In low- to middle-income countries (LMICs), most breast cancers present as palpable lumps; however, most palpable lumps are benign. We have developed artificial intelligence–based computer-assisted diagnosis (CADx) for an existing low-cost portable ultrasound system to triage which lumps need further evaluation and which are clearly benign. This pilot study was conducted to demonstrate that this approach can be successfully used by minimally trained health care workers in an LMIC country.We recruited and trained three nonradiologist health care workers to participate in an institutional review board–approved, Health Insurance Portability and Accountability Act–compliant pilot study in Jalisco, Mexico, to determine whether they could use portable ultrasound  to acquire images of palpable breast lumps of adequate quality for accurate computer analysis. Images from 32 women with 32 breast masses were then analyzed with a triage-CADx system, generating an output of benign or suspicious (biopsy recommended). Triage-CADx outputs were compared with radiologist readings.The nonradiologists were able to acquire adequate images. Triage by the CADx software was as accurate as assessment by specialist radiologists, with two (100%) of two cancers considered suspicious and 30 (100%) of 30 benign lesions classified as benign.A portable ultrasound system with CADx software can be successfully used by first-level health care workers to triage palpable breast lumps. These results open up the possibility of implementing practical, cost-effective triage of palpable breast lumps, ensuring that scarce resources can be dedicated to suspicious lesions requiring further workup. Most cancers therefore present as palpable masses5 identified either by patients or through clinical breast examinations. In a study from India, 85% of palpable masses in premenopausal women proved to be benign on fine-needle aspiration.6 Ultrasonography (US) is widely used in the management of palpable breast masses7; it is nonionizing, and it can be affordable. Although portable US systems are available,8 interpretation has generally been performed by experienced operators, usually radiologists or breast surgeons.In LMICs, 23% of new breast cancer cases occur among women age 15 to 49 years, compared with 10% of new breast cancer cases in high-income countries.9 or magnetic resonance imaging.10 All require experienced radiologists to interpret the images and use the CADe results as an aid for detection.16 No current CADe or CADx systems focus on breast cancer image analysis and diagnosis using low-cost portable US devices such as a triage-CADx system to distinguish those patients who need immediate additional medical attention and those who can be observed.Computer-aided detection (CADe) and computer-aided diagnosis (CADx) systems have been developed for breast cancer; however, most research has focused on detection with mammography and diagnosis using high-end cart-based US, automated whole-breast US,17 as well as for CADe of thoraco-abdominal lymph node and interstitial lung disease classification.18The triage-CADx system is based on convolutional neural networks (CNNs) with transfer learning. This architecture has been shown to provide state-of-the-art performance for CADx of lesions on mammograms20 and also using collected and anonymized images from Magee-Womens Hospital of the University of Pittsburgh Medical Center.21 Although we were able to demonstrate excellent triage performance as assessed by area under the receiver operating characteristic curve (AUC), these results were based on images obtained with high-end cart-based US systems typically available in high-resource settings, and trained radiologists or technologists specializing in breast US imaging captured the images.We previously showed the effectiveness of an earlier triage-CADx system using images from the American College of Radiology Imaging Network 6666 screening breast US studyThe purpose of this study was to demonstrate that nonradiologist health care workers in an LMIC setting can use a portable US system to acquire images of palpable breast lumps comparable to those obtained by trained radiologists and equally adequate for accurate triage with CADx.www.tensorflow.org), and the architecture is shown in 20 anonymized images from Magee-Womens Hospital of University of Pittsburgh Medical Center,21 and images collected from University of Southern California and University of California Los Angeles during phase I. This model was then tested on the images collected in Mexico by both the nonradiologist health care workers and the trained radiologist. The model is compliant with digital imaging and communications in medicine (DICOM) and can process US images stored as DICOM files on a picture archiving computer system or US imaging device. It can also process images stored in standard image formats such as JPEG, TIF, or PNG.The triage-CADx system  has been developed to operate as standalone software that can be run on a Windows 7.0 or higher operating system. The deep-learning CNN model used for this study is Inception-v3 using TensorFlow  equipped with an 8-MHz linear array transducer. Three minimally trained health care workers with no prior experience in US imaging performed image acquisition. The three trainees participating in the study were chosen to represent first-level health care workers (those who are comfortable with patients but do not have any radiology experience) and included a first-year medical student, a surgical nurse, and a gynecologic intern. The operators were trained for approximately 30 minutes by the lead investigator (S.M.L.), using a PowerPoint presentation. They were taught to ask each woman to point out the palpable mass and then draw a line with a marker across the longest portion. A second line was then drawn perpendicular to the first. They were walked through using the GE Vscan Dual Probe device and shown how to take orthogonal views of the lump with and without caliper measurements. The study was institutional review board approved and Health Insurance Portability and Accountability Act compliant. It was conducted in a resource-limited setting  in March 2016 using a low-cost portable US deviceOperators obtained two orthogonal views using the low-cost study machine  and also using the in-house machine  with L12-5 linear array probe. Three scenarios were examined: one, triage-CADx performance using in-house US device  and radiologist; two, triage-CADx performance using low-cost portable GE Vscan Dual Probe device with 8-MHz linear probe scanned by a radiologist; and three, triage-CADx performance using low-cost portable GE Vscan Dual Probe device with 8-MHz linear probe scanned by trainee.22 assessments  of the lesions scanned on the in-house Philips HD11XE machine. All women with a mass scored BI-RADS ≥ 4a underwent US-guided core biopsy according to the standard of care at the hospital.We recruited women with at least one self- or physician-identified palpable breast mass who were age ≥ 18 years presenting to a government hospital in Jalisco, Mexico, for evaluation in March 2016. Eligible participants had no history of breast surgery  at the site of the palpable abnormality that would interfere with the intended field of view. Additionally, participants could not have existing mastitis or other signs of inflammation of the breast. All participants were informed about the study and consented in Spanish before examination. The hospital radiologist (A.P.G.M.), with 3 years of experience, performed a targeted US examination and provided Breast Imaging Reporting and Data System (BI-RADS)The median age of the patients in the study was 46 years . Of the 32 masses, two (6%) were malignant: a known cancer (BI-RADS 6 lesion) and one BI-RADS 5 mass, both invasive ductal carcinomas. The 12 other biopsied masses, assessed as BI- RADS 4a or 4b, proved to be benign and included seven fibroadenomas, two simple cysts, one fibrocystic disease, one intraductal papilloma, and one phyllodes tumor. The size of the masses imaged ranged from 0.8 to 5 cm, with a median of 1.8 cm. Five lesions were not measured, because they were larger than the 5-cm transducer width.The women were able to identify their palpable lumps for the trainees, who then acquired the appropriate images using the handheld US. Images scanned by a trainee versus a trained radiologist using the GE Vscan Dual Probe are shown in The distribution of BI-RADS scores for the lesions is shown in The triage-CADx system correctly classified all 13 BI-RADS 2 and five BI-RADS 3 lesions as benign. This study demonstrates that first-level health care workers are capable of acquiring images with a portable US machine comparable to those of a trained radiologist. The triage-CADx system was applied to both the trainee and radiologist images with 100% sensitivity (two of two), 100% specificity (30 of 30), and an AUC of 1.0, enabling triage-CADx to correctly identify 30 benign lesions and the two cancers.At this site, the average wait for a diagnostic US is 9 months because of limited available trained radiologists. Many of the participants traveled up to 6 hours by bus to participate in the study, corroborating the need for a simple accessible local means of triage. This approach could potentially be applied among women participating in military deployment, remote scientific exploration, or space flights, where the ability to triage a palpable lump on site could conserve much-needed resources.In this study of 32 women with 32 palpable breast masses, inexperienced operators were able to obtain diagnostic images using low-cost portable US comparable to those acquired by a trained radiologist. On the basis of those images, triage-CADx software accurately classified all masses, including two malignancies.21 showed a hypothetic reduction in benign biopsies by 40% while maintaining a sensitivity of 92%. The greatest improvement in specificity came from downgrading masses assessed as low suspicion (BI-RADS 4a) by breast-imaging specialist radiologists. In the validation study at Keck Medical Center of University of Southern California and Harbor–University of California Los Angeles Medical Center, experienced breast-imaging radiologists obtained orthogonal views of palpable lesions using high-end US devices. Evaluation of 156 women with 170 palpable masses demonstrated that triage-CADx achieved a sensitivity of 100% and specificity of 85%,21 corresponding to a reduction in the number of benign biopsies of 69%.Our previous results using high-end US equipmentAlthough the number of masses in this preliminary study was small, these initial results are promising, showing a perfect separation of malignant and benign lesions, resulting in a sensitivity of 100%, specificity of 100%, and AUC of 1.0. In practice and with a larger number of patient cases, we expect these numbers to change. It is important to note that the triage-CADx system in this study was trained and validated using high-end cart-based US systems with transducer frequencies ≥ 12 MHz as recommended for breast imaging. Although the portable study machine  acquired images at a frequency of 8 MHz, the triage-CADx system was still able to deliver excellent results on these images.Phase II of this project is under way and includes the same study design using the GE Vscan  and a much larger number of participants; it will compare AUC results of the triage system with the standard-of-care radiologist performance on high-end US equipment with histopathologic confirmation.This approach is not intended for screening but rather for triage in the field of palpable breast lumps to better allocate scarce health care resources. This small series demonstrates the ability of first-level health care workers with minimal training to locate and capture US images of palpable lumps equivalent to those of trained radiologists. We also confirmed that the images from a portable handheld US device were adequate for analysis by the triage-CADx system. This ability will be further confirmed in our ongoing larger study.Although the device used in this study is more expensive than most LMIC settings can afford, lower-cost devices are becoming more available, and we have shown that our artificial intelligence platform generalizes across different hardware devices so that it can be applied to new hardware platforms as they come to market.We have demonstrated that a minimally trained health care worker can effectively capture images of palpable breast lumps that are equivalent to those obtained by trained radiologists and are suitable for triage-CADx analysis. In addition, a triage system trained to distinguish suspicious and benign breast lesions can be used with a low-cost portable US system. We are currently enrolling women in a larger prospective study in Jalisco and Tijuana, Mexico, with the goal of demonstrating that health care workers without radiology training will be able to use this device in nonurban areas to accurately distinguish palpable lumps that require biopsy from those that do not.


