the centre chamber with a blank fibrin gel  × 106 μm2 – a 14% increase. The effects of TCM conditioning were further shown when vessel diameters were analysed which is shown in the histogram in 62 Vessels close to the tumour implant were found to have dilated, increased in tortuosity, and formed bulbous structures. Angiogenic sprouting from host vessels was also observed and resulted in remodelling of the host venules as the tumour increased in size. Tumour vessels were observed to reach diameters as large as 200 μm, consistent with maximum diameters observed throughout this system. The effect of tumours on vasculature morphology was also investigated by Yamaura and Sato, who observed a 3-fold increase in vascular surface area and a 5-fold increase in vascular length, per unit volume of tissue.63,64 Together, these observations support those made throughout this study, and further support the supposition that TCM can indirectly induce a similar tumour vasculature morphology to vasculature produced through direct contact with tumour cells.The morphological effects of [2 : 1] EGM : TCM conditioning were quantified by analysing networks using AngioToolPD calculated using a commonly used permeability model for vasculature networks.59,60 From this, PD was calculated to be (3.9 ± 0.6) × 10−7 cm s−1 showing good agreement with previous values throughout literature, which typically quote permeability values between (1.2–15.5) × 10−7 cm s−1.16,60,65 Observation of an effective endothelial barrier by measurement of dextran perfusion exclusively through the vasculature gave confidence in this system's ability to effectively recreate functional vasculature networks. Flow through networks was further characterised by tracking the perfusion of 2 μm green, fluorescent beads through the vasculature  through the network. This allowed for observation of flow through the network and subsequent calculation of the permeability coefficient using the rate of leakage through the vessel walls. Perfusion across a series of locations was imaged periodically for 45 minutes ESI Fig. S3 vβ3 expression is influenced by the presence of TCM. NHLFs and HUVECs were grown in an off-chip, 24-well plate configuration which closely recreates the on-chip seeding configuration of vasculature cultures. This seeding configuration is also based on a common 2D branching assay used to evaluate the angiogenic ability of endothelial cells. Culturing cells off-chip allowed for straightforward immunostaining, detachment, and flow cytometry analysis, compared to cells grown on-chip which were not readily retrievable from the microfluidic chamber.The pro-angiogenic effects of [2 : 1] EGM : TCM conditioning on network morphology has been observed in the previous on-chip vasculature experiments. To quantitatively assess the effect of TCM conditioning on HUVEC-NHLF fibrin cultures, immunostaining and flow cytometry was used to determine how integrin αvβ3 and analysed using flow cytometry. HUVECs and NHLFs could be distinguished from one another due to the GFP HUVEC emission. Immunofluorescent αvβ3 integrin intensity values for each cell type were plotted as histograms and a peak fitting function was used to find the modal emission intensity. This allowed for the expression of αvβ3 to be accurately determined for each EGM : TCM ratio.ESIvβ3 upregulation. 4) compared to (2.2 ± 0.05 × 104). This further increased to 430% when the highest TCM concentration was used, giving a peak intensity of (8.1 ± 0.2 × 104). In contrast, NHLF expression intensity was relatively unchanged in response to [2 : 1] and [1 : 1] EGM : TCM media, however, an increase of 75% (up to 3.5 ± 0.6 × 104) was observed with a [1 : 2] EGM : TCM media ratio. Calculation of the percentage of cells positive for αvβ3 expression  appeared to be expressed on the surface of endothelial cells (blue arrows) as well as by the supporting NHLF cells (white arrows), the crescent-shaped cells surrounding the vessels. This was consistent with flow cytometry observations along with previous observations throughout literature, which have observed integrin expression in supporting stromal cells due to their binding to similar ECM proteins as vascular endothelial cells.27,28Overall, flow cytometry results throughout this fibrin-containing angiogenesis assay suggest that the presence of TCM results in the upregulation of integrin αin vivo tumour vasculature. Experimentation, therefore, progressed to determining the efficacy of using integrin αvβ3-targeted LS to improve tumour vasculature targeting, and further investigate novel methods of delivering targeted LS to tumours using MB-mediated sonoporation. Results from this investigation would not only demonstrate the potential of integrin αvβ3 targeting and MB delivery but also demonstrate the effectiveness of this tumour vasculature model as a tool for evaluating potential treatments.Results thus far have demonstrated the ability of TCM-conditioning to influence vasculature to display tumour vasculature-like properties. Analysis of vessel morphology and integrin protein expression has shown that networks conditioned with TCM show characteristics that align with those observed in vβ3, through observation of LS accumulation in tumour vasculature networks. Observing an increase in LS accumulation in networks displaying tumour-vasculature-like properties compared to healthy vasculature would demonstrate the potential of αvβ3 as a target for tumour vasculature. 2 μg mL−1 and 4 μg mL−1 antibody concentrations were each tested to observe whether an increase in the targeting moiety increased accumulation – providing evidence that the inclusion of αvβ3 targeting was driving LS accumulation. Isotype control antibodies were also tested at each concentration to observe whether any increased accumulation was a result of non-specific antibody binding, as opposed to binding specifically to integrin αvβ3. Isotype antibodies therefore show if an increase in fluorescent signal is due to an increased targeting efficacy or merely an artifact from increased non-specific binding. Biotin -neutravidin binding was used to bind biotinylated αvβ3 antibodies onto the surface of red fluorescent (DOPE-Atto647N) LS containing Biotin-PEG2000, as described in section 2.4. This method of binding antibodies to the surface of MBs and LSs has been well documented and used similar ratios to many previous studies.36,40,54,67This series of experiments focused on evaluating the effectiveness of targeting LS to integrin αvβ3 is known to be expressed more highly on vessels undergoing higher rates of flow due to the increased intramural pressure created by the reduced vessel diameters.68 As described in section 2.5, LS were perfused for an hour before fresh media was added to the inlet reservoir. A further hour was then given for media to perfuse the network and any remaining unbound LS are washed away. Imaging was then performed across several regions of the chips, using a 100× objective and z-stacks to capture entire profiles of vessels. Accumulation was quantified using the particle analysis tool in ImageJ. The average intensity was calculated for each z-stack then plotted as a single data point in a boxplot. −1 and 4 μg mL−1 targeted LS through healthy networks, and 4 μg mL−1 targeted