Decoding laminar information across deep brain structures and cortical regions is necessary in order to understand the neuronal ensembles that represent cognition and memory. Large animal models are essential for translational research due to their gyrencephalic neuroanatomy and significant white matter composition. A lack of long-length probes with appropriate stiffness allowing penetration to deeper structures with minimal damage to the neural interface is one of the major technical limitations to applying the approaches currently utilized in lower order animals to large animals. We therefore tested the performance of multichannel silicon probes of various solutions and designs that were developed specifically for large animal electrophysiology. Neurophysiological signals from dorsal hippocampus were recorded in chronically implanted awake behaving Yucatan pigs. Single units and local field potentials were analyzed to evaluate performance of given silicon probes over time. EDGE-style probes had the highest yields during intra-hippocampal recordings in pigs, making them the most suitable for chronic implantations and awake behavioral experimentation. In addition, the cross-sectional area of silicon probes was found to be a crucial determinant of silicon probe performance over time, potentially due to reduction of damage to the neural interface. Novel 64-channel EDGE-style probes tested acutely produced an optimal single unit separation and a denser sampling of the laminar structure, identifying these research silicon probes as potential candidates for chronic implantations. This study provides an analysis of multichannel silicon probes designed for large animal electrophysiology of deep laminar brain structures, and suggests that current designs are reaching the physical thresholds necessary for long-term (∼1 month) recordings with single-unit resolution. Spatiotemporal neuronal ensembles distributed throughout laminar structures in the brain such as the cortex and hippocampus are presumed to be the substrate for cognition and memory. Oscillatory activity and resulting power bands are transmitted throughout the layers of laminar structures as different inputs are driven at different frequencies depending on their area of origin . LaminarThe hippocampus is an example of a deep laminar structure that is highly involved in encoding episodic memory formation presumably via spatiotemporal ensembles of neurons . Once muRecent advances in high-density linear electrode arrays and wireless recording technology could tremendously enhance translational research studies of large-scale networks in species with large, gyrencephalic brains . IdeallyTechnology necessary to produce finer features at longer length has only recently became available, including stitching across the dye in the photomask process to utilize smaller feature processes. New multichannel silicon probes designed for large animal electrophysiology allow for simultaneous recordings of fields and spikes from multiple layers of laminar structures such as cortex and hippocampus, and we therefore tested various designs of laminar silicon probes suitable for large animals. Across probe designs, we compared neurophysiological characteristics and biomechanical compatibility based on the variables such as electrode site layout and probe thickness. Current technology in silicon probes appears to have reached the critical size/feature dimension while maintaining insertion stiffness, proving that single units in deep brain structures can be recorded for chronic periods without extensive damage to the neural interface. Further refinement of these probe technologies in combination with chronic drives and wireless technology may usher in a new high-density era for previously unexplored regions of cortex, hippocampus and other non-laminar deep structures in large animals and potentially humans.RRID: NSRRC_0012) and underwent the current studies at the approximate age of 5–6 months at a mean weight of 38 ± 3 kg . At this age Yucatan pigs are considered post-adolescent with near-fully developed brains, while young enough to be of a manageable weight for procedures and behavior  and 0.5 mg/kg of midazolam . Animals were intubated with an endotracheal tube and anesthesia was maintained with 2–2.5% isoflurane per 2 liters OAll animals underwent implantation of multi-channel silicon probes under sterile conditions similar to acute implantation procedure described previously . BrieflyChronic 32-channel silicon probes for large animal electrophysiology were designed with ATLAS Neuroengineering  and NeuroNexus . In addition, acute 64-channel silicon research probes were developed in a collaboration with SB Microsystems  and Cambridge NeuroTech . Designs of multichannel silicon probes are shown in All multichannel silicon probes were designed with one low-impedance channel placed 1–2 mm above the adjacent proximal channel, which was recorded for use as an internal reference . For dor2 (2 were coated with Pt (nsites = 155).32-channel silicon probes had custom designed individual sites arranged in groups of four closely spaced sites or tetrodes . Three e2 . The ind with Pt . The ave2 .32-channel silicon probes had custom designed individual sites arranged in groups of four closely spaced sites or tetrodes . Three e2 . The ind2 for NN32/EDGE150 and 4,000 μm2 for NN32/EDGE80 .32-channel silicon probes had a linear site layout, with the electrode sites placed 200 μm apart. The individual electrode sites were also strategically positioned at the edge of the probe . The tot2/EDGE80 . The ind2. The individual electrode sites were 165 μm2 (nsites = 63).64-channel silicon probes had a linear site arrangement, with the individual electrode sites positioned at the edge of the silicon probe . The ele 165 μm2 . The ind2. The individual electrode sites were 165 μm2 (nsites = 63).64-channel silicon probes had individual electrode sites arranged in poly-2 style . The ind 165 μm2 . The indElectrophysiological recordings with 32-channel silicon probes  were performed in awake behaving animals at various time points at up to 6 months post implantation. Wide bandwidth neural signals were acquired continuously, sampled at 30 kHz with FreeLynx digital acquisition system, amplified and either wirelessly transmitted to Digital Lynx 4SX acquisition system with Cheetah recording and acquisition software during behavioral space recordings or stored to an on-board microSD memory card during home cage recordings . Electrophysiological recordings with 64-channel silicon probes (CAMB64/EDGE and CAMB64/POLY-2) were performed acutely under isoflurane anesthesia, with wide band neural signals continuously acquired, sampled at 32 kHz and amplified with Digital Lynx 4SX acquisition system with Cheetah recording and acquisition software .1 (RRID: SCR_014480), which was developed for higher density electrodes, and manually refined with KlustaViewa2, or phy3 software packages. The Klusta packages are designed to construct putative clusters from all probe channels simultaneously by taking advantage of spike timing and