Tactile-kinesthetic-proprioceptive (TKP) input used to facilitate speech motor control is considered an active ingredient within speech motor interventions. Objective metrics identifying skill level differences across speech-language pathologists (S-LP) providing TKP cues are crucial for monitoring treatment delivery fidelity. The study examined three kinematic measures indicating accuracy and consistency of TKP inputs by 3 S-LPs with varying experience levels . Confidence interval measures were used to compare the accuracy of jaw movement amplitudes of the vowel /a/ made by a model participant versus S-LPs giving the TKP input. Generalised Orthogonal Procrustes Analysis (GPA) and cyclic Spatial Temporal Index (cSTI) were used to determine movement consistency. Results revealed passive jaw excursions induced by S-LP 2 and 3 to be not statistically significant from the model participant's active jaw movements. cSTI values decreased with advanced level of experience . GPA analyses revealed a similar pattern for S-LPs with more experience demonstrating lower mean RMS values . Findings suggest kinematic measures adapted from the motor control literature can be applied to assess S-LP skill differences in providing TKP cues. The use of tactile-kinesthetic-proprioceptive (TKP) inputs has an established presence in remediating speech sound disorders (SSDs) within many contemporary speech motor interventions  has been demonstrated in a number of behavioral studies  movement ranges can be used to distinguish experience levels of S-LPs providing those TKP cues. For the purpose of this pilot study, a model participant without speech problems was deemed to provide a more reliable context for testing the usefulness in assessing clinicians' skills. We hypothesize that, with more specialized training and years of experience with speech motor disorders, a S-LP should (a) demonstrate increased accuracy when predicting and cuing client jaw ranges for a given vowel  of the client's mean range of active jaw movements), (b) demonstrate increased consistency in finger-hand movements when providing TKP cues as determined by Generalised Orthogonal Procrustes Analysis (GPA), and (c) demonstrate increased consistency in induced (passive) orofacial movements using a cyclic Spatial Temporal Index (cSTI) measure. These measures will be explained in more detail in the method section.A 23-year-old female with no self-reported speech, language, hearing, or neurological difficulties served as the model participant-client in this study. Three female S-LP participants, with different levels of training and experience , provided TKP cues to the model participant's orofacial structures. S-LP 1 had recently graduated from an accredited S-LP program with eight months experience providing S-LP services to children with speech sound disorders. S-LP 2 and S-LP 3 had more than 15 years of experience and advanced training with speech motor interventions with both children and adults. The study was approved by the University of Toronto's Health Sciences Research Ethics Board, and all participants provided a written informed consent prior to participation.CalPos program available from the manufacturer [All kinematic and time-aligned acoustic data were collected with the Electromagnetic Articulograph 501 system (EMA AG501) . The EMAfacturer . The medfacturer , 24. Allfacturer . Sample-facturer ). The mofacturer  to extraSensors were attached onto the model participant's face at the following anatomical locations: mandible midline, nose bridge, behind the left and right ear mastoid, and two sensors 1.5 cm symmetrically on either side of the philtrum of the upper lip. Furthermore, six sensors were placed on the hand providing the TKP inputs in the following manner: one each on the thumb, index finger, and middle finger (placed on the nail plate) and three reference sensors on the dorsal part of the hand.active condition: the model participant repeated the stimuli (vowels in isolation and in sequence) approximately 10 times at a self-paced rate and (b) passive condition: the model participant's movements were generated solely by the action of the S-LP's hand movements; e.g., moving the jaw down  or rounding/retracting the participant's lips (more detail provided below). The order for participant-S-LP dyads was S-LP 3, S-LP 1, and S-LP 2.Speech stimuli were vowels /a, i, u/ produced in isolation and in a sequence combined with the consonant /t/ as in /ta, ti, ta, tu/ in succession. Movement data were recorded under two conditions: (a) Three kinematic measures were used to quantify accuracy and consistency of TKP cues provided by an S-LP. All of these measures have been modified and adapted from measures previously reported in the speech and limb motor control studies , 27.Measuring accuracy of jaw movements has been deemed of crucial importance in enhancing the critical role of jaw control in speech production , 29. DevTwo measures were utilized to quantify the consistency of TKP inputs provided by an S-LP: the Generalised Orthogonal Procrustes Analysis (GPA) and the cyclic Spatial Temporal Index (cSTI). For vowel /i/, the thumb and index fingers were placed above the upper lip at the intersection between levator anguli oris and the skin locations covering the location of zygomatic major muscles with slight pressure applied backwards towards the model participant  Figure . This reGeneralised Orthogonal Procrustes Analysis , 34 has The entire movement trajectory data from the S-LP's thumb (relative to the reference sensor on the participant's nose bridge) over successive trials of stimuli “ta-ti-ta-tu” was used for the GPA analysis. To extract movement data of the S-LP's thumb relative to the reference sensor, the latter was subtracted from the trajectory of the S-LP's thumb in 3 dimensions . Before applying GPA, each movement trajectory was time normalized to 1000 points using FFT  in a time-domain signal, while preserving the underlying frequency values of the signal itself) interpolation separately for each of the three dimensions, across 10 repetitions of /ta-ti-ta-tu/. A centroid or mean value was calculated for each movement trajectory and for each dimension . The movement trajectories were then linearly shifted such that the centroids are aligned with the origin (noise bridge sensor coil). The thumb movement data for each of the 10 repetitions of /ta-ti-ta-tu/ was then represented as a 1000∗3 matrix, with each of the rows representing 3D positions at a particular time and each column representing the position value along one of the three Cartesian coordinates across time. For convenience in describing the GPA method, we will follow the convention of referring to each of the 10 1000∗3 matrices as Si (where i represents the ith repetition). During the GPA process, we attempt to align repeated 3D movements. In our case, we have 10 repetitions. Each repetition contains a 1000 points of 3-dimensional data (hence a 1000∗3 matrix). Si is a convention to represent one such matrix. S1 would represent the first matrix representing the first repetition, S3 would represent the third matrix representing the third repetition, and so on.j goes from 1 to 1000), rms of the ith trajectory (rms(i)) from the mean is defined as square root (((Si(1) − Smean(1))2 + (Si(2) − Smean(2))2 … (Si(1000) − Smean(1000))2)/1000). The mean RMS is therefore defined as (rms(1) + rms(2) … rms(10))/10 for 10 repeated trajectories) residual between each of the shapes and their mean consensus trajectory. The RMS residual for one trajectory is calculated by summing the squared distances of each data point in the trajectory to the corresponding data point in the consensus trajectory, averaging over the number of data points (1000) and calculating the square root. The mean of this RMS residual over all the movement trajectories derived from a S-LP's thumb sensor coil gives us a measure of the intrinsic error in the thumb movement pattern (or shape) of that S-LP. Additionally, to account for differences in S-LP's range of hand motion, we also report displacement-normalized RMS residuals (nRMS).For the GPA analysis, one movement trajectory of the 10 repetitions of /ta-ti-ta-tu/ was selected as a common consensus trajectory or exemplar (M), and the remaining nine movement trajectories were rotated to align with this exemplar. Matrix rotation is performed by finding an orthogonal matrix Qi for each movement trajectory Si which minimizes the value of ||Si∗Qi-M||. The solution for finding such an orthogonal rotation matrix Qi has been published elsewhere . For theHowever, GPA assesses consistency in the shape of the S-LP's finger space movement paths, and we need a measure to capture end product consistency of a S-LP's finger-hand movement trajectories . In the current study, cyclic STI or cSTI  was usedz-score transformed) normalized. From these records, 50 standard deviations were obtained at 2% intervals on the normalized axis and summed to give the cSTI score [Kinematic consistency of passively induced upper lip movements was indexed using the cSTI, with movement cycles operationally defined as peak-to-peak or valley-to-valley trajectory cycles related to the Euclidean distance between the two sensor coils placed 1.5 cm symmetrically on either side of the model participant's philtrum on the upper lip. For cSTI, cycles were limited to displacement records for /ti/ for which the S-LP was inducing lip retraction gestures in the model participant. Lip gestures corresponding to /tu/ (for lip rounding) were not used in the cSTI analysis due to motion artifacts caused by lip muscle protrusion and bunching (resulting in sensor coil rotation) from the S-LP's fingers pulling the lips closer. The lip retraction displacement records for /ti/ gestures were time (to 1000 points) and amplitude  using an online biostatistics software .Descriptive statistics (Mean and Standard Deviations (S.D.)) for passive jaw excursions (in mm) induced by S-LP 1, 2 and 3 compared to active jaw movements made by the model participant are provided in t-tests indicated that, for S-LPs 2 and 3, passive jaw excursions induced were not statistically different from actual active jaw movements made by the model participant  orofacial movements in a model participant varied as a function of the S-LP's training and experience. These findings suggest that kinematic measures adapted from the speech and limb motor control literature can be successfully applied to quantify S-LP skill levels in providing TKP cues. The broader clinical implications of these findings will be discussed next.The act of speaking requires coordination of the speech subsystems  within time and space. Past literature has shown that the multiple degrees of freedom of movement in speech production are constrained through organized functional synergies, allowing for variability in movement patterns , 38. HenFundamental to intervention for clients with SSDs are skills requiring attunement to task-specific visual information while observing a client's ability to execute a speech motor movement, assessing where difficulties are occurring, and planning intervention by selecting speech motor targets using TKP inputs . In our More specifically, for S-LP training, the S-LP needs to learn how to perform the motor act underlying the TKP cues, that is, learn to move his/her hand/fingers to a specific spatial location , timed with the movement within the intended range . This isContemporary motor control literature considers the consistency with which behaviors are repeatedly executed to be one of the critical elements of skilled motor expertise , 44. VarThe GPA is said to reflect the precision or computational difficulty with which the motor system executes the desired movement paths. The higher mean RMS residual values in S-LP 1 (least experienced) may imply a greater difficulty in planning and executing complex thumb movement paths as