LS through TCM conditioned networks. The graphs alongside these images, shown in −1 αvβ3 antibody (T-LS 2 μg mL−1) resulted in an average accumulation intensity of 600 ± 70, showing a significant increase compared to the previous condition. LS were observed to accumulation diffusely throughout the vasculature, not appearing to bind to any regions specifically. Perfusions performed with LS conjugated with 2 μg mL−1 isotype antibody (IC-LS 2 μg mL−1), found a similar degree of accumulation (600 ± 120), suggesting the increase in adhesion is primarily due to the non-specific binding of antibodies to the vessel walls. Perfusion of LS targeted with 4 μg mL−1 αvβ3 antibodies (T-LS 4 μg mL−1) was previously observed to accumulate on the surface of the vasculature as perfusion occurred , LS with the same lipid composition but without any antibodies present, showed very little accumulation within the vasculature networks and resulted in an average fluorescent intensity of 180 ± 20. Targeted LS conjugated with 2 μg mLoccurred . This in−1 isotype control (IC-LS 4 μg mL−1) results which also showed an increase in accumulation compared to 2 μg mL−1 perfusions but did not result in regions of significant LS binding. This, therefore, suggests that several of the lower value intensities observed for T-LS (4 μg mL−1) perfusions were due to non-specific binding, whereas accumulations above 2000 were due to specific αvβ3 integrin binding. Performing a Mann–Whitney U-test revealed no statistical significance between 2 μg mL−1 isotype or targeted LS accumulations, whereas 4 μg mL−1 targeting showed ** (p < 0.01) significance when tested against the 4 μg mL−1 isotype control. As the accumulation results from perfusions using LS targeted with 2 μg mL−1 did not indicate that this concentration of antibody was having an impact on targeting, it was decided that experiments moving forwards would only use LS targeted with the higher 4 μg mL−1 αvβ3 antibody concentration. LS were now perfused through TCM-conditioned networks to observe whether upregulation of integrin αvβ3 resulted in increased rates of LS accumulation. Post perfusion imaging, performed after an hour of washing and shown in vβ3 in similar off-chip culture models, this suggests that integrin αvβ3 may be being expressed more consistently and homogeneously across [2 : 1] EGM : TCM networks. Together with the observations of TCM network morphologies, suggests that this may be a result of the network being induced to remain in an angiogenic state rather than stabilise into mature, healthy vessels. The disorganised, tortuous network structures produced through [2 : 1] EGM : TCM conditioning may also have influenced intensity values by causing irregular flow and presenting locations in which liposomes could readily accumulate. Together, the increase in median and maximum LS accumulation values with [2 : 1] EGM : TCM conditioned networks agrees well with flow cytometry observations which observed an increase in integrin αvβ3 expression intensity as well as an increase in the number of cells positive for integrin expression. Whilst a series of follow-up experiments using drug-loaded LS is required to further quantify the therapeutic effect of using integrin αvβ3 targeting, these results provide strong evidence that integrin αvβ3 targeting could facilitate the increased accumulation of LS in tumour-associated vasculature.These regions resulted in fluorescent intensity values of typically 2000 or above. Regions analysed below this value were found to show accumulations more consistent with non-specific antibody binding. This was reflected in the 4 μg mL−1 targeted LS were conjugated to the surface of MBs again using biotin–neutravidin binding and incorporating biotinylated DSPE-PEG-2000 lipids into MBs. This formed what is referred to as targeted LS–MB conjugates (T–LS–MB). Along with the complete exposure condition of T–LS–MBs perfused through TCM networks then burst with US, two further control conditions were tested. The first control looked to observe the impact of bursting the MBs with US by allowing T–LS–MBs to perfuse through the networks without the use of an US trigger. Differing LS accumulation intensities would then demonstrate the impact of MB-mediated sonoporation on LS deposition within the vasculature. The second control condition sought to observe the impact of using targeted conjugates by exposing healthy networks to the complete, T–LS–MBs and US exposure condition. As MBs are significantly larger than LS and their increased surface area will make them more susceptible to shear stresses from fluid flow. Increased adhesion will therefore be required for MBs to remain successfully bound to the vessel surface compared to LS. This may affect the previously observed targeting efficacy found when using 4 μg mL−1 integrin αvβ3 antibodies.To investigate the use of MBs as a potential method of improving the delivery of targeted LS to tumour vasculature, 4 μg mLPerfusion experiments were repeated in the same configuration previously used with US now being incorporated. Microfluidic chips were insonated after MBs were observed to fully populate the vasculature networks. vβ3 is being expressed regularly throughout the network, agreeing with previous TCM conditioning observations. However, the low-intensity values suggest that minimal numbers of LS were free to accumulate within the networks. This shows that a lack of US trigger prevents significant numbers of LS from being released from the MB surface. It is difficult to determine whether the use of 4 μg mL−1 targeting was sufficient to allow for MB adherence to the vessel surface as it is possible that some regions of LS accumulation are a result of MB accumulation and subsequent dissolution. However, the low intensities would suggest that targeting adhesion is insufficient in allowing significant numbers of MBs to persist within the network. Alternatively, low numbers of unbound LS in the MB solution may be resulting in the observed accumulation intensities, which would result in the frequent observation of low numbers of LS accumulated within the vasculature.Observation of LS accumulation after perfusion of T–LS–MBs in TCM networks without an US trigger found relatively few LS across the vessel surfaces. Regions of LS accumulation were commonly observed however the magnitude of LS accumulation in each region was much lower than previously observed. The high frequency of LS regions suggests that integrin α−1 targeted LS perfusions. Quantification of accumulations found an average intensity of 6800 ± 1200, a 3-fold increase in intensity compared to the same perfusion performed in healthy vasculature. This result provided compelling evidence that the inclusion of 4 μg mL−1 integrin αvβ3 antibodies successfully facilitated the targeting of MB-LS conjugates and allowed for significantly improved LS delivery when MBs were burst with US. Intensity values for this complete exposure condition were found to consistently exceed control values, resulting in a high *** (p > 0.001) statistical significance value when subject to a non-parametric Mann–Whitney U-test. Images taken after US exposure were unable to observe any immediate effects of MB sonoporation on endothelial integrity using this current imaging and