the spatial arrangement of electrode sites (RRID:SCR_001622).Neural signals acquired from the 32 or 64 channels on the silicon probes were bandpass filtered (0.1 Hz to 9 kHz) in real time prior to sampling. Off-line spike detection and sorting was performed on the wideband signals using the Klusta packagede sites . After mRRID:SCR_001622) and processed using a combination of custom and modified scripts from the freely available Matlab packages FMAToolbox , Chronux , and EEGLAB   .Histological analyses were performed to identify electrode tracks on brain tissue from male Yucatan miniature pigs. At the study endpoint, transcardial perfusion was performed under anesthesia using 0.9% heparinized saline followed by 10% neutral buffered formalin (NBF). After further post-fixation for 7 days in 10% NBF at 4°C, the brain was dissected into 5 mm blocks in the coronal plane and processed to paraffin using standard techniques , 2018. ETissue sections were dewaxed in xylenes and rehydrated to water via graded ethanols before being immersed in 1% LFB solution  at 60°C for 4 h. Excess stain was then removed by immersion of sections in 95% ethanol. Differentiation was performed via immersion in 0.035% lithium carbonate for 10 s followed by multiple immersions in 70% ethanol until the gray and white matter could be clearly distinguished. Slides were rinsed and counterstained via immersion in preheated 0.1% CV solution  for 5 min at 60°C. After further rinsing, slides were differentiated in 95% ethanol with 0.001% acetic acid, followed by dehydration, clearing in xylenes and cover slipping using cytoseal-60.Tissue sections were dewaxed in xylenes and rehydrated to water via graded ethanols before being immersed in Weigert’s Working Hematoxylin solution, prepared by mixing equal parts of Weigert’s Iron Hematoxylin A  and Weigert’s Iron Hematoxylin B  for 10 min. After rinsing in distilled water, tissue sections were stained for 3 min in Van Gieson’s solution . After further rinsing, slides were differentiated in 95% ethanol with 0.001% acetic acid, dehydrated, cleared in xylenes and coverlipped.RRID: AB_2336826) was performed for 30 min, also at room temperature. Lastly, the 3,3′-diaminobenzidine (DAB) peroxidase substrate kit  was applied according to manufacturer’s instructions. All sections were counterstained with hematoxylin, dehydrated in graded ethanols, cleared in xylenes, and cover slipped.Immunohistochemistry (IHC) labeling was performed according to previously published protocols , 2018. BRRID:SCR_002798). Single unit waveforms recorded with silicon laminar probes are displayed as mean ± SD. Amplitudes of single units are displayed as mean ± SEM. Impedance of silicon probes is shown as mean ± SEM.The data was analyzed using Graphpad Prism software, version 7  in awake behaving pigs, and for neuropathological changes induced by their placement in the porcine hippocampus over time.Using silicon probes of various designs, the laminar structure of pig dorsal hippocampus was examined electrophysiologically under awake behaving conditions . Since t2) placed 1–2 mm above the most-proximal probe site (depending on the silicon probe design)  of the ventricle. Designing silicon probes with an internal reference for intra-hippocampal (or other deep brain structures) recordings may help to reduce noise and improve the signal quality over time. We estimated how an introduction of the internal reference affects the noise during awake and anesthetized hippocampal recordings by comparing power of hippocampal oscillations at the level of the pyramidal CA1 layer referenced to either an internal or skull screw reference . All mul design) . For dor design) . Under a design) . The skuWe evaluated the stability of neurophysiological signals recorded from pig hippocampus over time in a sub-sample of these probes. Changes in hippocampal signals were evaluated using power of LFP signals over time . Since tNext, we evaluated the amplitude and number of single units recorded from the porcine hippocampus over time . Since tnsingleunits = 96), it decreased over time to 47 ± 6 μV  at D7 and 36 ± 2 μV  at D14. In addition, at D21 post implantation, single units were detected only from the narrower design of silicon probe (NN32/EDGE80). The average amplitude of single units was 28 ± 6 μV , just above the threshold for spike detection (nsingleunits = 40 vs. NN32/EDGE80: nsingleunits = 17). However, after 3 weeks (D21), no units were detectable with NN32/EDGE150 silicon probes, while the NN32/EDGE80 still recorded single units (nsingleunits = 3), suggesting a greater amount of tissue damage produced by the silicon probe with a larger cross-sectional area  demonstrated (as expected) a degree of tissue disruption with associated hemorrhage around the probe track , multiple histological changes were observed surrounding the silicon probe track. Notably, in one case the choroid plexus could be visualized entering the alveus, presumably having been translocated during the insertion as has previously been visualized   affects stability of neurophysiological signals recorded chronically (over time), we histologically evaluated tissue damage in the porcine hippocampus produced by silicon probes of various designs . Microscbe track . In addibe track . While, n n = 2) .To increase unit yield and to improve longevity of neurophysiological signals  over time and to reduce pathological tissue response to chronic silicon probe implantations, novel 64-channel flexible silicon probes were developed in a collaboration with SB Microsystems and Cambridge NeuroTech and tested acutely (under anesthesia) in pigs.4 square microns [as is the case for a 100 μm thick and 250 μm wide probes , we aimed to custom-design novel multichannel silicon probes with minimal cross-sectional area suitable for insertion. The goal was to reduce the profile of the probe, potentially reducing damage to the neural interface and inflammation. Increasing a probe’s thickness imparts greater mechanical stiffness of the silicon probe which benefits insertion, but causes a further compliance mismatch between probe and brain tissue, potentially contributing to gliosis and local cell death for chronic implementations. In order to test stiffness/insertion abilities empirically, mock versions of CAMB64 silicon probes were created to have a fixed width of 80 μm, while the thickness of the mock probes varied in a range of 25–100 μm. While 25 μm thick mock silicon probes could be inserted into cortex, they failed to advance to the hippocampal structure. In contrast, mock silicon probes with thickness of >35 μm were capable of insertion into the hippocampus. Based on the insertion ability and acute neuropathology, the thickness of 35 μm was selected for production of initial research silicon probes, giving a minimum dimension for the probes as stiffness increases linearly with width.To potentially reduce tissue damage even further, we aimed to design silicon probes to have a full-length silicon rather than a standard “silicon plus guide tube” design. By reducing the tissue impact during insertion, smaller, less