required for TKP inputs in a therapeutic context. The data from the more experienced S-LPs demonstrated that they executed their finger/hand movements with greater precision, indicated by the lower RMS values. In a study by , principFrom a clinical standpoint, consistency of induced movements in target structures such as the lips and jaw  is as important as consistency of S-LP hand movements. Experimental data suggests that somatosensory signals arising from cutaneous afferents in the facial skin  play a crucial role in speech motor learning and adaptation , 49. ForThe importance of establishing treatment fidelity to determine treatment efficacy has been clearly established . In speeVerrel et al.  postulatThe study has several major limitations. External generalizability of these results is severely limited by the small sample of participants. A demonstration of these measures and outcomes with a larger group of participants is imperative. Furthermore, using a healthy model's actual facial movements to judge appropriate TPK cueing, although appropriate for the context of this pilot study, does not allow for generalization to clients with impaired speech production skills. In the field of speech-language, pathology assessment of functional speech outcomes is critical. In therapy where these cues are typically used, such measures are indeed critical for assessing progress in a client's speech production abilities. However, this information was not gathered in the present study as the focus was specifically on the accuracy and consistency measures obtained from the S-LP's hand movements and S-LP-induced passive jaw and lip movements on a model participant's face. This individual was not actually speaking during measurements of these variables (other than to establish the jaw movement range), again because our focus for the present study was not on the acoustic outcomes but on the delivery of tactile cues. This is why, for the purpose of this pilot study, we used an adult model participant without speech, language, hearing, or neurological issues so it would be possible to access the specific features of tactile cue delivery without possible confounding issues presented by a motor speech disorder. This presented a more reliable context for testing the feasibility of assessing clinicians' skills. In a healthy adult participant with perfectly intact and “normal” functioning sensory-motor systems, we do not expect any significant changes in speech acoustics as a function of accuracy and consistency of TKP cues, especially with a limited number of trials (10 repetitions of target items). However, in the context of a developing sensory-motor system in a child or an impaired speech system in either adults or children , the auditory-to-speech motor mapping required for the achieving accurate speech output may be more susceptible to inaccurate and inconsistent TKP input by an S-LP. The findings from the current study suggest that kinematic measures adapted from the motor control literature can indeed be applied to assess S-LP skill differences in providing TKP cues. Utilizing these objective measures to capture S-LP skill levels, future studies must be conducted on populations with speech disorders to establish the impact of S-LP skill levels on functional speech outcomes.Lastly, the cross-sectional design used provides no indication of the longitudinal development of motor skill in clinicians and limits causal inference. Future studies should track S-LPs' motor skill levels as a function of training in a longitudinal study using a population with specific speech disorders and possibly within a therapeutic context. This may one day allow us to track S-LPs' motor skill levels objectively and set fidelity standards for providing specialized speech treatments.Overall, we found that accuracy in estimating and subsequently implementing a client's jaw movement range, consistency in finger-hand movements when providing TKP cues, and the consistency of the resulting-induced (passive) orofacial movements in a model participant varied as a function of the S-LP's training and experience. These findings suggest that kinematic measures adapted from the speech and limb motor control literature can be successfully applied to quantify S-LP skill levels in providing TKP cues. These preliminary findings are a first step for further explorations of the feasibility of these measures in evaluating S-LP functional skill levels during training and for determining if a clinician is ready to treat patients without supervision.


Breakup of droplets at bi-layer bifurcating junction in polydimethylsiloxane (PDMS) microchannel has been investigated by experiments and numerical simulation. The pressure drop in bi-layer bifurcating channel was investigated and compared with single-layer bifurcating channel. Daughter droplet size variation generated in bi-layer bifurcating microchannel was analyzed. The correlation was proposed to predict the transition between breakup and non-breakup conditions of droplets in bi-layer bifurcating channel using a phase diagram. In the non-breakup regime, droplets exiting port can be switched via tuning flow resistance by controlling radius of curvature, and or channel height ratio. Compared with single-layer bifurcating junction, 3-D cutting in diagonal direction from bi-layer bifurcating junction induces asymmetric fission to form daughter droplets with distinct sizes while each size has good monodispersity. Lower pressure drop is required in the new microsystem. The understanding of the droplet fission in the novel microstructure will enable more versatile control over the emulsion formation, fission and sorting. The model system can be developed to investigate the encapsulation and release kinetics of emulsion templated particles such as drug encapsulated microcapsules as they flow through complex porous media structures, such as blood capillaries or the porous tissue structures, which feature with bifurcating junctions. Emulsions are ubiquitous in the daily life and have been extensively applied in food industry, cosmetics, pharmacy, and biochemical reactions ,2,3. EmuDroplets can be split in active or passive manners, while the passive fission is more widely applied mainly because of ease of implementation and low cost. Geometrically mediated breakup of droplets relates with the fluid flow resistance and geometries in respective channels of the microfluidic system. A variety of geometries have been demonstrated in passive fission where a droplet is split as it flows past T-junction , arbitraNevertheless, the droplet breakup dynamics in confined geometries such as the proposed structure has not been adequately studied, and it demands in-depth investigation. For example, pressure drop is the driving force of fluid flow in bifurcating microchannel for the formation and fission of droplets. High-pressure drops may lead to high energy consumption. The examination of pressure drop between single- layer and bi-layer bifurcating microchannel is thus necessary. The criterion based on Capillary number for the transition between the breakup and non-breakup regime has been investigated for single- layer bifurcating channel when the mother droplets interact with the tip of the bifurcation . The valIn this study, the formation of single emulsion droplets at upstream T-junction and splitting of droplets at downstream bi-layer bifurcating junction have been investigated via numerical simulation and experiments. First, the pressure drop in bi-layer bifurcating channel was investigated and compared with counterpart in the form of single-layer. Subsequently, daughter droplet size variation was confirmed from experimental study of bi-layer bifurcating microchannel, hypothesis was made to relate the size variation with the different pressure distribution in upper and lower channels, and the hypothesis was verified. Analysis was performed to determine the daughter droplet volume ratio at various Capillary numbers in bi-layer bifurcating channel and compared with that in single-layer bifurcating channel. Finally, a correlation was proposed to predict the transition from breakup to non-breakup of droplets in bi-layer bifurcating channel. Understanding of the mechanism of droplet fission in the microstructure can lead to more versatile control over the emulsion formation and fission, and the release kinetics when the emulsion-templated microparticles are used for encapsulation and releasing application. The novel droplet fission approach provides insights for the design and fabrication of higher order emulsions with custom-made volume and shape, inspiring new biomedical and industrial applications based on microfluidic emulsification platform. QDP, and oil is injected as the continuous phase at a constant average flow rate QCP . The mixture was degassed in a vacuum desiccator before it was poured into the epoxy mold and cured at room temperature for 24 h . Deionized (DI) water was used as the dispersed fluid. The interfacial tension of 14.26 mN/m between water and oil was measured by a goniometer . The properties of the test fluids are shown in sion see c, which sion see d. The coα denotes volume fraction, t and V denote time and flow velocity respectively, and the subscript CP represents continuous phase. A 3-D numerical model of the droplet formation and fission in the bi-layer bifurcating microchannel was established. The two-phase flow problem was solved using the VOF (Volume of Fluid) method by CFD software Ansys Fluent 15.0 . In this method, the interface between water and oil phase was tracked by solving the following convection equation ,40: (1)∂P is the pressure, σ is the interfacial tension between two phases. The gravity effect was neglected because the gravitational force is negligible when the drop diameter is in the order of micrometers [Ca, represents the ratio of viscous force to interfacial tension and can be defined by,The governing equations for incompressible two-phase Newtonian fluids can be described by continuity equation (Equation (2)) and Navier–Stokes equation (Equation (3)), the cell-averaged density and dynamic viscosity can be obtained from Equations (4) and (5).rometers . Capilla−6 s and the solution converges when the residual is below a tolerance set as 1.0 × 10−6. No-slip condition was applied at the solid boundaries of the walls of the microchannel. Zero gauge pressure was applied at the outlet of the microdevice. The flow velocity was specified at each inlet of the microdevice. The numerical data were analyzed by the Ansys CFX-Post Processor 15.0. The simulations were performed using numerical grids composed of tetrahedral elements generated by ANSYS package ICEM CFD 15.0  algorithm was used in the transient calculations for droplet generation and fission. The momentum equation was discretized using second order upwind scheme, and the pressure interpolation was achieved by Pressure Staggering Option (PRESTO!) scheme. An iterative solver was deployed to solve the control-volume discretized equations. The iterative time step is 1015.0 see . The accP1 between continuous phase inlet and outlet, and the pressure drop P2 between dispersed phase inlet and outlet have been calculated and plotted against Capillary number spanning a range from 0.018 to 0.054. P1 is higher than P2 for both microsystems, due to the longer distance from the continuous phase inlet to the outlet . Flow resistance is therefore proportional to channel length, and higher flow resistance can arise from either increase of radius of curvature or included angle of channel, leading to higher pressure drop with the same flow rate. With the same volumetric flow rates at inlets, pressure drop is directly proportional to flow resistance, the value of which is larger in single-layer microstructure, because two lapped segments ΔP=QEmphasis will subsequently be given to droplet formation and fission in bi-layer