experimental setup. The use of membrane permeability probes such as propidium iodide would improve the ability to identify any sonoporation effects on endothelial cell membranes. Observation of the opening of gap at endothelial cell–cell contacts was not observed in this study in contrast to observations made throughout previous studies.36,41 However, the imaging modalities and timescales used throughout this study differ significantly from those used previously. A worthwhile follow-up experiment could employ the use of a cell detachment agent, such as EDTA, to induce cell detachment and contact opening to serve as a positive control.Perfusion and subsequent bursting of T–LS–MB conjugates in healthy vasculature networks were found to result in significantly increased numbers of LS compared to the previous control condition. LS were found to be distributed across many regions of the vasculature surface, resulting in an average fluorescent intensity value of 2200 ± 400. Despite these perfusions being performed in healthy networks, this intensity value was higher than the 1400 ± 200 value found after perfusions through [2 : 1] EGM : TCM networks without an US trigger. This demonstrates the impact of bursting MBs on the extent of LS accumulation. Finally, exposing networks to T–LS–MBs in TCM networks with an US trigger was observed to significantly increase LS accumulation. Post perfusion imaging observed large swathes of LS across several points on the vessel surface, consistent with observations made throughout 4 μg mLin vivo studies have observed the opening of the blood brain barrier (BBB) with the use of MBs and focused US, suggesting the integrity of the endothelium is affected significantly by sonoporation.69,70 A study by Tung et al. observed that the BBB was only opened by non-linear MB oscillation when the MB diameter was similar to the capillary diameter but in all instances of inertial cavitation.70 As the vessel diameters in the vasculature networks in this study were more comparable to precapillary vessels (>25 μm) than capillaries (<10 μm), this may explain the lack of MB impact on cell–cell junction integrity. Continued vasculature integrity after US and MB exposures suggested that any events resulting in irreversible sonoporation were not sufficient enough to compromise precapillary vessel function. However, whilst the overall vascular integrity appeared unaffected by MBs and US, the transient opening of cell–cell contacts by MB sonoporation was not investigated. A potential mechanism for the increased accumulation of LS conjugated to MB is the sonoprinting of LS onto the surface of the endothelium. De Cock et al. and Roovers et al. first described the process of sonoprinting as a mechanism by which nanoparticle-loaded MB facilitate the deposition of nanoparticles onto 2D cell monolayers and tumour spheroids.71,72 In spheroids, this allowed for the increased release of drugs from nanoparticles attached to the outer layers of the spheroids. These observations are also consistent with previous observations by Bourn et al., which observed the increased deposition of doxorubicin-loaded LS when conjugated to MBs – increased drug accumulation and decreased spheroid viability was observed as a result.40 Several follow up experiments could be performed to further investigate the MB-related phenomena observed throughout this study and previous studies. It is suggested that very similar experiments previously conducted on simplistic 2D systems could be repeated using this system to observe the impact of improving the physiological relevance of the system. Propidium iodide, dead cell staining, and calcium fluctuations could all be implemented to probe the effects of MBs on cell–cell contacts, vessel permeability and overall endothelial integrity.73It is also suggested that the lack of gap formation may be a consequence of using a physiologically accurate vasculature model which is complete with supporting ECM and stromal components. Observations of junction opening are often made in systems comprising of endothelial cells seeded onto polycarbonate or PDMS surface, which minimally supports the adhesion of cells compared to a functional basement membrane layer. In contrast to this, many Whilst the use of indirect tumour conditioning in this study prevented the direct evaluation of tumour cell viability, increased accumulation, and uptake of therapeutics in the vasculature can still be used as an indicator of improved drug delivery. The use of TCM conditioning also enabled the routine production of tumour-associated vasculature compared to previous tumour vasculature systems which have relied on cancer cell-vasculature co-cultures, demonstrating the advantages of this relatively simplistic system.vβ3 expression revealed properties consistent with that observed in in vivo tumour vasculature. The microfluidic system developed to culture vasculature allowed for perfusion of model therapeutics exclusively through the vessels, with rates of flow and shear stress similar to those found within capillaries. Targeting LS with 2 μg mL−1 integrin αvβ3 antibodies was found to result in similar rates of accumulation compared to isotype controls of the same concentration, suggesting this concentration was not significant enough to facilitate specific targeting. Whereas 4 μg mL−1 targeted-LS were found to result in increased rates of LS accumulation, revealing a 3-fold increase compared to 4 μg mL−1 isotype controls. Perfusion of 4 μg mL−1 targeted-LS through [2 : 1] EGM : TCM networks observed a further 60% increase in accumulation intensities compared to healthy networks. Together with the observation of TCM-induced integrin αvβ3 upregulation in off-chip vasculature cultures, provides evidence that TCM induced αvβ3 upregulation facilitates the uptake of targeted LS in microfluidic vasculature networks. The destruction of T-MB-LS within the vasculature was observed to significantly increase LS accumulation in TCM networks compared to healthy networks, finding a 3-fold increase. Overall, these results indicated that the delivery of drug-loaded LS to tumours could be enhanced through the combinatory use of integrin αvβ3 targeting and MB-mediated LS delivery. Whilst initial results appear promising, several additional experiments are still required to further elucidate the efficacy of integrin targeting and MB-mediated drug delivery.This study presents a novel method of producing perfusable vasculature cultures which display tumour-associated properties. Characterisation of vessel area, diameter, and integrin αTogether, experiments throughout this study have demonstrated the ability of this microfluidic vasculature system to evaluate potential anti-cancer therapeutics.https://doi.org/10.5518/1092. The processed data required to reproduce these findings are available to download from https://doi.org/10.5518/1092.The raw data required to reproduce these findings are available to download from There are no conflicts of interest to declare.LC-023-D2LC00963C-s001