brittle silicon probes may also increase the survival rate of neurons in close proximity to the travel path. In order to produce longer silicon probes without the need for the extension tube, novel research silicon probes were manufactured using projection (“stepper”) lithography rather than contact lithography, allowing us produce 0.5 μm resolution features and changing the width/channel count trade-off in our favor. However, the use of projection lithography for these large devices (up to 40 mm in length) meant that the 5X master reticle exceeded the maximum allowed size, and required us to develop a process for stitching multiple exposures of multiple reticles across the process wafer. This novel lithography process allowed for the full length of the probe to be manufactured in silicon. The mock version of these new probes was capable of insertion into the dorsal hippocampus without support of metal guide tubes, which also resulted in less damage to cortical tissue en route to the hippocampus. The new lithography technology used to create novel research silicon probes also allowed for a doubling of the number of sites on the probe, leading to the first 64-channel silicon probes for large animals that are made exclusively from silicon wafer at lengths capable of reaching much of the large animal brain. In addition, the individual electrode sites on CAMB64/EDGE and CAMB64/POLY-2 silicon probes were composed of gold (Au) coated with an organic polymer, lowering their impedance and decreasing the noise during recordings in comparison to other silicon probes used .While a linear design is fit for a wide range of applications, isolation of single unit activity may be difficult with only a linear site arrangement. To utilize commercial spike sorting software available at the time , individual sites on silicon probes with a linear design had to be artificially grouped into sets of four (tetrodes) in order for single units to be sorted. Previously, single units recorded with 32-channel silicon probes of the earlier designs could not be properly isolated with spike sorting software available at the time, partially due to potential overlap between putative units between the artificial tetrodes.To address this issue, we initially custom-designed silicon probes (ATLAS32/TET and NN32/TET) to have most of the individual sites arranged in four sites placed close together (tetrodes), allowing for high-quality cell discrimination in hippocampal recordings . Extra eThe advent of laminar spike-sorting software removed the necessity for specific tetrodes to be recreated in the silicon probes, however, probe geometry is still an important part of the spike-sorting process . We therSilicon multichannel probes designed and used for large animal hippocampal electrophysiology allow for a longer area of coverage in contrast to single site recordings, and reveal laminar structure and correlation of unit activity with field behavior. We have performed a retrospective analysis of chronically implanted pigs from our experiments in order to characterize the differences in electrophysiology over time with various probe sizes and geometries from different manufacturers. In addition, we have examined the neural interface out to 6 months post-implantation in order to assess whether neuropathology in the large animal hippocampus resembles that previously reported in rodents. Differing probe geometries were examined in a new higher density research probe in order to assess their ability for sorting units. Long-length silicon probes may also replace the need for multiple acute insertion experiments if the animal is chronically implanted and/or a drive is utilized.Our initial attempts at chronic implantations using probes that were developed for acute work and prior to modern spike sorting methods for laminar probes were successful only for field recordings. Units that appeared during the implantation surgery were either not present or were significantly attenuated even 24 h post implantation. Histological examination suggested that the insertion damage to the neural interface, and sometimes even the cytoarchitecture, was significant with these larger dimension probes. In addition, attenuation of unit amplitude over time as well as field power in various frequency ranges is potentially explained by both cellular and collagen encapsulation of the probe as demonstrated at 6 weeks and 6 months post implantation. We therefore redesigned these probes to increase laminar coverage, and plated the electrodes on the edge of the probe in order to take full advantage of the contacts as has previously been described . This chWe also tested various designs of silicon probes available from and developed with manufacturers for the ability to separate single units. Linear, tetrode plus linear and poly-2 site arrangement designs were evaluated for single unit vs. multi-unit cluster isolation. Spacing between individual electrode sites played a significant role as 200 μm spacing (as on NN32/EDGE style probes) increased the number of multi-units sorted. This could affect the precision of research studies focused on activity of hippocampal neurons, for example characterization of place cells or interneurons. Interestingly, some CA1 units spanned not two, but three electrodes, suggesting that large putative pyramidal cells that run parallel to the probe can be detected across greater distances than those previously reported, and further suggesting that lateral electrode separation for analysis in these structures may be helpful. Although most of the probes with a linear design site arrangement  were able to record neuronal activity of individual neurons, silicon probes with a tetrode (ATLAS32/TET and NN32/TET) and a poly-2 (CAMB64/POLY-2) design site arrangement had better cluster separation with currently available sorting algorithms, as the proportion of single units to multi-units recorded with a given probe increased. Previous acute examinations in large animals have also noted the usefulness of the parallel geometry in isolating units, even prior to the advent of new sorting techniques . New sorMock probe testing of the CAMB64 25 mm-long probes demonstrated that 35 μm shank thickness for 80 μm wide probes strikes an ideal compromise between ease of tissue penetration and appropriate targeting vs. the need to maintain small device dimensions. This allowed for production and testing of the 30 mm long 64-channel probes described above, which yielded substantially more units in the poly-2 style probe in acute testing. The balance between maximal stiffness, length and electrode density with minimal cross-sectional area in order to reduce damage to the neural interface remains a significant challenge in large animals. Designs such as the NeuroNexus’ NN32/EDGE probe and the new CAMB64 probe approach these challenges with different design solutions, but appear to have reached a critical threshold where the full depth can be reached with enough electrodes for sorting, and allowing for single unit detection out to a month post implant without being driven. Future