bifurcating microchannel at various flow conditions by adjusting the flow rate of continuous phase and dispersed phase. The captured images from experimental recordings are shown in Ca = 0.054 (see t = 5.760 s). The two parts are dislodged and finally detached from each other (see t = 5.976 s). The breakup of a mother droplet at bi-layer bifurcating junction is affected by shear stress distribution, as illustrated in The experiments allow for observation of droplet formation and splitting in 2-D view. Numerical simulation has been conducted to supplementarily unveil the droplet fission associated breakup dynamics in 3-D perspective, and the temporal evolution of droplets with dispersed phase flow rate of 6 L/min and continuous phase flow rate of 18 μL/min is compared with experiments when .054 see . The intCa = 0.018, while it is reduced to 171.5 μm at flow rate of 24 μL/min when Ca = 0.054. Quantitative comparison of size between daughter droplets in different layers can be found from Ca = 0.045. It indicates that asymmetric cutting of droplets using one bi-layer bifurcating junction can passively break up droplets into daughter droplets with two sizes, each of which has controlled monodispersity. Multiple sizes can be achieved with scale-up of the bi-layer bifurcating junctions. Assuming volume ratio of the daughter droplets generated by bifurcating junction is represented by f, the results of volume ratios at different Capillary numbers are shown in The droplet size in lower channel is larger than that in upper channel, indicating formation of unequally sized daughter droplets as shown in We attribute the droplet size variation to be related with the different pressure distribution in upper and lower channels. The presence of stepwise constriction leads to elevation head  [The critical capillary number for breakup ion (11) ,(11)Cacf ranges from 0.68 to 0.78 in the cases as depicted in f = 0.68 and 0.78 respectively were also added in f = 0.73. The phase diagram indicates that non-breakup occurs when Ca is below 0.012, the mother droplet migrates into the lower channel because of lower flow resistance and exits from the microdevice. It is also of interest to note that transition from breakup regime to non-breakup regime can be triggered by modifying the radius of curvature of bifurcating channels. For example, at the same flow rate when Ca = 0.054, when r = 5 mm for both channels, the droplet splits up into two daughter droplets after striking the stagnation point of bi-layer bifurcating junction ), preventing droplets from entering the channel. Combing Equations (9)–(11), we can obtainayer see . The drotion see . In contIn this study, we have developed novel droplet fission approach using bi-layer bifurcating microchannel. The flow of emulsions and associated droplet breakup dynamics through bi-layer bifurcating junction have been investigated numerically and experimentally. Monodispersed daughter droplets are formed in upper and lower channel, while the average droplet size varies in different layers, arising from elevation loss formed at bi-layer bifurcating junction. Flow resistance can be tuned by controlling radius of curvature, and channel height ratio so that production of daughter droplets of distinct sizes can be achieved. Compared to bi-layer bifurcating microchannel at the same flow conditions, droplet fission is achieved at cost of high-pressure drop in single-layer bifurcating microchannel. The geometrically mediated droplet fission offers versatile control over droplet size distribution via asymmetric splitting. The microdevice is structurally simple and compact. Xurography process enables fabrication in facile way with low cost, obviating the need of expensive facilities such as clean room. The high scalability of the PDMS device can allow adding bifurcated junction per demand to create higher order emulsions, useful for synthesizing particles or capsules with customized inner compartments for encapsulation and release applications. Full exploitation of microfluidic production and control of emulsion droplets is severely limited if only a certain droplet size can be produced by single device. In contrast, our novel droplet fission platform allows for much better flexibility in forming droplets with distinct sizes while each size has good monodispersity. This would facilitate the use of droplets as micromixers or chemical reactors. Potential applications using the 3-D bi-layer bifurcating microstructure also include drug encapsulation with adjustable multiple dosages for drug delivery.


Inhibitory receptors are key regulators of immune responses. Aberrant inhibitory receptor function can either lead to an exacerbated or defective immune response. Several regulatory mechanisms involved in the inflammatory reaction induced by monosodium urate crystals (MSU) during acute gout have been identified. One of these mechanisms involves inhibitory receptors. The engagement of the inhibitory receptors Clec12A and SIRL-1 has opposing effects on the responses of neutrophils to MSU. We review the general concepts of inhibitory receptor biology and apply them to understand and compare the modulation of MSU-induced inflammation by Clec12A and SIRL-1. We also discuss gaps in our knowledge of the contribution of inhibitory receptors to the pathogenesis of gout and propose future avenues of research. Crystal-induced arthropathies are a group of disorders that are triggered by crystal deposits in articular and periarticular tissues . MonosodInhibitory receptors play key roles in regulating almost every aspect of an immune response mainly by blocking activating pathways –5. The iMonosodium urate crystals are a crystallized form of uric acid, a product of purine metabolism . Uric acMonosodium urate crystals are a potent pro-inflammatory stimulus for professional phagocytes, the main drivers of acute gouty arthritis , 9. WhenMonosodium urate crystals activate a unique subset of signaling pathways in the neutrophil [reviewed in Ref. ]. These One intriguing feature of acute gouty arthritis is its spontaneous resolution, typically within 7–10 days . NeutropNegative regulatory mechanisms not only dampen a gout attack but also influence its initiation by modulating the threshold of activation of leukocytes. These negative regulatory pathways are downstream effectors of inhibitory receptors expressed on the surface of the cell. Each leukocyte expresses a diverse repertoire of inhibitory receptors . The chaInhibitory receptors are essential for the maintenance of immune homeostasis as well as the termination of an immune response by blocking signaling pathways that lead to cellular activation . AberranInhibitory receptors are composed of a variable number of extracellular ligand-binding domains, a transmembrane domain and a cytoplasmic tail that contains at least one immunoreceptor tyrosine-based inhibitory motif (ITIM) . The extOnce engaged by a ligand, inhibitory receptors signal through their ITIM(s)  that is Inhibitory receptors regulate many aspects of leukocyte development and function . Some ofin vitro. Although these experimental approaches only offer us a snapshot of the involvement of these inhibitory receptors in the continuum of the cellular and chemical events of an inflammatory response, they provide insight into how these receptors work. Experiments in knock-out mice complement in vitro studies by revealing the outcome of the integration of activating and inhibitory signals in a specific inhibitory receptor-deficient background.To understand the role of inhibitory receptors in an inflammatory response, the modulation of their expression and their clustering, the phosphorylation of their ITIM motif(s), and the recruitment of protein partners are studied Studies in neutrophils identified two inhibitory receptors associated with MSU-induced inflammation, namely, MICL  and the IgSF receptor known as SIRL-1 . The ligation of these receptors regulates MSU-induced activation of neutrophils in different ways underscoring the ability of inhibitory receptors to differentially regulate leukocyte function.via alternative mechanisms. They can also bind non-glycan ligands . CLEC12A is devoid of the known amino acid required for glycan and calcium binding in its CTLD, rendering the identification of its natural ligands a challenge. Murine CLEC12A binds various mouse tissues, the identity of which remains to be determined (CLEC12A belongs to the C-type lectin superfamily of proteins. It harbors an extracellular CTLD linked to its transmembrane domain by a neck domain and one ITIM motif (VTYADL) in its cytoplasmic tail –28. CTLDtermined , 35. Regtermined , 37. It termined , 39. As termined . This obIt is now widely accepted that one of the key roles of CLEC12A is to negatively regulate myeloid cell function and that it is associated with several inflammatory diseases –44. The in vitro were corroborated in vivo in a knock-out mouse model of CLEC12A  has the opposite effect. In the RBL-2H3 cell in vitro model of FcεRI activation, for instance, ligation of SIRL-1 with anti-SIRL antibody inhibited IgE-induced degranulation [Table S. aureus but not the production of ROS, thereby underscoring a certain degree of functional selectivity (SIRL-1 negatively regulates innate immune responses toward various stimuli. As is the case with CLEC12A, the cell-surface expression of SIRL-1 diminishes when certain stimuli activate monocytes and neutrophils , 48. Theectivity .S. aureus or LPS (SIRL-1 also inhibits neutrophil activation in response to MSU. The ligation of SIRL-1 with anti-SIRL suppresses MSU-induced formation of NETs . This ins or LPS , 50. It The evidence that CLEC12A and SIRL-1 modulate MSU-induced neutrophil activation was obtained from experiments performed with naked crystals. MSU thus trigger activating signaling pathways by directly interacting with components of the cell surface. Since MSU are composed of a rugged, crystalline surface that is negatively charged, it is difficult to envisage how such a surface can exhibit the same level of specificity as a conventional “hand in the glove” receptor–ligand interaction. One receptor through which MSU activates signaling in human neutrophils is the Fc receptor FcγRIIIb . Since tCLEC12A and SIRL-1 elegantly illustrate the ability of inhibitory receptors to have distinct effects on leukocyte activation. Whereas the ligation of SIRL-1 with a specific antibody leads to the downregulation of cellular effector functions, the antibody-induced ligation and internalization of CLEC12A enhances cellular activation. This implies that the downregulation of the cell-surface expression of CLEC12A releases its inhibitory effect and consequently lowers the threshold of activation of neutrophils toward MSU. These observations suggest that CLEC12A may be constitutively phosphorylated in resting cells to increase the threshold of activation. The phosphorylation status of the ITIM of CLEC12A remains, however, to be determined in resting and stimulated cells. With regards to SIRL-1, it is most likely constitutively phosphorylated since it associates with SHP-1 in resting monocytes. This notion is supported by the inhibition of the MSU-induced formation of NETs after the ligation of SIRL-1. Since the anti-SIRL-1 antibody inhibits MSU-induced formation of NET, it is an agonistic antibody. By contrast, the anti-CLEC12A antibody does not seem to have agonistic effects since it enhances cell activation by binding CLEC12A. Although the role of CLE12A and SIRL-1 in gout remains to be fully characterized, these two receptors may regulate neutrophil activation in a temporal manner since NET formation is a later response to MSU than the mobilization of calcium.In vivo studies will also be informative. With regards to CLEC12A, for instance, it may also contribute to the resolution of inflammation in gout since the arthritis phenotype in CLEC12A knock-out mice persists