In situ Raman spectra reveal strong molecular carboxyl-metal cation coordination at the negatively charged electrode surface, hindering the formation of molecular junctions for electron tunnelling. This work validates the critical role of localized cations in the electric double layer to regulate electron transport at the single-molecule level.The nature of molecule-electrode interface is critical for the integration of atomically precise molecules as functional components into circuits. Herein, we demonstrate that the electric field localized metal cations in outer Helmholtz plane can modulate interfacial Au-carboxyl contacts, realizing a reversible single-molecule switch. STM break junction and I-V measurements show the electrochemical gating of aliphatic and aromatic carboxylic acids have a conductance ON/OFF behavior in electrolyte solution containing metal cations (i.e., Na A common approach to design single-molecule switch is to use molecular backbones in response to external stimulus, but often requires complex organic synthesis. Here, Tong et al. show how to in situ control of the molecule-electrode contact using electrochemical gating to realize a reversible switch. The construction of a reliable molecular switch is an important step because of its key role in information storage, logical data manipulation, and signal processing4. Over past three decades, a common approach to design single-molecule switch is to use a molecular backbone that can change the molecular conformations or spin/redox states upon exposing to external stimulus, such as light6, mechanics8, pH10, chemical reactants11, magnetism15, and electricity22. However, this often requires complex organic synthesis to obtain a stimuli-responsive molecular scaffold that can switch the tunnel junctions between two stable conductance states. Most of the external stimulus methods cannot be used in applications that require all-electric drive circuit components23. Alternatively, interfacial molecule–metal contacts can also significantly determine electron transport through the molecular junctions27. To date, the pre-setting anchoring groups in molecular backbones has been the typical way to manipulate the molecule–electrode interaction and electron transport. However, in situ control of the molecule–electrode contacts toward electrical functional components in the same molecular backbone remains technically challenging.With the increasing demand for the miniaturization of electronic devices, the use of chemically identical nanometer-sized molecules as functional components has become a thriving subfield of nanoscience29. So far, the research reports with electrochemical gating have mainly focused on regulating electron transport by altering the energy alignment between the Fermi level of electrodes and molecules29, ignoring the potential-dependent structure of electric double layer might change the molecule–electrode contacts. This arises from the most used molecular anchoring groups, such as thiols, can be covalently bonded to electrodes with little effect on the electrochemical potentials. The carboxylic acid group is one of the most used anchoring groups to fabricate molecular junctions32. Recently, break-junction measurements have proven that the formation of carboxyl-linked molecular junctions relies on the interaction through deprotonated −COO− groups33. In addition, it is reported that the deprotonated −COO− groups can coordinate with the alkali metal ions and transition metal ions35. Meanwhile, electrochemical potentials can effectively change the charge states of the electrode surface to tune the distribution of ions in the electric double layer at the interfaces, which in turn affect the interfacial dissociation equilibrium of −COOH ⇌ H+ + −COO− and carboxyl-metal cation interactions. This may provide a unique opportunity to realize reversibly controlled molecule–metal contacts for tuning electron transport, but has not been explored and reported so far.Electrochemical gating is one of the most feasible and effective methods to tune interfacial electronic structures, which has been widely applied in studying electron transport across single molecules as a function of electrode potential in different electrolyte solutions+, K+, Mg2+, and Ca2+), single-molecule electrical measurements shows the conductive states of aliphatic and aromatic carboxylic acids molecular junctions transform from a detectable state (ON state) at 0 V to below the minimum detection limit of the currently used amplifier range (OFF state) at −0.5 V vs. Pt. Cyclically switching the applied potentials in both STM-BJ and I–V tests leads to the conductance ON/OFF ratio of 4-(methylthio) benzoic acid (4-MTBA), 3-(methylsulfanyl) propanoic acid (MPA) and terephthalic acid (TPA), compared to almost no conductance change without the metal cations in solutions. In conjunction with Raman spectra and DFT simulations, it is found that the adsorbed molecules change from carboxyl-metal cation coordination, protonation to deprotonation, as the applied potential changes the electrode surface from negatively to positively charged. Therefore, a reversible single-molecule switch could be realized in the carboxylic acid-linked junctions through electrochemical control of the localized ions.To test the above-mentioned hypothesis, herein, we have employed in situ STM break junction (STM-BJ) and Raman spectroscopy to probe the electrochemical potential-dependent molecular conductance and structures of carboxylic acids molecules with methyl sulfide (SMe) adsorbed on Au(111) electrode. In the electrolyte solution containing metal cations  can coordinate with the −COO− groups at negatively charged Au(111) electrode. These might hinder the formation of Au–COO− contacts when an Au STM tip is driven into and suspended above SAMs. In contrast, at the positively charged Au(111) surface, the local hydroxide anions can deprotonate the SAMs, and the electrostatic repulsion can disrupt the carboxyl-metal cation coordination, leaving abundant of −COO− groups that could bind to Au tip to form molecular junctions for electron transport.The main idea of our work is to exploit the electric field-localized cations in outer Helmholtz plane (OHP) to control the forms of carboxylic acid molecules  assembled on Au(111) substrate, which can significantly affect the Au–carboxyl contacts with the Au STM tip during the formation of molecular junctions. By controlling the electrochemical potential with a potentiostat, the electrode surface can be easily charged positively or negatively. Then the anions or cations in the solution can be concentrated in OHP as counter ions, compensating the negatively or positively charged electrode surface according to Gouy–Chapman–Stern model. As schematically illustrated in Fig. Eads = −1.23 eV) is significantly weaker than that of deprotonated 4-MTBA with COO− (Eads = −2.66 eV). Similarly, the deprotonated 4-MTBA adsorb on the 25H2O/Au(111) through −COO− group, whose two O atoms strongly bind to surface Au. When adding Na+, the hydrated metal cation makes −COO− more than 4 Å from the Au surface on the Na-25H2O/Au(111), resulting in weaker adsorption. These support the feasibility of controlling Au–carboxyl contact by local cations.To substantiate this hypothesis, we first performed density functional theory (DFT) and ab initio molecular dynamics (AIMD) calculations to analyze the interaction between carboxylic acid molecules and Au electrode in different configurations using the Vienna Ab Initio Simulation