chronic implantation of the CAMB64 probes will further test whether the trade-off of device width for increased channel count increases single unit yield, and for how long. These probes are also not at the minimal feature dimensions for this technique, as electrode yield in the initial testing run was also a consideration. Future dimensions of 80 μm wide, 64-channel probes may also increase yield by reducing damage to the neural interface, but this remains to be tested as well.Passive laminar silicon probes (as described above) are but one solution to the problem of laminar recordings in large animals . Other pWe hope that this study will help to facilitate adoption of novel silicon multi-channel probes suitable for chronic implantations in large animals, by comparing silicon probes available for use in large animal electrophysiology, as well as comparing them to a new design and process. NeuroNexus EDGE style probes (NN32/EDGE80) were determined to yield the largest number of units of the available probes for acute and chronic recordings from laminar structures due a linear edge site arrangement, 6 mm of coverage, and potentially reducing damage to the neural interface upon insertion. In addition, cross-sectional area was found to be one determinant of silicon probes’ performance. Novel CAMB64 silicon probes with a poly-2 design were found to have an even better single unit separation and a denser sampling of the laminar structure than existing linear probes. By increasing channel density, we were able to better visualize laminar structure and create offset geometries that enabled better unit sorting. Channel density, site arrangement, and the physical profile of the silicon probe are all important factors to consider when designing probes for acute and chronic implantations to study laminar structures over time in awake behaving animals. We hope these results will lower the threshold for adoption in chronic implantations, by demonstrating consistent yields in laminar electrophysiological recordings in large animals using commercially available probes. We hope that in combination with new wireless technologies allowing for freely moving behavior, this will support new discoveries in both hippocampal and neocortical neurophysiology. In addition, better detection and understanding of laminar circuitry and changes in human disease  are needed, and therefore viable electrodes need to be tested first in translational large animal models prior to clinical use.This study was carried out in accordance with the recommendations of the University of Pennsylvania, an AALAC accredited institution. The protocol was approved by the University of Pennsylvania IACUC animal care committee.AU and JW: conceptualization and visualization. AU, PK, TH, BJ, and JW: methodology. AU, CA, PK, and JW: software. AU, CA, PK, HC, VJ, and JW: validation. AU, CA, CC, and KG: formal analysis. AU, CA, PK, KG, and JW: investigation. DC and JW: resources. AU: data curation and writing-original draft preparation. AU, CA, KG, and JW: writing-review and editing. JW: supervision, project administration, and funding acquisition.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.


The hydrodynamic and thermal behavior of the electroosmotic flow of power-law nanofluid is studied. A modified Cauchy momentum equation governing the hydrodynamic behavior of power-law nanofluid flow in a rectangular microchannel is firstly developed. To explore the thermal behavior of power-law nanofluid flow, the energy equation is developed, which is coupled to the velocity field. A numerical algorithm based on the Crank–Nicolson method and compact difference schemes is proposed, whereby the velocity, temperature, and Nusselt number are computed for different parameters. A larger nanoparticle volume fraction significantly reduces the velocity and enhances the temperature regardless of the base fluid rheology. The Nusselt number increases with the flow behavior index and with electrokinetic width when considering the surface heating effect, which decreases with the Joule heating parameter. The heat transfer rate of electroosmotic flow is enhanced for shear thickening nanofluids or at a greater nanoparticle volume fraction. The development of the micro-electric mechanical system (MEMS) has received great attention because of its innovative application in chemical, medical, and biological-related industries. For instance, Lab-on-a-chip, which conducts experiments by processing bioliquids or chemical solutions on a microchip, has become increasingly popular . ElectroThe hydrodynamic behavior of EOF of different fluids is investigated in different microchannels, such as slit , rectangThe thermal characteristics have to be carefully considered due to the involvement with fluid flow and the applications in the cooling systems of a MEMS. The Joule heating effect that is an inherent phenomenon in EOF resulting from the ohmic resistance of the electrolyte dominates the heat generation of fluid flow. It results in the change of temperature-dependent electrical/transport properties, such as viscosity and electric conductivity of the working liquids, which, in turn, influences the hydrodynamic behavior of EOF. Concerning the Joule heating-induced heat transfer of EOF, the models in a microcapillary  and in a2O3, is much greater than that of a pure fluid, and hence, the thermal performance of nanofluid is dramatically enhanced in the presence of these nanoparticles. Furthermore, the nanofluid is stable, which has no additional problems like pressure drop, sedimentation, or blockage in microchannels. Choi firstly proposed the model of nanofluid 1/2. Accordingly, the dimensionless P-B equation and the boundary conditions are obtainedTo facilitate the discussion, the following scales are introduced ξ ≤ 0.025 V), the known Debye–Hückel linearization principle /2 as effμ denotes the effective viscosity coefficient. For a fully developed, incompressible and laminar EOF of power-law nanofluid, it is assumed that there is only an axial velocity w = w n, electrΩ is discretized to a grid system of 121 × 121. The numeric algorithm shows that the numerical solutions can achieve the accuracy of −8 between the numerical velocity and analytical velocity of Newtonian nanofluid EOF. Furthermore, the iterative criterion is given as ΔL < 10−10 to make sure the EOF is fully developed. For the EOF of Newtonian nanofluid, in K = 10, ϕ = 0.06, and S = 3. To clearly show the comparison, 25 grid points of the numerical solution are plotted in For validating the numerical algorithm proposed above, a test of grid dependence is conducted. As a result, the volumetric domain K = 10. ϕ = 0. ϕ = 0.06. It is noted from these two groups of figures that the velocity shows homogeneous abatement across the channel as the volume fraction of nanoparticles increases no matter what fluid rheology is considered. This is because the nanoparticles in fluid enhance the effective viscosity of power-law nanofluid in response to the sear rate and cause greater dispersion of the velocity distribution. As observed in [erved