for a significantly longer period of time than in wild-type mice (To develop a more complete picture of the immunopathogenesis of gout, the constellation of inhibitory receptors that regulate myeloid cell responses toward MSU needs to be identified. Moreover, it is essential to identify the ligands for these receptors to better understand their functional and temporal role during homeostasis and inflammation as well as their mode of action. Pyz et al.  reportedype mice .The dampening of MSU-induced neutrophil activation by the SIRL-1 antibody supports the notion that targeting myeloid inhibitory receptors is a promising and new therapeutic option for the treatment of gout. Inhibitory receptors could also be targeted with compounds that preserve their expression once the mechanisms underlying their internalization and degradation are characterized. Targeting inhibitory receptors that regulate the threshold activation of neutrophils during the early stages of a gout attack would be beneficial since there is a therapeutic window of opportunity prior to the peak of a gout attack during which patients feel a slight pain. Colchicine is one such drug that effectively reduces inflammation if administered during the early phase of an acute gout attack. A mechanism through which colchicine may dampen a gout attack is by preserving the cell-surface expression of CLEC12A on neutrophils .It is difficult to predict whether targeting endogenous inhibitory pathways will cause less adverse effects than currently used drugs. Considering the limited choice of anti-inflammatory drugs to treat gout due, in part, to contraindications in many cases, it is timely to consider developing additional drugs to treat gout attacks . A recenIt is an exciting time for inhibitory receptor research since we have better tools to study their biology and a clearer picture of how they work facilitating the investigation of their function and role in disease. At least two other inhibitory pathways in lymphocytes are targeted for the treatment of rheumatoid arthritis and cancer  providinMF and PHN contributed equally to the writing of the manuscript.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.


Grandparenting can be a rewarding and health-promoting experience for older adults. However, grandparent-caregivers often experience greater stress and poorer health relative to non-caregiving grandparents. Further, little is known about grandparents caring for a child with a rare, chronic illness. This study aimed to extend knowledge of the expectations, roles, and experiences of grandparents providing care to a child affected with an inherited metabolic condition. The sample included 23 grandparent-mother dyads from the Inherited Diseases, Caregiving, and Social Networks Study. The grandparent sub-sample ranged from 49 to 79 years of age (Mage = 64), the majority were female (83%) and married (74%), and almost half (48%) were retired. Social network assessments were analyzed to determine concordance between mother- and grandparent-reports of grandparents’ role in the child’s caregiving network. Fifteen mother-grandparent dyads (65%) agreed on grandparents’ role in the child’s network, with the majority of those (93%) considering the grandparent to be very close and important (versus less close or excluded from the caregiving network). Grandparents whose self-reports of their role in their child’s caregiving networks were consistent with mother-reports appeared more likely to report that they spend enough time caregiving than those whose reports were inconsistent. Content analysis of grandparents’ interviews provided supporting information about the joys and regrets of their grandparenting experience and perspectives on caregiving expectations. This research leverages multi-informant social network and qualitative data to illuminate grandparents’ role in the caregiving networks of chronically ill children and adaptation to non-normative grandparenting experiences.


This review focuses on novel carbosilane dendrimers containing branches with Si-C and Si-O-C bonds. Introduction of organic moieties into the dendrimers is performed by hydrosilation of carbon-carbon double/triple bonds. Versatile organic or organometallic moieties are introduced onto the peripheral regions of dendrimers by coupling and complexation reactions, which clearly demonstrates their potential for variation.  Dendrimers of unique architecture containing a single kind of multi-functionality have been of increasing interest during the last three decades . Their set al. starting from a carbosilane core with one to four ethynyl groups  , and its photoluminescent properties were observed  Scheme .MALDI TOF mass spectroscopy provides valuable information about the unimoleculirity of lower generation dendrimers; but it gives no conclusive information about its purity, so GPC was chosen to study the purity of the dendrimers. The GPC chromatogram shows very narrow peaks corresponding to that of the respective generation of the dendrimer .4 with Si-O-C branches are discussed [nSiHCl3-n; n = 1-3) [Dendrimers of various siloxane core moieties with characteristic branching points and peripheral groups have been prepared ,21. In tiscussed . The synn = 1-3)   at the regular retention time. Furthermore, Diels-Alder reactions of the anthracenes on the dendritic periphery with 1,4-benzo-quinone and 1,4-naphtoquinone were performed with good results [N-ethylmaleimide, 1,4-naphtoquinone, and tetracyanoethene has been performed under refluxing toluene conditions. Diels Alder reaction of hexadienyl dendrimer gives the first generation with 12 bicyclo groups on the periphery but the 2nd generation is not synthesized with a single type of substituent due to the lack of enough surface to accomodate 36 enes (Scheme 9).Dendrimers with anthracene groups on the dendritic periphery have been prepared . Diels-A results . A dendr results . Diels-AVersatile dendrimers of a siloxane core  and bicyA photo switching device was prepared from dendrimers with azobenzene terminal groups. The UV absorbance of the dendrimer decreases with increased irradiation time from the UV absorbance spectrum and heat treatment, however LB monolayers do not show absorbance shifts, indicating that the azobenzene dendrimer cannot be photoisomerized. Terpyridine end-functionalized dendrimers are prepared and the terpyridine groups on dendrimers are coordinated to the ruthenium ions under the mild condition to produce a paramagnetic ruthenium complex This review focused on the divergent growth methods for carbosilane dendrimers with Si-C and Si-O-C moieties from carbosilane and cyclic siloxane cores. There are very few reactions for the syntheses of these dendrimers. The highly branched dendrimers are prepared from a small core up to the 7th generation and additions of various functional groups to them are reported. The siloxane dendrimers are prepared from the reaction of Cl-Si bonds of dendrimer with bulky alcohols. The repetitive cycles of hydrosilation and alcoholysis are performed for the synthesis of carbosilane dendrimers. Versatile dendritic macromolecules are prepared by using the terminal functional groups of the dendrimer. Several organic and/or organometallic moieties are introduced in the peripheral region of the carbosilane and carbosiloxane dendrimers by the simple coupling and/or complexation reactions.


The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Point mutation knock-in using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) is currently the most promising technology for this purpose. Despite some progress in applying this technique to zebrafish, there is still a great need for improvements in terms of its efficiency, optimal design of sgRNA and ssODNs and broader applicability. The papers discussed in this Editorial provide excellent case studies on identifying problems inherent in the mutation knock-in technique, quantifying these issues and proposing strategies to overcome them. These reports also illustrate how the procedures for introducing specific mutations can be straightforward, such that ssODNs with only the target mutation are sufficient for generating the intended knock-in animals. Two of the studies also develop interesting point mutant knock-in models for cardiac diseases, validating the translational relevance of generating knock-in mutations and opening the door to many possibilities for their further study. Summary: With the co-publication of three research articles describing CRISPR/Cas9-based approaches for knocking in mutations in zebrafish , this Editorial reflects on the current state of genome editing for disease modeling in zebrafish. Danio rerio) is to generate accurate models of human genetic diseases to recapitulate their clinical features, to understand the molecular mechanisms that underpin them, and to model treatments and disease management approaches. Before the advent of precise genome editing, understanding a missense mutation within a protein-coding human gene in zebrafish necessitated designing an mRNA expression vector or a transgenic construct. These approaches did not actually replace the normal (wild-type) zebrafish gene with an altered human one. Additionally, expression of a human (trans)gene raises the question whether that gene is, in fact, functional in fish. Does the mutant gene function in fish? Are expression levels sufficient? Is expression in the right place and at the right time? These answers may be elusive.One of the great potentials of zebrafish  molecule to guide the Cas9 endonuclease to the genomic site of choice. Single-stranded oligodeoxynucleotides (ssODNs), or oligos for short (as referred to herein), are currently the donor templates of choice for mutation knock-ins A. These Our own group previously reviewed zebrafish studies aimed at developing precise point mutation techniques using CRISPR/Cas9 , includiThe paper by Boel et al. focuses on the technical aspects of knock-in optimization . The firBoel and colleagues also investigated whether asymmetric homology arm design for 120 nt oligos can improve knock-in efficiency . No singin vitro findings of tp53 gene  could function as a modifier allele, resulting in a congenital heart defect. The pbx4 A131V allele did not produce a clearly discernable phenotype, but it was also not completely functional because its presence increased the severity of heart defects when combined with a null pbx4 allele (kcnj8 V65M Cantú syndrome mutants. Their abcc9 G989E knock-in had a phenotype similar to that of the kcnj8 V65M knock-in mutants, confirming a more generalized genotype-phenotype correlation (The main aim of these two studies was to model heart disease in zebrafish. Therefore, the mutations the authors introduced needed to produce a tractable, disease-relevant phenotype. Farr et al. aimed to test whether the zebrafish 4 allele . Tessadorelation . These nIn sum, these three papers highlight various technical optimizations that can achieve robust and reproducible knock-in mutations, leading to zebrafish models with greater fidelity to the human diseases they are modeling. Lessons from these papers will be instructive to other investigators by providing important factors to consider when designing CRISPR/Cas9-based knock-ins in zebrafish. Although the generation of knock-in mutations continues to pose challenges, its successful implementation promises to be of tremendous value to the broader model organism community to study complex genetics that contribute to disease, in genes and in non-coding regions of the genome. By incorporating these mutants into high-throughput drug screening pipelines, the zebrafish holds great potential to provide rapid, cost-effective preclinical therapeutic data in a uniquely whole-organism vertebrate context that can streamline confirmatory murine studies and ultimately inform future clinical trials for patients with genetic disorders.