Package (VASP) software  electrode in the same solution37. As adding 0.1 mM 4-MTBA in the solution, the PZC negatively shifts by about 10 mV because of the molecular adsorption. When changing the electrolyte to HClO4 or Ca(ClO4)2, a similar PZC could be found  by immersing a dry and clean Clavilier-type Au(111) half-bead single-crystal electrode in electrolyte solution with potential control following the reported papers4, which is higher than that of PZC, indicating a positively charged surface. Figure 0G0  is assigned to the formation of Au atomic contacts during the stretching process of the tip39. As the tip further stretches, there is another step feature appeared at 10−3.0G0 after rupturing Au atomic contacts (red curves). This corresponds to the formation of molecular junctions of 4-MTBA at the positively charged Au surface. Thousands of these conductance-displacement traces are collected to construct 1D conductance histograms, showing an obvious conductance peak at around at 10−3.0G0 in the one-dimensional (1D) conductance histograms in Fig. −6.0 to 10−0.3G0 show an obvious stretching state centered around 10−3.0G0 after rupturing Au atomic contacts in the two-dimensional (2D) conductance histogram  distribution with a Gaussian-fitted peak at 0.4 nm  electrodes was firstly set at 0 V vs. Pt in 0.1 mM 4-MTBA + 50 mM NaClOram Fig. , the ste−3.0G0 in Fig. +, Mg2+, and Ca2+; While in the solution with non-metal cations , these conductance peaks can still be observed in Fig. 4Cl solution might arise from the strong specific adsorption of halide ions and hydrolysis reaction  substrate to negatively charge the surface, interestingly, these step features and conductance peaks appeared at 1011 substr4 aqueous solution is firstly examined by using cyclic voltammograms (CVs). As shown in Fig. 41. A linear correlation between anodic (red square) and cathodic (blue square) current peaks with the scan rates is found in the insert. This proves that the reversible peaks arise from the interfacial 4-MTBA assembled on the Au(111). In addition, such reversible current peaks can also be observed in the CVs using SCE and Pt as the reference electrodes in 50 mM NaClO4 and 50 mM HClO4 solutions  substrate. A Clavilier-type Au(111) half-bead single-crystal electrode immersed in 0.1 mM 4-MTBA + 50 mM NaClO−3.0G0 appears gradually and becomes intense as the potential increases. Such potential-dependent characteristics of conductance peaks can also be observed in the solutions containing other metal cations of K+, Mg2+, Ca2+ , the applied potentials and cations have little effect on the conductance peaks . As schematically illustrated in Supplementary Fig. 2 shell are dropped on Au(111) to work as the Raman-signal amplifiers. Three-dimensional finite-difference time-domain simulations revealed that the electromagnetic field strength in the gap between NPs and Au (111) surface could be enhanced up to 7–8 orders of magnitude under 633 nm laser illumination for detecting the surface species42. In addition, the chemically inert silica shells can protect the Au core from the analytes and environments to avoid generating interference Raman signal from the Au core.To verify the mechanism of local cations on Au–carboxyl contacts, shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS)4 with 0.1 V intervals. The 600–1800 cm−1 spectral range contains all the most significant peaks of 4-MTBA ), consistent with previous reports46. As potentials increase, it becomes weak, while two bands at 845 and 1365 cm−1 ascribed to COO− bending (β(COO−)) and symmetric stretching (νs(COO−)), which appear and become intense above −0.2 V. These indicate the molecular -COOH groups dissociates into −COO−. When changing the electrolytes to NaClO4, interestingly, an obvious Raman band at 1387 cm−1 can be observed at −0.5 V in Fig. 23Na NMR experiments show that Na+ ions presented near the surface play an important role in stabilizing the high coverage of the carboxylate on the Au surface34. In addition, the calculated Raman spectra of 4-MTBA-hydrated Na+ coordination complex also present an obvious band at 1396 cm−1 related to the C = O symmetric stretching (νs(COO−Na+)) in Supplementary Fig. −1 is ascribed to the νs(COO−Na+), consistent with reported carboxyl-metal cation complex systems45. This proves that the local Na+ cations can strongly coordinate with the -COO− groups at negatively charged Au(111) surface. Similarly, a much intense Raman band ascribed to carboxyl-Ca2+ symmetric stretching (νs(COO−Ca2+)) blue shift to 1397 cm−1, and the Raman band of ν(COOH) disappears at −0.5 V in Fig. 2+ with −COO− groups. The same variation characteristics of spectral peaks can also be confirmed in the calculated spectra in Supplementary Fig. Figure νs(COO−Na+) and νs(COO−Ca2+) decrease to disappear. Meanwhile, the νs(COO−) band rises and becomes intense; While the Raman intensity of ν(COOH) firstly increases and reach maximum around PZC, then decreases both in Fig. νs(COO−Ca2+), νs(COO−) and the conductance peak intensities against the potentials of Au(111) substrate are shown in Fig. −. Compared to in HClO4 solution, the local metal cations can displace the proton in the carboxyl group to form the ligand complex at the very negatively charged surface, which inhibits the Au–COO− contacts to form molecular junctions.As the potential of the electrode increases, the Raman intensities of −3.0G0 repeatably appears at 0 V, but disappears at −0.5 V in the conductance histograms. Furthermore, other aliphatic and aromatic carboxylic acids, 3-(methylsulfanyl)propanoic acid (MPA) and terephthalic acid (TPA), also shows the same interesting switching phenomena upon changing the potentials from −0.5 V to 0 V  was also used for the break-junction experiments, there is also no conductance peak observed at −0.5 V  substrate are swept between 0 and −0.5 V. Figure I–V curve recorded upon the formation of molecular junctions. Obviously, the current jumps to a low value or a high value during a negative or positive potential sweep, respectively. These can be attributed to the breakdown and formation of molecular junctions. The relative current difference is about 3.83 nA, comparable to single-molecule conductance in the break-junction measurements. When the gate potential is lower than −0.41 V, tip current is turned off for all I–V parallel tests  surface, local concentrated hydrated metal cations  in outer Helmholtz plane (OHP) can coordinate with the -COO− groups of the self-assembled monolayers (SAMs). This inhibits the formation of Au–COO− contacts for electron transport when an Au STM tip is driven into or suspended above SAMs (OFF state); At positively charged Au(111) surface, the electrostatic repulsion can disrupt the carboxyl-metal cation coordination, and local hydroxide anions can deprotonate the SAMs, leaving abundant of -COO− groups that can bind to Au tip to form molecular junction for electron transport (ON state). The present work offers a detailed molecular picture of the effect of localized cations on carboxyl molecules at the electrochemical interfaces, paving a new avenue for realizing reversible single-molecule switches via gate electrodes.In summary, a local-cation-controlled reversible single-molecule switch has been successfully demonstrated