in , althougϕ = 0) and when K = 10, the temperature distributions of shear thinning fluid, Newtonian fluid, and shear thickening fluid at S = 0 are displayed in S = 3 are displayed in S irrespective of the fluid type. Furthermore, the dimensionless temperature increases with the flow behavior index n, indicating that the difference of heat transfer characters caused by different shear rates of fluid type is remarkable, especially for shear thinning base fluid.Without consideration of nanoparticles in fluid  has a uniform influence on the temperature distribution, which leads to the increasing (decreasing) tendency of Nusselt number with electrokinetic width. Furthermore, the decrease in flow behavior index results in the pronounced abatement of Nusselt number, meaning that the heat transfer rate of shear thinning nanofluid is much smaller than the heat transfer rate of Newtonian fluid and shear thickening fluid. This is consistent with the prediction in [The variations of the Nusselt number with the electrokinetic width are shown in ction in .K = 10. It shows that the Nusselt number is inversely proportional to the Joule heating parameter, and the decreasing rate becomes smaller as nanofluid changes from shear thickening to shear thinning. Since it is considered that Joule heating has a spatially uniform effect, the greater Joule heating parameter corresponds to relatively uniform heat distribution. Consequently, the convective heat transfer rate is reduced as presented in ϕ significantly improves the heat transfer rate.Nu and the variation of Bejan number Be with flow behavior index n for different volume fractions of nanoparticles are depicted in K = 20 and S = 3. The combined effects of nonlinear rheological behavior and nanoparticle volume fraction on Nusselt number are investigated. To note, the dependence of Nusselt number on the flow behavior index shows nonlinear. For shear thinning base fluid, the Nusselt number is more amenable to the change in flow behavior index n. The increase in viscosity for shear thinning fluid resulting from the decrease in flow behavior index reduces the Nusselt number, and thus, the heat transfer rate of shear thinning nanofluid is obviously less than that of shear thickening nanofluid. Furthermore, the variation of the Nusselt number with the volume fraction of nanoparticles is almost linear. It shows that the heat transfer performance of nanofluid is clearly enhanced compared to the conventional fluid regardless of the base fluid rheology. These results are also witnessed in Be > 0.5 implies that the primary irreversibility is contributed to the heat conduction when S = 3.The variation of the Nusselt number K, flow behavior index n, nanoparticle volume fraction ϕ, and Joule heating parameter S on the velocity field, temperature field, Nusselt number, and Bejan number have been studied. The following conclusions can be drawn:The velocity is reduced due to the increase in viscosity of nanofluid resulting from the addition of nanoparticles.ϕ truly enhances the dimensionless temperature and Nusselt number by changing the thermal conductivity and shear stress of velocity, no matter what type of base fluid is considered. Accordingly, the Bejan number decreases with increasing nanoparticle volume fraction and flow behavior index.The volume fraction of nanoparticles K for S ≥ 0 and a decreasing function for S < 0.Nusselt number is an increasing function of the electrokinetic width S increases. The heat transfer rate of shear thickening nanofluid is much larger than that of shear thinning fluid.The Nusselt number shows abatement, and the temperature exhibits greater variation along the channel as Joule heating parameter In the present paper, a numerical investigation for hydrodynamically and thermally fully developed EOF of power-law nanofluid in a rectangular microchannel has been conducted. The interactive influences of electrokinetic width To sum up, nanoparticle volume fraction and Joule heating effect show significant influence on the thermal behavior of EOF of power-law nanofluid. It should be noted that the dependence of the Nusselt number on the flow behavior index is nonlinear. The Nusselt number becomes a weak function of electrokinetic width as the electrokinetic width gets larger. The heat transfer rate of shear thinning nanofluid is more amenable to the change in nanofluid rheology. Therefore, the heat transfer of power-law nanofluid has to be carefully treated, and one should make a distinction when manipulating different types of nanofluid in practical problems. This understanding of the mechanism of thermally fully developed EOF can be referred to in more efficient control of fluids and optimization of microfluidic devices.


Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [3H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding.Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the V The continuous exposure of the central nerves system to HIV-1 viral proteins, inflammation, and antiretroviral agents results in neuropathological and neurocognitive deficits observed in the patients with HAND7. Transactivator of transcription (Tat) protein, one of seven HIV-1 viral proteins, has been shown to play a critical role in HIV-1 viral replication as well as the development of HAND8, which can be exacerbated by concurrent cocaine abuse9. Thus, developing an intervention strategy in the early stage of HIV-1 infection would prevent the development of neurocognitive dysfunction in HIV-1 infected individuals.About thirty-seven million people are currently living with HIV-1 infection worldwide, leading to a significant global public health problem. While the effective antiretroviral therapies significantly reduced the mortality rate in the patients with HIV-1 infection, nearly 50% of HIV-1 infected patients have various degrees of neurological complications that are referred to as HIV-1-associated neurocognitive disorders (HAND)11, and motivation13. Dopamine (DA) transporter (DAT) is a presynaptic membrane protein that reuptakes the released DA from the synaptic cleft back into cytosol, maintaining a stable DA homeostasis. The DAT activity is directly inhibited by HIV-1 Tat protein and cocaine, which synergistically enhances synaptic DA levels9. The dysregulation of DA system is a mediating factor of HAND as well as a factor in cocaine abuse15. Using computational modeling and experimental approach, we have identified several key residues on human DAT (hDAT), which are crucial for Tat-hDAT interaction and dynamic transport process9. Furthermore, we have demonstrated that in vitro exposure to Tat reduced reuptake of DA via hDAT in cells18 and rat striatal synaptosomes19. The inhibitory effect of Tat on DAT function results from Tat directly interacting with DAT21. Single point mutations of hDAT at tyrosine88 , lysine 92 , histidine547  differentially alter basal DA uptake but attenuate the Tat inhibitory effects on DA transport18. For example, DA uptake is decreased in K92M and increased in H547A, respectively; however Y88F mutant preserves basal DA uptake18. Notably, the