N-Glycolylneuraminic acid (Neu5Gc)-terminated glycans are present in all animal cells/tissues that are already used in the clinic such as bioprosthetic heart valves (BHV) as well as in those that potentially will be xenografted in the future to overcome end stage cell/organ failure. Humans, as a species lack this antigen determinant and can react with an immune response after exposure to Neu5Gc present in these products/cells/tissues. Genetically engineered source animals lacking Neu5Gc has been generated and so has animals that in addition lack the major αGal xenoantigen. The use of cells/tissues/organs from such animals may improve the long-term performance of BHV and allow future xenografting. This review summarizes the present knowledge regarding structural complexity and tissue distribution of Neu5Gc on glycans of cells/tissue/organs already used in the clinic or intended for treatment of end stage organ failure by xenografting. In addition, we briefly discuss the role of anti-Neu5Gc antibodies in the xenorejection process and how knowledge about Neu5Gc structural complexity can be used to design novel diagnostics for anti-Neu5Gc antibody detection. N-glycolylneuraminic acid (Neu5Gc), have been identified and they may contribute to the xeno-rejection process  in 1965 , but since its discovery in the 80's the deaminated neuraminic acid, KDN , is also included in the family of sialic acids  is another major type of sialic acid and is also expressed in deuterostomes. The initial characterization of Neu5Gc biosynthesis was explored by Schauer in the 1960's showing that Neu5Ac was converted by CMP-N-acetylneuraminic acid hydroxylase (CMAH) to the N-glycolyl form by addition of an oxygen atom to the N-acetyl group  of proteins involving enzymatic glycosylation of the protein backbone Kobata, . The varN- and O-linked glycosylation. N-linked glycans are usually attached via an N-acetylglucosamine (GlcNAc) to Asparagine (Asn). They are classified into three types, the high mannose (oligomannose), complex, and hybrid types. N-glycan biosynthesis is initiated via the synthesis of the Man5GlcNAc2 core unit on the dolichol pyrophosphate lipid anchor, which is then re-oriented to the luminal side of the endoplasmic reticulum (ER) membrane and extended to a Glc3Man9GlcNAc2 sequence. Transfer of the Glc3Man9GlcNAc2 oligosaccharide to the consensus sequence (N-X-S/T) in acceptor polypeptides is performed en bloc by the oligosaccharyltransferase (OST). N-glycans are further modified in the late ER and Golgi apparatus generating a plethora of N-glycan structures. The processing is possibly determined by the function of the glycan structures and the compartment where they are localized, resulting in a species- or even cell type-specific diversity of N-linked glycans , but other O-glycans may be linked to Ser/Thr via GlcNAc, fucose, glucose, mannose, or xylose   integrated in the outer layer of the lipid bilayer and the saccharide chain exposed to the cell environment. In contrast to glycoproteins that carry several different saccharide chains, only one single glycan is attached to each ceramide. As for protein-linked glycoconjugates, glycolipid structural complexity is vast. Immunogenic determinants are linked to various core saccharide chain types   to Gal and α2,6-linked to GalNAc  as opposed to anti-peptide antibodies may be explained by the contribution of entropic factors to binding, which is not solely reliant on enthalpic factors  have been identified by mass spectrometry in In investigations of glycolipids of naïve animal heart valves and pericardia, an unexpected finding was the lack of Neu5Gc-terminated gangliosides in pig heart valves  antibodies, the immunodominant group of which is Neu5Gc, were originally identified based on their ability to agglutinate erythrocytes of many animal species  for detection of HD antibodies using flat-bottomed 96-well plates coated with purified Neu5Gc-LacCer  aimed at detecting and quantifying a broader portion of the anti-Neu5Gc repertoire  responsible for the biosynthesis of Neu5Gc have been generated and may be used as source animals for future xenografting including procurement of tissues for bio-prosthesis manufacturing.In addition to the αGal antigen determinant, glycans with terminal Neu5Gc residues may constitute an immunogenic barrier for xenografts into humans. However, firm evidence for the role of Neu5Gc antibodies in xenograft rejection is lacking. Because the immune biology of the anti-Neu5Gc response is slightly different from both the ABO and anti-Gal antibody responses, further studies are needed to better define the exact role of the Neu5Gc antibody repertoire in the xenorejection process. Because carbohydrate antigens are quite resistant to destruction/removal by the procedures used in the manufacturing of bioprosthetic products of animal origin, these antigen determinants must be considered when using live as well as chemically modified animal cells/tissues/organs for treatment of end stage human organ failure. Animals genetically engineered to silence the CMP-All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


NPHP3 were found on one allele, in combination with a synonymous SNV , 18 base pairs from the exon–intron boundary within exon 15 of NPHP3. The only mRNA transcript amplified from wild-type whole blood showed complete splicing out of exon 15. Urine samples obtained from control subjects and the father of family 2, who carried the synonymous SNV variant, were therefore used to culture hUREC and allowed us to obtain kidney-specific mRNA. Control kidney mRNA showed retention of exon 15, while the mRNA from the patient’s father confirmed evidence of a heterozygous alternate splicing of exon 15 of NPHP3. Analysis of RNA derived from hUREC allows for a comparison of kidney-specific and whole-blood RNA transcripts and for assessment of the effect on function of putative splice variants leading to end-stage kidney disease.The majority of multi-exon genes undergo alternative splicing to produce different mRNA transcripts and this may occur in a tissue-specific manner. Assessment of mRNA transcripts isolated from blood samples may sometimes be unhelpful in determining the affect on function of putative splice-site variants affecting kidney-specific mRNA transcripts. Here we present data demonstrating the power of using human urine-derived renal epithelial cells (hUREC) as a source of kidney RNA. We report clinical and molecular genetic data from three affected cases from two families all with end-stage renal disease by 15 years of age. In both families, heterozygous variants which are predicted to effect function in  Next-generation sequencing techniques, including whole-exome sequencing (WES), has seen a huge surge in gene discovery, exemplified by gene discovery in various forms of inherited renal diseases. Indeed, ciliopathy exome capture and WES approaches have been successful in identifying numerous new genetic forms of nephronophthisis, an autosomal recessive renal ciliopathy –3. SimilNephronophthisis is a chronic interstitial nephropathy and an autosomal recessive form of childhood kidney failure. It has numerous genetic causes, although the underlying molecular genetic change remains elusive in around 50% of cases, due to either unknown genetic causes or hidden variants in known genes. A molecular genetic diagnosis is important to understand genotype/phenotype correlations, prevent the need for a renal biopsy, and to perform screening of at risk individuals.NPHP3 found in families with previously undiagnosed end-stage renal disease (ESRD).Here we present data demonstrating the power of using human urine-derived renal epithelial cells (hUREC) as a liquid biopsy of the kidney in order to isolate tissue-specific RNA, to demonstrate the functional effect  of a genomic DNA variant in www.LOVD.nl/NPHP3 . Full details of methodology are provided in Supplementary Data.All patients consented to this study. Ethical approval was obtained from the UK National Research Ethics Service Committee Northern and Yorkshire 09/H0903/36). Following informed and written consent, blood samples were obtained from affected patients and their relatives and healthy gender and age-matched controls. WES was performed as previously described  and targ/H0903/36In the first family, a 14-year-old presented chronic kidney disease stage 4 which progressed to unexplained ESRD by 15 years of age ) in combination with a synonymous SNV . The variants were present in compound heterozygous state with the frameshift variant inherited maternally and the synonymous change inherited paternally  in NPHP3, segregating from the mother, combined with the above-mentioned synonymous SNV in NPHP3 , segregating from the father , and therefore may have been implicated in abnormal splicing of exon 15 of NPHP3. In silico tools, including Human Splicing Finder suggested a functional effect, with probable disruption of exonic splicing motifs , should prompt a review of synonymous variants within the same gene to see if a synonymous variant may be the second allele which affects function.This report suggests that synonymous changes in known disease causing genes (such as the known NPHP genes) cannot automatically be filtered out from WES datasets before an in silico assessment of influence on splicing has been determined. The significance of synonymous variants in escribed . Here tw defects . In the NPHP3 by determining alternate splicing in kidney RNA isolated from urine-derived renal epithelial cells. This system will allow for a comprehensive comparison of kidney-specific and whole-blood RNA transcripts and for the ex vivo assessment of the functional effect of putative splice variants.In conclusion, we confirm the functional effect of a synonymous single-nucleotide polymorphism in Supplemental Methods and Figures