in carboxylic acid molecular junctions with electrochemical STM-BJ, 4 solution.All electrochemical measurements were carried out at a CHI-660E potentionstat using a homemade three-compartment glass cell with Pt as reference electrode and counter electrode. A Clavilier-type Au(111) half-bead single-crystal electrode was used as the work electrode. The solutions in electrochemical measurements were deaerated and protected by argon. The open circuit potential between the Pt wire and SCE is 0.48 V in 0.1 mM 4-MTBA + 50 mM NaClOConductance measurement by using STM break-junction (STM-BJ) technique was performed on the home-modified Nanoscope IIIa STM . In a four-electrode electrochemical system, the same Pt ring and Pt wire were used as counter electrode and reference electrode in all electrochemical STM-BJ experiments, respectively. The naturally formed (111) facet at an Au bead substrate and mechanically cut Au STM tip  were used as work electrodes. To minimize the ionic leakage current in electrochemical environments, Au tip is coated with thermoset polyethylene glue with a little exposed tip apex. Prior to each experiment, the substrate was electrochemical-polishing and flame-annealing. The single-molecule conductance measurements were performed in a solution containing 0.1 mM 4-MTBA + 50 mM electrolytes. The molecules can adsorb on Au(111) through the anchoring groups of methyl sulfide (SMe) to form a self-assembled monolayer.The procedure of the STM-BJ method is briefly described as follows: Firstly, the STM tip is driven toward the substrate to a preset current value (50 nA) via piezoelectric control. Then an external pulse voltage is applied on z-piezo to bring STM tip into the substrate surface to ensure tip contact with the substrate. Then the tip is pulled away from the substrate at a constant speed of 20 nm/s. During the process, molecular junctions can be formed. Meanwhile, the current of the tip is recorded at a sampling rate of 20 kHz. Thousands of tip current-displacement curves are collected to construct the conductance histogram without data selection. The conductance measurement is carried out at a bias voltage of 50 mV.I–t tests with STM feedback loop turned off is briefly described as follows: First, the STM tip is stabilized at a fixed distance set according to the tunneling parameters (It = 0.8 nA and Vbias = 50 mV). Then we turn off the STM feedback loop and record the tip current for 20 s. When no molecule bridges the two electrodes, there is only the base current. Due to thermal, mechanical drift or molecular motions, the tip can contact the molecules. Once a molecular junction is formed, the current suddenly increases, which means that one or more molecules bridge the two electrodes. The I–t curves are recorded with STM feedback loop of 0 at the substrate potentials of 0 and −0.5 V.The procedure of the I–V measurements is described as follows: Firstly, the STM tip is driven toward the substrate to a preset current value (50 nA) via piezoelectric control. Then we turn off the STM feedback loop, an external pulse voltage is applied on z-piezo to bring STM tip 1 nm into the substrate surface to ensure tip contact with the substrate. Next, the tip is withdrawn until the tip current value reaches the single-molecule conductance value . When the single-molecule conductance is detected, the tip will be fixed over the substrate. The tip current is recorded during the substrate potential is swept from 0 to −0.5 V.The procedure of 2 nanoparticles synthesized following previous reports55, dropped on the Au(111) electrode as Raman-signal amplifiers.Raman experiments were carried out on a confocal microscope Raman system (Renishaw InVia). The excitation wavelength was 632.8 nm, and a ×50 microscope objective with a numerical aperture of 0.55 was used in all Raman measurements. In situ electrochemical Raman experiments were carried out in a homemade Raman cell with potential control at an CHI-660E potentiostat. The 120 nm Au @ ca. 2 nm SiO57 software based on the density function theory (DFT). The effects between electron exchange and correlation were described by the Perdew–Burke–Ernzerhof (PBE) functional, which was one of the most well-established generalized gradient approximation (GGA)58. The interactions between electrons and ions were represented by projected augmented wave (PAW)60 potentials with a cutoff energy of 450 eV. The Brillouin-zone integration61 was approximated by 1 × 2 × 1 k-point sampling grid. The convergence criterion for the energy calculation was set to 1.0 × 10−4 eV, while the force tolerance of structure optimization was set to 0.01 eV Å−1. The van der Waals interaction was described by DFT-D3 method62. To verify the impact of the model, a series of Au models were constructed by DFT calculation. A lattice cell of a three-layer 2 × 2 Au(111) surface and a three-layer 4 × 2 Au(111) surface with more than 15 Å vacuum layer were applied to prove the tiny influence of model size on the trend of adsorption energy. In order to approach the experimental environments, another three-layer 2 × 2 Au(111) surface with a thinner vacuum layer was constructed to simulate the presence of gold electrodes at both terminals. The results showed that the thickness of the vacuum layer also has barely effect on the trend of adsorption energy for this system. Furthermore, the three-layer (2 × 2) Au(111) surface under 25 H2O molecules environment with or without Na+ were used to optimize the adsorption structure of 4-MTBA molecule by AIMD simulations. The AIMD simulations were performed in the canonical ensemble (NVT)64 with the Nose-Hoover thermostat at 300 K for a time period of 5 ps with a time step of 0.5 fs. Then the 4-MTBA was removed from the equilibrated 4-MTBA/aqueous phase/Au(111) system for constructing aqueous phase/Au(111). And a standard DFT calculation was used to optimize the 4-MTBA/aqueous phase/Au(111) and aqueous phase/Au(111) system.All the theoretical calculations were performed by Vienna Ab Initio Simulation Package (VASP)Supplementary InformationPeer Review File


Solanum tuberosum L.) suffers from ID, some argue that the potential genetic gains from using inbred lines in a sexual propagation system of potato are too large to be ignored. The aim of this research was to assess the effects of inbreeding on potato offspring performance under a high latitude and the accuracy of the genomic prediction of breeding values (GEBVs) for further use in selection. Four inbred (S1) and two hybrid (F1) offspring and their parents (S0) were used in the experiment, with a field layout of an augmented design with the four S0 replicated in nine incomplete blocks comprising 100, four-plant plots at Umeå (63°49′30″ N 20°15′50″ E), Sweden. S0 was significantly (p < 0.01) better than both S1 and F1 offspring for tuber weight , tuber shape and size uniformity, tuber eye depth and reducing sugars in the tuber flesh, while F1 was significantly (p < 0.01) better than S1 for all tuber weight and uniformity traits. Some F1 hybrid offspring (15–19%) had better total tuber yield than the best-performing parent. The GEBV accuracy ranged from −0.3928 to 0.4436. Overall, tuber shape uniformity had the highest GEBV accuracy, while tuber weight traits exhibited the lowest accuracy. The