mutational effects on normal DA uptake and Tat inhibitory effect on DAT function are associated with alterations of transporter conformational transitions9.Normal dopaminergic transmission is important for maintaining different brain activities including attention, learning, memory22. Recent studies have demonstrated that novel SRI-compounds exhibit a partial antagonistic role in DAT function as allosteric modulators25. We have reported that SRI-30827, one of the novel allosteric modulators, blocks Tat interaction with DAT26. Thus, identifying the specific binding sites on hDAT for Tat and its role in DA transport process could be beneficial to attenuation of the inhibitory effect of Tat on DAT-mediated dopaminergic transmission. On the other hand, through an allosteric modulatory mechanism, the inhibitory effect of Tat on DAT function can also be diminished by targeting the specific DAT residues that are distinct from Tat binding sites. However, based on our computational structural models for Tat binding with hDAT, the interaction of Tat with hDAT involves multiple residues of DAT21 and our previous results obtained from single point mutations of DAT only present the role of a particular residue in Tat-DAT interaction. Therefore, this study investigated the mutational effects of Y88F/H547A and Y88F/K92M/H547A on basal DAT function, Tat inhibitory effect on DA uptake, and dynamic DA transport process.We have demonstrated that Tat protein inhibits DAT function in an allosteric modulatory mechanism20, hDAT residues Y88, K92, and H547 could form hydrogen bonds with residues K19, P18, and R49 of HIV-1 Tat, respectively. According to our previous computational and experimental results, single mutation of either hDAT Y88 (Y88F), K92 (K92M) or H547 (H547A) could significantly attenuate the binding between DAT and Tat18. As shown in Fig. Based on the constructed hDAT-Tat binding model in our previous workmax of [3H]DA uptake, respectively, while Y88F preserved normal DA uptake18. The current study determined the multiple mutations of these residues on DA uptake and DAT binding sites. As shown in Table 5 cells), the Vmax values were increased by 92% in Y88F/H547A  = 2.33, p < 0.05) and dramatically decreased in Y88F/H547A/K92M  = 16.2, p < 0.001), respectively. Additionally, compared to WT hDAT (1.21 ± 0.17 µM), the Km value in Y88F/H547A was increased by 250%  = 2.54, p < 0.05) but not altered in Y88F/K92F/H547A.We have demonstrated that single point mutations on hDAT at His547 (H547A) and Lys92 (K92M) displayed a 196% increase and a 71% decrease in the V3H]WIN35,428 binding sites share pharmacological identity with the DA uptake site and is also part of the cocaine binding domain28. Kinetic analysis of [3H]WIN35,428 binding was conducted to determine the effects of multiple mutations of hDAT on the DA binding site. As shown in Table max nor Kd values of [3H]WIN35,428 were altered in Y88F/H547A, whereas Y88F/K92M/H547A displayed a 61% decrease in the Bmax value (t(14) = 2.4, p < 0.05) and a 199% increase in the Kd value (t(14) = 4.4, p < 0.001). Thus, due to the significant reduction on DA uptake for Y88F/K92M/H547A, only the Y88F/H547A mutant was included for the rest of experiments.In the hDAT, the [3H] DA uptake in WT hDAT and Y88F/H547A (Table 50) for DA . In contrast, compared to WT hDAT, Y88F/ H547A displayed an increase in IC50 for cocaine  and GBR12909 . We also evaluated the mutational effect of Y88F/H547A on the IC50 of DA, cocaine, and GBR12909 for inhibiting [3H]WIN35,428 binding  and cocaine  = 3.73, p < 0.001]. No difference in IC50 GBR12909 inhibiting [3H]WIN35,428 binding was observed between WT hDAT and Y88F/H547A.To characterize the influence of Y88F/H547A on DA transport and the specific binding site in hDAT for DA, cocaine, GBR12909, and amphetamine (AMPH), we determined the ability of these DAT inhibitors or substrate to inhibit . No significant mutation × PMA treatment interaction  was found. Addition of PMA reduced the Vmax values in WT hDAT by 51%  and Y88F/H547A by 60% , respectively, compared to their respective vehicle controls. Similarly, two-way ANOVA analysis on the Km values revealed significant main effects of mutation  and PMA treatment . No significant interaction of mutation × PMA treatment was observed. The addition of 1 µM PMA reduced Km values by 53% in WT hDAT  and 63% in Y88F/H547A , respectively. These results suggest that the double mutation of Tyr88 and His547 attenuates the single mutant H547A-increased sensitivity of PMA-induced decrease in DA uptake.The activation of PKC by phorbol 12-myristate 13-acetate (PMA), a PKC activator, decreases DA uptake through DAT3H]DA uptake observed in WT hDAT18. Since the triple mutant (Y88F/K92M/H547A) displayed an extremely low Vmax of [3H]DA uptake, only the double mutant (Y88F/H547A) was tested for its effect on Tat-induced inhibition of DA uptake. Based on the computational modeling predictions . As shown in Fig. 1–86 (rTat1–86) significantly reduced the specific [3H] DA uptake in WT hDAT by 31% relative to its control . However, the Tat-induced reduction of [3H] DA uptake in WT hDAT was attenuated in Y88F/H547A compared to its control , suggesting that the double mutation of Tyr88 and His547 attenuates Tat’s inhibitory effect on DA uptake.Our previous studies demonstrated that single point mutation of Tyr88 (Y88F) or His547 (H547A) attenuated Tat-induced inhibition of DA uptake and [3H]WIN35,428 binding. Addition of Zn2+ is able to partially reverse an inward-facing state to an outward-facing state32. On the basis of this principle, the addition of Zn2+ to WT hDAT would inhibit DA uptake, whereas in a functional mutation of DAT, Zn2+ might diminish the preference for the inward-facing conformation and thus enhance DA uptake. As shown in Fig. 3H]DA uptake in WT hDAT and Y88F/H547A revealed significant main effects of mutation , zinc , and mutation × zinc interaction . The addition of zinc significantly decreased the [3H]DA uptake in WT hDAT and its mutant in a concentration dependent manner. Compared to the respective controls (in absence of Zn2+), the addition of Zn2+ (10 and 100 µM) produced a similar reduction of [3H]DA uptake in WT  but a greater reductions  in Y88F/H547A (ps < 0.01). This suggests that the double mutation of Y88/H547 does not affect zinc-induced regulation of DA uptake. In contrast, as shown in Fig. 3H]WIN35,428 binding revealed significant main effects of mutation , zinc , and mutation × zinc interaction . Compared to the respective controls (in absence of Zn2+), addition of Zn2+ (10 and 100 µM) significantly increased [3H] WIN35,428 binding in WT hDAT . The Zn2+ (10 and 100 µM)-induced increase in [3H]WIN 35,428 binding in WT hDAT was significantly diminished in Y88F/H547A , suggesting that Y88F/H547A attenuates zinc-induced increase in [3H]WIN35,428 of hDAT by altering transporter conformational transitions.Tat protein has been shown to regulate DA transport allosterically, which may lead to alteration of transporter conformational transitions3H]DA in PC12 cells expressing WT hDAT and Y88F/H547A  = 59.38, p < 0.0001], time , and mutation × time interaction . Post hoc analyses showed that compared to WT hDAT, DA efflux levels were significantly elevated at 1 and 10 min .To further determine the effect of Tyr88/His547 mutation on transporter conformational transitions, we examined basal efflux levels of [47A Fig. . twenty 34. AMPH-stimulated [3H] DA efflux in PC12 cells transfected with WT hDAT and its mutants was determined under a stable baseline of DA efflux  = 1.92, p = 0.14, one-way ANOVA). As shown in Fig. 3H]DA efflux in WT hDAT and mutants in a concentration dependent manner. Two-way ANOVA analysis on the Vmax values revealed significant main effects of mutation , AMPH  and mutation × AMPH interaction . As shown in Table max values of [3H]DA efflux in response to AMPH were reduced in H547A (0.22 ± 0.04) and Y88F/H547A (0.20 ± 0.03) compared to WT hDAT . No difference in Km was found among WT hDAT and all mutants.AMPH acts as a substrate for DAT which subsequently reverses the transporter leading to DA effluxlux Fig. , showing3H]DA uptake in WT and Y88F/H547A  and Y88F/H547A . Next, we performed the kinetic analysis of [3H]DA uptake in WT and Y88F/H547A in presence of AMPH . As shown in Fig. max of [3H]DA uptake reveled significant main effects of mutation  = 9.50, p < 0.01) and AMPH  = 11.99, p < 0.01). No significant interaction of mutation × AMPH treatment was found  = 1.61, p = 0.22). Post hoc analyses show that the Vmax value in Y88F/H547A (18.9 ± 2.81 pmol/min/105 cells) was higher than that in WT hDAT  = 2.49, p < 0.05, n = 5/group) in the absence of AMPH. AMPH decreased the Vmax in both WT hDAT  = 2.76, p < 0.01) and Y88F/H547A  = 2.58, p < 0.05) compared with their respective controls. There was a significant difference in the magnitude of the AMPH-induced decrease in Vmax values between WT hDAT and Y88F/H547A (t(8) = 1.86, p < 0.05). Furthermore, two-way ANOVA analysis on Km values revealed significant main effects of mutation  and AMPH ,whereas no significant interaction of mutation × AMPH treatment was found . In the absence of AMPH, the Km values of [3H]DA uptake was greater in Y88F/H547A (5.12 ± 0.76 µM) than that in WT hDAT , which was attenuated in Y88F/H547A (7.24 ± 1.15 µM).Double mutation of Tyr88 and His547 could induce DAT conformational change by altering AMPH interaction with DAT. We first examined the affinity for AMPH to inhibit WIN35,428 binding, increased basal DAT-mediated DA efflux, and reduced AMPH-stimulated DA efflux. Collectively, these findings suggest that targeting DAT residues Tyr88 and His547 may have potential therapeutic benefits on Tat-induced dysregulation of dopaminergic system seen in patients with HAND.Our recent studies demonstrated that single hDAT mutants, Y88F, K92M, and H547A differentially regulate DAT reuptake but attenuate Tat’s inhibitory effect on DAT function. This study sought to determine whether multiple mutations on these hDAT residues (Y88F/H547A) and (Y88F/K92M/H547A) influence the direct interactions between Tat and DAT. There were three major findings. First, compared with WT hDAT, Y88F/H547A enhances V35. This YYY motif has hydrophobic contact with a typical U-turn loop associated with extracellular loop 6 (EL6) and transmembrane 10 (TM10a). Any structural changes affecting the stability of the YYY motif could destabilize the R85-D476 salt bridge of hDAT, and the salt bridge is key for the conformational conversion39. H547 is expected to significantly affect the hDAT stability because H547 is adjacent to Y548 of the YYY motif35, which is consistent with the observed increase in DA uptake by the H547A mutation18. On the other hand, the side chain of K92 residue (TM1b) forms a favorable salt bridge with the side chain of D313 (TM6a) during the molecular dynamics simulation on the hDAT-DA binding complex, and the bridge is also essential for the conformational conversion of hDAT required for the DA uptake process40. For this reason, disrupting the K92-D313 salt bridge will likely impair or slow down the DA uptake process, which has been validated pharmacologically by mutations on K92 (K92M)17 and D313 (D313N)41. In addition, the aromatic ring on the side chain of Y88 is sandwiched by TM1b and extracellular loop 4 (EL4) in a hydrophobic region of hDAT. Therefore, mutation of Y88 to phenylalanine retains the normal reuptake function of hDAT and demonstrated no difference compared to WT hDAT17. However, Y88F increased the Km of DA binding by about 50%, suggesting that Y88F may slightly alter the local structure around Y8817. In the current study, the double mutant Y88F/H547A retains the increased Vmax observed in H547A, suggesting a critical role of the mutated H547 residue in regulating transporter activity. In contrast, the combination of the mutated residues Y88, K92, and H547 does not follow a simple math addition, because compared to WT hDAT, a 96% reduction of Vmax was observed in Y88F/K92M/H547A, whereas the Vmax was decreased by 71% in K92M17. These results suggest that the inclusion of the K92 mutation in the K92-D313 salt bridge is a critical factor in the differential DAT kinetics in the triple mutation of hDAT. As Y88 is spatially adjacent to R85 and K92 in helix TM1b, the double mutations on both Y88F and K92M are expected to induce more structural disturbance to the R85-D476 and K92-D313 salt brides, thus further decreasing Vmax of the transporter. Therefore, triple mutant Y88F/K92M/H547A has a lower Vmax compared to, single mutant K92M. Although including K92M mutation significantly decreases the Vmax for DA uptake in the triple mutant Y88F/K92M/H547A compared to Y88F/H547A, no significant change in the Bmax of [3H]WIN 35,428 binding is found in the double or triple mutants, suggesting that the multiple mutations may alter DAT re-uptake function rather than the substrate binding sites on DAT. This conclusion is also supported by the increased Km in Y88F/H547A and no change in Km in Y88F/K92M/H547A. However, single point mutations of Y88, K92, and H547 differentially affect the Vmax with no changes in the Km18. One possible explanation is that allosteric modulation of the dynamic DA transport process may be responsible for the enhanced Vmax and its associated DA uptake potency.According to our computationally modeled structures, Y548-Y470-Y551 is a stable motif (denoted as the YYY motif for convenience) essential for the conformational conversion of hDAT required for the DA transport process3H]DA to inhibit DA uptake relative to WT hDAT, which is similar to that in Y88F and H547A18. However, Y88F/H547A increased the DA uptake potency for cocaine and GBR12909, which is consistent with the single mutation of Y88F relative to WT hDAT17. Similar to Y88F17, Y88F/H547A displayed decreased potency for DA to inhibit [3H]WIN35,428 binding. However, the inhibition potency of [3H]WIN35,428 binding for cocaine was decreased in Y88F/H547A but increased in Y88F17. Y88F/H547A, similar to Y88F17, retained the [3H]WIN35,428 binding potency for GBR12909. GBR12909 is a selective inhibitor of DA uptake, binding to the piperazine acceptor site without