The aim was to appraise and synthesize studies evaluating the clinical and cost effectiveness of conservative interventions for chronic lower extremity musculoskeletal (MSK) conditions and describe their characteristics, including the type of economic evaluation, primary outcomes and which conditions.The search strategy related to economic evaluations of lower limb MSK conditions that used conservative therapies. Eight electronic databases were searched , as were the reference lists from included articles. The quality of articles was appraised using a modified version of the economic evaluations’ reporting checklist (economic) and The Cochrane Collaboration’s tool for assessing risk of bias .n = 25; 93%) were most common. The main approaches adopted were cost–utility analysis  or cost–effectiveness analysis . Two studies involved interventions including footwear/foot orthoses; for heel pain  and overuse injuries . Fifty per cent of economic evaluations adopted the EQ-5D-3L as the primary outcome measure for quality of life and quality-adjusted life year calculations.Twenty-six studies were eligible and included in the review. Economic evaluations of conservative interventions for OA or pain affecting the knee/hip (Economic evaluations have been conducted largely for exercise-based interventions for MSK conditions of the hip and knee. Few economic evaluations have been conducted for other clinically important lower limb MSK conditions. A matrix presentation of costs mapped with outcomes indicated increasing costs with either no difference or improvements in clinical effectiveness. The majority of economic evaluations were of good reporting quality, as were the accompanying clinical studies. This is a comprehensive systematic review of economic evaluations of conservative treatments for common lower extremity musculoskeletal conditions.Economic evaluations of hip and knee OA dominate cost-effectiveness literature for lower extremity musculoskeletal conditions.The reporting quality of clinical and economic evidence for lower extremity musculoskeletal conditions is generally good.Worldwide, >20% of the population have a musculoskeletal (MSK) condition . These cThe overall aim of economic evaluations in health care is to aid decision-makers to make efficient and equitable decisions . EconomiSystematic reviews are useful to assess evidence of effects, adverse effects and health-related quality of life and to identify gaps in research . SystemaAccordingly, the aims of this review were to identify and critically appraise the current evidence of clinical and cost effectiveness of conservative interventions for the treatment of lower extremity MSK conditions to determine whether there is sufficient evidence to inform policy and practice  and to ia priori (PROSPERO 2015: CRD42015024441 , SF-12v2 [1 CUA (SF-6D)], AQoL-6D (1 CUA) and HUI-3 (1 CUA). Each of these tools produces utility values that can be used in the calculation of quality-adjusted life years, essential for comparisons across different diseases. Three studies collected only clinical measures of health for hip OA and knee OA and were therefore restricted to CEA methodology (i.e. cost per unit of improvement in condition-specific outcome measures) , 42, 47.et al. [et al. [et al. [The reporting quality of economic evaluations and related clinical studies was generally good . This is unsurprising given the prevalence of hip/knee OA problems in populations  and the et al. [post hoc economic evaluation using information collected during the clinical study. In addition, consideration should be given to whether weaker reporting quality might also be, in part, a consequence of translation .et al. [To make decisions about resources, it is useful to present information on costs and outcomes for each of individual intervention arm with the comparator. For example, Barton et al.  involvedThe outcome measures adopted by included studies were largely appropriate for the evaluations of interventions’ clinical and cost effectiveness. Provided that sufficient validation and evaluation of measurement properties have been undertaken, patient-reported outcome measures provide a means by which to assess and quantify the health consequences of health care for patients with specific conditions. In contrast, economic evaluation requires comparability across different disease conditions via use of a common metric. Quality-adjusted life years provide a common metric and can be calculated using a preference-based single index measure for health. These can be collected using generic preference-based measures, such as EQ-5D (used by the majority of included studies) and also by conversion to SF-6D from SF-36 and SF-12. However, the measurement properties of the generic preference-based measure (i.e. EQ-5D) for specific conditions should be known/evaluated before use in that specific clinical context. The lack of specificity of generic preference-based measures has been highlighted as a concern . Given tThe paucity of evidence about cost effectiveness of conservative, non-pharmacological and non-surgical rehabilitative interventions for the range of lower limb MSK conditions is a concern. Consistent pressures on demand for health care worldwide, coupled with a changing landscape owing to demographic and health-care developments, make the need for evidence concerning clinical and cost effectiveness more pertinent. Including economic evaluation in clinical trial design will build the evidence base about clinical and cost effectiveness. Presenting the evidence in a form such as the matrix used for this review aids decision-makers in considering clinical and economic evidence together. The ideal intervention would be in A1, where it would be both more effective and use fewer resources, but C1 is typically where new treatments map. Often, a new intervention offers improvements in outcomes but generally will also cost more (i.e. increased resource use). The studies included in this review mainly fall into C1 and C2. Presentation of information about clinical and cost effectiveness in a matrix is intended to facilitate discussions about ways to achieve maximal health gain through resource allocation decisions. C1 indicates greater costs with greater effectiveness. To make use of this, decision-makers should also consider specific health-care system implications (for costs) at their local system level, and country-specific cost-effectiveness thresholds (explicit or implicit). C2 costs more and does not deliver outcomes any better than the comparator ; therefore, it would be advisable not to introduce this intervention.We intended this systematic review to be broad in scope, to encompass any type of economic evaluation of any conservative intervention for any lower extremity MSK condition of mechanical aetiology. To focus on a specific disease and a specific physical location using a PICO-type  strategy would have narrowed the returned titles but at the cost of restricting confidence that all relevant studies had been identified.There are limitations to this systematic review that are worth highlighting. Restricting studies to conservative treatments excluded co-provision of treatments . This was purposeful in order to determine the reported clinical effectiveness of conservative treatment. It might be that co-provision of treatment would be more aligned to real-world health-care practice and should be considered. The desire to consider clinical effectiveness meant excluding economic evaluations of interventions undertaken in general populations. Research of this nature is often focused on preventative measures, and the economic interest is prediction of prevented demand and avoided costs, rather than management of existing health-care budgets given current demand for health care.The dominance of exercise-based interventions for MSK conditions of the hip and knee, with few economic evaluations of other clinically important lower extremity conditions, such as foot and ankle disorders, highlights a gap in the literature and therefore current knowledge. Common MSK conditions of the lower leg, such as Achilles tendinopathy or plantar fasciitis, are prevalent  and haveSupplementary DataClick here for additional data file.


N-isopropylacylamide and acrylic acid in the presence of a cross-linker N,N′-methylenebisacrylamide. The electrostatic interactions between the cationic cetyltrimethylammonium bromide (CTAB) stabilized Au NRs and anionic microgel particles were expected to occur in order to prepare stable Au NRs-microgel composite particles. The optical and structural characterization of the composite was achieved using UV-Vis spectroscopy, Field emission scanning electron microscopy (FESEM), Transmission electron microscopy (TEM) and dynamic light scattering (DLS). TEM image shows that Au NRs are attached on the surface of the microgel particles. Dynamic light scattering measurements prove that the composite particles are temperature responsive, which means the particles undergo a decrease in size as the temperature increases above its phase transition temperature. In vitro cytotoxicity of the composite materials were tested by 3--2,5-diphenyltetrazolium bromide (MTT), Lactate dehydrogenase (LDH), and hemolysis assay, which showed non-toxicity (biocompatibility).In this paper, we report an easy route for preparing new metal nanorod-polymer composites consisting of gold nanorods, Au NRs, and temperature responsive copolymer “microgel” particles. The microgel particles of ~200 nm in size, which contain carboxylic acid groups, were prepared by surfactant-free emulsion polymerization of a selected mixture made of  N-isopropylacrylamide) (PNIPAM) and its copolymers or microgels/nanogels  have been an area of interest due to the fact that this material has the capacity to alter or respond to external stimuli  microgels with a hydrodynamic diameter of ca. 200 nm were synthesized by surfactant-free emulsion polymerization. The copolymer products were characterized by NMR. The 1H NMR spectra of the copolymer is shown in 3 protons of the NIPAM units were observed in the 1H NMR spectra of the copolymers, and the signal from 1.9 to 2.6 ppm ppm was caused by the polymer backbone assigned to the (–CH2–CH–) protons. The peak at 12.0 ppm was attributed to the (–COOH) protons of the acrylic acid groups present in the copolymer.The copolymer poly microgel particles and cationic surfactant (CTAB) stabilized Au NRs. pH of the as-synthesized microgel solution was adjusted to pH 9 so that a proton is removed in the carboxyl groups present in the microgel particles. At pH 9, the polymer is highly swollen due to osmotic effect and electrostatic repulsion between the AAc residues, which both favour incorporating the positively charged Au NRs into the negatively charged voids of microgel particles through electrostatic interactions. A schematic diagram of the formation of Au NRs-microgel composite particles is shown in −1 , from the CH3–N+ moiety of CTAB (−1 (CH3 asymmetric stretching), 1649 cm−1 , and 1546 cm−1 , [−1 in the microgel sample  and the swollen (Vswollen) state. Comparing the relative swelling volume shows that both components behave very similarly. No significant difference in swelling behavior from the pure microgel particles was observed. The as-prepared colloidal solution of Au NRs-microgel composites were highly stable and did not show any sign of aggregation of precipitation even after storage for several months at ambient temperature. The temperature-responsive swelling properties of the composite particle were studied using DLS experiments. As expected, there is a significant change in size induced by the change in the temperature. As shown in The MTT assay evaluates the mitochondrial function by measuring the ability of viable cells to reduce MTT into a blue formazon product . LDH is Furthermore, hemolytic activity was carried out by using the blood agar plate method. Hemolysins are enzymes that lyse red blood cells and are formed after cell deterioration ,46. The This discussion centers on a versatile and easy method for the assembly of gold nanorods onto the microgel particles based on electrostatic interactions. This method includes the introduction of carboxylic groups and their subsequent deprotonation of microgel particles and then uptake of cationic Au NRs through the presence of attractive Coulomb interactions in a solution. The as-prepared composite particles are temperature-responsive and undergo a decrease in particle size as the temperature increases above the phase transition temperature. The as-prepared Au NRs-microgel composite samples were stable for months when stored at ambient temperatures. When using in vitro studies, the Au NRs-microgel composite particles show no toxicity to the given cell lines. Therefore, it could find important applications in both drug delivery and photothermal therapies.