F1 full sib’s GEBV accuracy was higher, on average, than that of S1. Genomic prediction may facilitate eliminating undesired inbred or hybrid offspring for further use in the genetic betterment of potato.Inbreeding depression (ID) is caused by increased homozygosity in the offspring after selfing. Although the self-compatible, highly heterozygous, tetrasomic polyploid potato ( Solanum tuberosum L.) is a tetrasomic polyploid (2n = 4× = 48 chromosomes) that accumulated during its history of cultivation many deleterious mutations in its genome, which leads to inbreeding depression in the offspring ensuing from self-fertilization; inbreeding depression may also arise from a reduction in intralocus interactions after selfing [n = 2× = 24 chromosomes) potatoes, these mutations, which depend on the cultivar, are often found in the pericentric regions, and most of the deleterious recessive alleles that affect plant survival and vigor are in chromosome regions with high recombination rates. Hence, crossing should be able to purge damaging, large-effect deleterious recessive mutations.Species having three or more chromosome sets show polyploidy. Polysomic polyploid species arose by the multiplication of chromosome sets from a single or related species . A self- selfing . CrossbrMedicago sativa L.). It has been argued that this rapid vigor loss results from selfing an alfalfa accession or cultivar with a high frequency of loci bearing three or four distinct alleles [Inbreeding depression reduces fitness because of a likely increase in deleterious homozygous alleles in the offspring after selfing. Inbreeding may lead to fixing favorable alleles to increase overall genetic value, but inbreeding due to drift might increase the frequency of homozygosity for unfavorable alleles and the loss of heterozygosity, thus leading to inbreeding depression . Inbreed alleles .The coefficient of co-ancestry measures the degree of relationship between individuals, while the coefficient of inbreeding tells the probability that two alleles at any locus are identical by descent. Co-ancestry analysis facilitates, therefore, the assessment of relationships among individuals and estimates—through its coefficient—the states of loci in their gametes . MalecotInbreeding affects various characteristics in a heterozygous outbreeding crop such as tetrasomic potato, but its effects are complex. Potato tuber yield and seed setting suffer significantly from inbreeding depression . In this1 hybrid diploid offspring may result from crossing inbred pure lines following a genome design. In this approach, the decision making during the inbred line development and crossing them to obtain F1 hybrids was based on genome analyses.Hybrid vigor or heterosis refers to the greater growth, survival, fertility or yield in the hybrid offspring than in their parents . MendozaGenetic diversity assessments provide insights about the changes in the cultigen pool due to crop improvement. Hirsh et al.  demonstrManihot esculentus). According to their simulation, although selection increases the frequency of favorable alleles, augmented inbreeding along selection reduces under directional dominance the gain in genotypic values. Purging selection among inbred offspring appears to be only effective in early inbreeding cycles (S1 or S2) but not in subsequent inbreeding cycles (S3 onward) due to the decreasing relatedness of the training set from selection candidates, thus lowering the accuracy (r) of the genomic prediction of breeding values.Removing deleterious alleles (thus reducing the genetic load) may be a target in vegetatively propagated crops, which often show limited recombination in their genome. For example, Momo and Jannink  used a n0 (cultivars used as parents), S1 (inbreeding of S0) and F1 (crossing two S0) generations and to assess if genomic prediction accuracy is affected by inbreeding in full-sib S1 and F1 offspring.The aims of this research were to determine the inbreeding effect in potato tuber yield and other tuber characteristics under a cool, very long daylength by comparing S1 and S1 seed, respectively, at SLU greenhouse in Alnarp (southern Sweden). The cultivars ‘Colleen’, ‘Melody’, ‘Queen Anne’ and ‘Rudolph’ produced S1 seed, while there were 28 F1 seed, of which 20 were derived from crossing with any of the above four cultivars. Two of these F1 were derived from ‘Queen Anne’ × ‘Colleen’ and ‘Queen Anne’ × ‘Melody’.The crossing block 2020 of the potato breeding program (Svensk potatisförädling) of the Swedish University of Agricultural Sciences (SLU) included 10 released cultivars and 6 breeding clones that set seeds after crossing by hand. Plants from each cultivar and breeding clone were used for hand-crossing with unrelated pollen or for self-fertilization to obtain F0) producing S1 seed, their S1 generation and the two F1 hybrids noted above among themselves. The field layout was an augmented design [0 replicated in 4-plant plots as cultivar checks in the nine incomplete blocks. Each of the nine incomplete blocks also included 100 plots (consisting of four plants) of the S1 and F1 clones. The spacing was 0.7 m between rows and 0.3 m spacing among plants within the plot. The tubers used for planting ‘Rudolph’ did not sprout well, and this cultivar was not further included for data analysis because of the number of uneven lost plants in each of the nine blocks.The field trial at Umeå  included the four cultivars  was used for characterizing S0, S1 and F1 with about 2000 SNPs, previously used in genomic prediction of breeding values for cultivars released in western Europe along with Svensk potatisförädling clones [Ten tuber characteristics were evaluated in the field trials, including tuber weight , uniformity of both tuber size and shape, tuber eye depth and tuber flesh reducing sugar, which was measured using potato glucose strips . Tuber ug clones . These S0, S1 and F1 sufficed for obtaining GEBVs without losing information [The number of SNPs used for genotyping Sormation ,29, thouith genotype in the jth block, CF was calculated asr is the number of blocks . In the analysis of variance of the augmented design, the degrees of freedom for the error should be above 10 following this relationship [0 vs. S1) and average heterosis (S0 vs. F1) as defined by Gardner and Eberhart [1 vs. F1).The analysis of variance of the field trial used the following equation:tionship :r>[10c−1Eberhart  and for Genomic predictions and the estimated breeding values (GEBVs) were obtained by fitting the GBLUP model:r et al.  for the r et al. . FinallyThe prediction ability of the GBLUP model was studied by means of a cross-validation for each trait. Fifty random partitions were generated, 70% of the observations were assigned to the training set and the remaining 30% to the testing set, and Pearson’s correlation coefficient between observed and predicted phenotypes was obtained. Computations were performed using the BGLR package  in R 3535 using 1 and 4 S1 offspring. The berry set was 79%, 82%, 86% and 100% for cultivars ‘Rudolph’, ‘Colleen’, ‘Queen Anne’ and ‘Melody’, respectively. The seed set after the self-fertilization of four cultivars was 113 ± 42, which was lower than the seed set in the F1 hybrid offspring (149 ± 29). This result suggested that inbreeding influenced the seed set but not the crossing outcome.The 2020 crossing block in Alnarp used 434 of their flowers to obtain 170 berries (ca. 39% crossing success) of 28 unique Fp < 0.05) for all tuber characteristics evaluated  than the inbred offspring (S1). ‘Colleen’, ‘Queen Anne’ and ‘Rudolph’ are early-maturity cultivars, while the high-yielding ‘Melody’ is a mid-season cultivar. The average total tuber weight of S1 was a quarter less than that of S0. This result indicated significant inbreeding depression for this important productivity characteristic of potato. The two F1 were on average significantly lower (p < 0.001) than the parents, which suggests a lack of average heterosis in the hybrid offspring. Nonetheless, there were some F1 offspring (15–19%) whose total tuber yield was in each cross above that of S0, thus revealing transgressive segregation for tuber yield in the heterogeneous F1. The average total tuber yield of F1 was significantly (p < 0.001) above that of S1. S0 had the highest weight among the largest tubers and S1 the lowest in a very long day site (about 14.5–ca. 21 h during the cropping season). This result confirmed that inbreeding also affects producing large tubers in potato.There were significant differences . However, tuber size uniformity and tuber eye depth were significantly (p < 0.001) affected by inbreeding  lower for tuber size uniformity and had deeper eyes in the tuber than S0. On average, there were non-significant differences (p > 0.05) for tuber eye depth between S1 and F1. The lowest reducing sugars in the tuber flesh were noted in S0, which was significantly lower (p < 0.01) than in both S1 and F1 which did not differ (p > 0.05) for this characteristic.Tuber shape uniformity was not affected by inbreeding, as indicated by the non-significant Scontrast . The avebreeding , as well1 offspring of ‘Colleen’ or ‘Queen Anne’ were the training populations for ‘Queen Anne’ × ‘Colleen’ and ‘Queen Anne’ × ‘Melody’ F1s  for total tuber yield when a non-related population was used for training the GEBV model for either S1 or F1. The exception was when using Melody S1 as training population for predicting total tuber yield in ‘Queen Anne’ × ‘Colleen’. The largest ρ estimates  for tuber size uniformity and tuber eye depth were for ‘Queen Anne’ × ‘Colleen’ F1 and ‘Colleen’ S1, as well as when using ‘Queen Anne’ S1 to predict GEBV for tuber size uniformity in related half-sib ‘Queen Anne’ × ‘Colleen’ F1. The best ρ estimates (>0.20) for reducing sugars in the tuber flesh were in ‘Queen Anne’ and ‘Rudolph’ S1 as well as for ‘Queen Anne’ and ‘Colleen’ S1s predicting the GEBV for this characteristic in their half-sib ‘Queen Anne’ × ‘Colleen’, which had a negative ρ estimate when using F1 itself for determining GEBV.ody’ F1s . ‘Rudolpstimates  were forTo the best of our knowledge, the novelty of this research includes bringing for the first time a segregating breeding population of tetrasomic polyploid potato to a latitude approaching the Artic Circle (66°14′ N for Norrbotten County in Sweden). Previously, advanced breeding clones, along with released cultivars, were included in field trials at Umeå . One of the major concerns for growing potato in such high Nordic latitudes is the adaptation to the short cropping season (about 90 days) under long daylength (14.5–21 h), with relatively low temperature (12.9–16 °C) and mean monthly sunshine hours ranging from 221 to 287. Tuberization, affected by daylength, early maturity and plant growth, as measured by its canopy and vine senescence, is a complex quantitative trait and its multi-genic nature  under short days in Peru. It is worth highlighting that ID was only noted in six (out of ten) Andean cultivars [S. tuberosum [Inbreeding depression significantly affects potato productivity in high Nordic latitudes . This reuberosum  grown unuberosum ,41 in Anultivars  and one uberosum  found thuberosum  advocateuberosum , high-yi1 offspring and the fact that a few breeding clones had tuber yield above that of the highest-yielding parent suggests significant within-family variation for this characteristic. This variation could result from highly heterozygous, diverse parents, which calls for rethinking the concept of specific combining ability (SCA) as a specific individual hybrid combination within the family rather than as the performance of a cross combination versus others. This can be estimated as the deviation of the individual genotypic value from the SCA of the cross.The lack of average heterosis for tuber yield in the two FThe visual evaluation of uniformity for tuber shape and size may be influenced by tuber size in potato. The percentage of large tubers decreased due to inbreeding, thus affecting the assessment of tuber shape. Small tubers tend to be round, while large tubers are more distinct and often round and long.Genomic prediction based on genotyping and together with genome-wide single-nucleotide polymorphisms (SNPs), co-ancestry and phenotypic data is a powerful tool to capture small genetic effects dispersed over the potato genome, thus allowing one to estimate with some accuracy an individual’s breeding value . Allele 1 and F1 full-sib offspring  estimated by Endelman et al.  and Selgmodeling . Modelinmodeling , while mmodeling . Furthermodeling  for the 1 offspring, Atlin [1 offspring might expose deleterious homo-allelic effects and facilitate identifying promising germplasm based on their allelic value. Of course, potential parents derived from this germplasm may become heterozygous after well-thought intercrossing by genome design. Likewise, Atlin [Although potato breeders seldom produce Sg, Atlin  indicatee, Atlin  stated t1) of a recurrent selection scheme, but it should be based on related training populations.Inbreeding depression affects tuber characteristics in potato. GEBVs may be useful for eliminating undesired offspring, likely carrying deleterious alleles for productivity, quality and tuber uniformity, in the early stages (e.g., first clonal testing generation or T


Mucosal closure techniques are the backbone of interventional endoscopy, requiring focused and dedicated development. We present a novel closure technique that is easily applicable, economical, and effective for large mucosal defects.An 85-year-man underwent gastric endoscopic submucosal dissection for adenocarcinoma of the stomach, which resulted in an ulcer measuring around 35 × 25 mm in size , and thVideo 1 Endoscopic closure of a postendoscopic submucosal dissection ulcer using the novel “CLiPS technique.”A modified anchoring clip was created by cutting the jaws off a large caliber reopenable clip (opening width 16 mm) and smoothing the edges with a file . A clipThe principle of the “CLiPS technique” is reduction in the defect size by strong and stable approximation of the edges using the pulley system. The anchoring clip supports the line and makes it independent of the scope, thereby increasing maneuverability of the scope. Our technique requires no additional accessories or special endoscopes. It does not require scope reinsertion and enables free maneuverability; it may be used in full-thickness closure. Further studies with more patients and larger defects should be considered.Endoscopy_UCTN_Code_TTT_1AO_2AG