There is an error in Fig 2. The wrong image was included as part of the final revision process such that lipid data for female controls were duplicated and shown as also representing female LERKO. The authors have provided the correct version here after a re-analysis of the original samples to confirm the results presented in Fig 2, as the original raw image underlying Fig 2 is no longer available. Raw data for all figures (including the data for the supplementary figures and tables) are available except for:Fig 1C: The panel for Western blot analysis of ERα in male liver lysate.Fig 2: All the raw data for this figure as stated above.S1 Fig: Coomassie-blue staining of membranes for male samples.The original female liver samples from the study have been re-analyzed for liver lipids. The conclusion that analysis of lipid content revealed no observable differences between female LERKO and control mice is confirmed. Representative images are shown in Liver samples from female LERKO and Control mice  were analyzed at the core facility of morphological phenotype analysis (FENO), Karolinska Institutet, by Oil-Red-O staining. Briefly, frozen liver tissues were immersed in OCT Cryomount , then frozen and sectioning at six micrometer. Frozen liver cryo sections were air dried, incubated in Oil-Red-O working solution for 3 min then washed in distilled water. Sections were subsequently lightly counterstained with hematoxylin.Image analysis of lipid content was performed with the Histoquant module in 3DHISTECH Panoramic Viewer. Five areas were selected from the slides for each mouse liver, and the percentage of the area covered with Oil-Red-O was calculated for each selected area. The Cumulated percentage of the areas covered with Oil-Red-O staining was then calculated for each mouse.S1 FileA. Female liver tissue sections were analyzed for lipid content. Lipid staining reveals similar amounts of lipid droplets in livers of control (CT) and LERKO animals. Representative images are shown. B. Percentage of areas with Oil-Red-O staining in the livers from female CT and LERKO mice (n = 3).Liver lipids in LERKO and control female livers (TIF)Click here for additional data file.


Histological analyses were performed with Masson’s Trichrome and Picrosirius Red stains, and antibodies against Melan-A and αSMA. The collagen content was measured in xenografts by quantitative polarization microscopy. In patient hepatectomy samples, activated HSteCs and their pathological matrix were localized surrounding the malignant lesions. In the mouse xenograft model, the number of hepatic metastases was increased when human HSteCs were co-inoculated. Histological analyses revealed a significant recruitment of HSteCs near the micro/macrolesions, and an increase in fibrillar collagen production. Our results show that HSteCs can provide a permissive microenvironment and might increase the therapeutic resistance of metastatic UM. Uveal melanoma (UM) is a malignant intraocular tumor that spreads to the liver in half of the cases. Since hepatic cells could play a role in the therapeutic resistance of metastatic UM, the purpose of our study was to investigate the pro-invasive role of hepatic stellate cells (HSteCs) in metastatic UM at the micro- and macro-metastatic stages. We first performed an immunostaining with the alpha-smooth muscle actin (αSMA) to localize activated HSteCs in UM liver macro-metastases from four patients. Their accumulation of collagen was assessed with Masson’s Trichrome stain. Next, we inoculated metastatic UM cells alone or with human HSteCs in triple-immunodeficient mice, in order to determine if HSteCs are recruited as early as the micro-metastatic stage. The growth of metastatic foci was imaged in the liver by  Uveal melanoma (UM) is the most common primary intraocular tumor in adults , and is Considering that activated HSteCs play a pivotal role in the metastatic growth of other tumors with a hepatic tropism, we first investigated their localization in liver macro-metastases resected from UM patients . Using αAn accumulation of collagen was seen around these αSMA-positive cells, as highlighted by the Masson’s Trichrome stain MT; . In PatiPrkdc Il2r Gamma (NCG) and NOD scid Gamma (NSG) triple-immunodeficient mice or when the hTERT-HSC cell line versus primary HSteCs were co-injected with UM cells. We established a mouse xenograft model of metastatic UM by injecting metastatic UM cells with human HSteCs into the spleen of triple-immunodeficient mice Table 2; . Using aAs the main objective of our study was to assess the synergic interactions between UM cells and HSteCs in metastatic growth, we then co-inoculated the TJU-UM001 cell line with different ratios of human HSteCs in triple-immunodeficient mice . Fluoresp = 0.0033).Activated HSteCs were recruited by both micro-  and macro-metastases  and metastases between 50–500 µm (p = 0.0054), even at the 10:1 ratio. These results confirmed the early role of stellate cells in favoring the growth and invasion of UM cells in the liver.We also analyzed the size in diameter of the metastases, to see if the ratios of HSteCs impacted their distribution between micro-and macro-metastases D. Once ap = 0.0512). Since the host’s HSteCs can also contribute to the collagen deposition near the malignant lesions, we investigated the collagen micro-architecture into the spleen, where there are no murine stellate cells. In this case, all spleens injected with both human HSteCs and UM cells showed a significant increase in collagen content and presented regions of altered fibrillar collagen architecture compared with the spleens injected only with UM cells (p < 0.0001). Given that activated HSteCs were recruited in the vicinity of metastases of all sizes, and that collagen deposition was observed with the Masson’s Trichrome stain, we characterized in more detail the collagen content within the metastatic microenvironment using quantitative polarization (QPOL) microscopy . SurprisOverall, our data demonstrate that HSteCs are activated by UM cells as early as the micro-metastatic stage, and their synergic interactions lead to modifications of the hepatic stroma and an increase of the metastatic growth.In recent years, components of the microenvironment of solid tumors, such as stromal cells and their pathological extracellular matrix, have gained attention because they can significantly impair the therapeutic response. Our findings support the hypothesis that HSteCs are the most reactive stromal cells of the liver in presence of invading UM cells. Indeed, we showed that matrix-producing stellate cells are abundantly present in UM patients’ hepatic macro-metastases. We thus established a mouse xenograft model of metastatic UM to assess if stellate cells were involved early in the growth of UM cells in the liver. We demonstrated that activated stellate cells were recruited at both the micro- and macro-metastatic stages, and they synthesized fibrillar collagen in the vicinity of metastases. In addition, the metastatic load was increased in immunodeficient mice when human HSteCs were co-injected, confirming that their synergetic interactions increased the liver susceptibility to UM invasion.Coupland and collaborators previously identified “hepatic fibrosis” and “HSteC activation” among the most differentially regulated biological processes when they compared secretomes of UM cells and choroidal melanocytes by proteomic profiling . In addiWe previously demonstrated a higher secretion of the pro-fibrogenic interleukins IL-6 and IL-8, and an increased expression of transmembrane integrins when metastatic UM cells were co-cultured with HSteCs . Our his2. This study followed the tenets of the Declaration of Helsinki and was approved by our institutional human experimentation committee .Liver metastasis tissues were resected from UM patients, after their written informed consent was obtained  or NOD scid Gamma  triple-immunodeficient mice (One million of fluorescent UM cells (tdTomato or Green Fluorescent Protein) were inoculated alone or with HSteCs  into the spleen of NOD CRISPR ent mice . Six weeFive-µm liver sections were colored with the Masson’s Trichrome (MT) stain. Tissue sections were also processed for immunohistological analysis with antibodies against Melan-A  and αSMA  in a Dako Autostainer Plus Link, according to the manufacturer’s protocol using the EnVision peroxidase procedure with the DAB or Magenta chromogen (Agilent Dako) . The dem−1; Thorlabs, Newton, NJ, USA) positioned directly under the illumination source and above the condenser and a circular analyzer [http://rsb.info.nih.gov/ij/). For colorimetric image analysis, acquisitions were done using the Axiocam 105 and the Picrosirius Red signal was isolated from the resulting RGB image by subtracting the green and blue channels from the red channel. A threshold was then applied to the computed image using the Otsu’s method and the total area of collagen regions was calculated.Liver sections were colored with the Picrosirius Red stain. The polarized signal was then imaged and quantified using both white and monochromatic red lights in QPOL as described previously ,47. Briep < 0.05) using one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test .Data are presented as box-and-whisker plots. The central box is the interquartile range with the median indicated by the horizontal line, and whiskers extend to the lowest and highest data values. Data were analyzed for statistical significance (We demonstrated that the co-inoculation of metastatic UM cells with human HSteCs increased the number of metastatic lesions smaller than 500 µm in our experimental model of UM liver metastasis. An accumulation of pathological extracellular matrix was co-localized with the activated αSMA-positive stellate cells in the vicinity of metastases. This matrix remodeling and contraction may increase the intratumoral interstitial fluid pressure, which will be detrimental for the uptake of anticancer drugs. New combined therapies targeting both the metastatic UM cells and the fibrogenesis of stellate cells are thus warranted.