influencing DA release42; while cocaine is a competitive inhibitor for DAT and prefers to an outward-facing transport state, leading to a decrease in DA transport44. According to our computational modeling prediction, both hDAT residues Y88 and H547 are the critical binding sites on DAT for Tat20. Therefore, our findings demonstrate that through allosteric modulation, mutation of Y88 and H547 alters the inhibition potency for DA uptake and DAT binding site for substrate and DAT inhibitors40.We further determined the effect of double mutant Y88F/H547A on the basal DA uptake via hDAT and binding potency for DA, GBR12909, and cocaine. Y88F/H547A had decreased the potency for WIN35,428 binding relative to WT hDAT. Second, Y88F/H547A augments basal DA efflux when compared to WT hDAT, which opposes our previous reports showing that neither Y88F nor H547A affected the basal efflux18. The elevated basal DA efflux in Y88F/H547A may be attributed to its elevated Vmax of DA uptake and DA accumulation. Taken together, Y88F/H547A, via an alteration of DAT conformational transitions, may influence the binding structure of hDAT-Tat complex, thereby disrupting Tat-induced inhibition of DA uptake. Studying the multiple mutation of DAT residues in Tat-DAT intermolecular interaction will greatly contribute to our ongoing proof of concept studies using novel allosteric modulators to establish their potential for therapeutic application in HAND.A major finding is that the double mutant Y88F/H547A completely attenuates the Tat-mediated inhibition of DAT reuptake function, which is consistent with our previous reports on Y88F- or H547A-induced attenuation of the Tat’s effect49. Our computational modeling has demonstrated that the dynamic DA uptake process includes outward-open, outward-occluded, and inward-open states21, which can be reversed by DA substrate and DAT mutation51. The current results indicate that the potency for AMPH inhibition of [3H]DA uptake was not altered in Y88F/H547A compared with WT hDAT, suggesting that the mutation of Tyr88 and His547 does not alter the interaction of AMPH with hDAT. However, AMPH-mediated DA efflux was reduced in H547A and Y88F/H547A, suggesting that mutations of Tyr88 and His547 residues alter the conformational equilibrium of the DAT transport transitions. Furthermore, AMPH decreases the Vmax of DA uptake in both WT hDAT and Y88F/H547A, which is consistent with the previous report51, however, the magnitude of the AMPH-mediated reduction of the Vmax in Y88F/H547A was greater than that in WT hDAT. Interestingly, the AMPH-mediated reduction of the Vmax was accompanied by increased Km values in WT hDAT but not Y88F/H547A. Given that DAT-mediated DA transport is accompanied by reuptake of two Na+ and Cl− ions, AMPH influx would transport Na+ through DAT and enhance its levels at the inner face of the transporter52. Therefore, AMPH decreases DA transport but increases Km values in WT hDAT, whereas altered transporter conformation by Y88F/H547A may attenuate the increased Km. Taken together, the results along with attenuated zinc-regulated [3H] WIN35,428 binding and increased basal DA efflux in Y88F/H547A further support that mutations of Tyr88 and His547 cause an alteration in conformational transitions, thereby disrupting the inhibitory effect of Tat on DAT reuptake function.AMPH, a substrate of DAT, competitively inhibits the reuptake of DA and elicits DAT-mediated DA efflux by reversal of the transporter18. An allosteric modulatory mechanism may contribute to Y88F/H547A-mediated attenuation of Tat-induced inhibition of DA transport. Understanding the multiple mutation effects of the identified DAT residues might provide a basis for a novel approach for developing compounds to attenuate Tat binding to DAT by an allosteric mechanism in HIV-1-infected individuals.In conclusion, our data further demonstrate DAT Tyr88 and His547 as important residues for DAT function and the Tat-DAT interaction. Double mutation of these residues preserves single mutant H547A-mediated enhancement of DA uptake and diminishes Tat-induced inhibition of DA transport53 and the constructed structure of hDAT-DA complex21. Protein docking and molecular dynamics simulations were employed to identify the conformation of hDAT-Tat complex. The energy-minimized complex structure used in this work were extracted from long-time equilibrated molecular dynamics simulation trajectories in our previous work35.We performed computational modeling and simulations to model the binding structure of hDAT with HIV-1 clade B type Tat based on the nuclear magnetic resonance (NMR) structures of Tat18. Single mutants, Y88F, K92M, and H547A, are expected to abolish hydrogen bonds between hDAT and Tat. Double (H547A/Y88F) and triple (H547A/Y88F/K92M) mutations were generated based on the wild type hDAT (WT hDAT) sequence  by site-directed mutagenesis. We then used the synthetic cDNA encoding hDAT subcloned into pcDNA3.1 + as a template to generate mutants using QuikChange™ site-directed mutagenesis Kit . We performed the DAT sequence for confirming the sequence of the mutant construct at University of South Carolina EnGenCore facility. Plasmids DNA were propagated and purified using a plasmid isolation kit .We selected the mutations of Y88, H547, Y88/H547 and Y88/K92/H547 based on predictions of computational modeling and simulations Fig.  and our 2 incubator. Once cells were growing to 100% confluence on plates, cells were seeded at a density of 1 × 105 cells/cm2 in 24-well plates and transfected with WT hDAT or mutants by Lipofectamine 2000 (Life Technologies). Twenty-four hours after transfection, intact cells or cell suspensions were used for experiments.In the current study, we used rat pheochromocytoma cells , Manassas, VA), which were maintained in Dulbecco’s modified eagle medium  supplemented with 15% horse serum, 2.5% bovine calf serum, 2 mM glutamine and antibiotics, 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were cultured at 37 °C in a 5% COmax) or Michaelis-Menten constant (Km) of [3H]DA uptake in intact PC12 cells transfected with WT hDAT or mutants as reported previously17. Twenty-four hours after transfection, cells in 24-well plates were washed twice with Krebs-Ringer-HEPES (KRH) buffer  and incubated with nomifensine  as nonspecific uptake for 10 min at room temperature. A fixed concentration of [3H]DA  mixed with one of six concentrations of unlabeled DA  were applied to cells at room temperature for additional 8 min. The specific DAT-mediated DA uptake was calculated by subtracting non-specific uptake from total uptake (in the absence of nomifensine). The reaction was terminated by removal of solution from wells and quickly washed three times with ice cold KRH buffer. Cells were then lysed in 500 μL of 1% SDS for an hour and radioactivity was measured using a liquid scintillation counter .In order to determine whether DAT mutants alter DAT function, we evaluated the maximal velocity  as the previous reports30. Intact PC12 cells transfected with WT or Y88F/H547A were preincubated with or without PMA for 30 min at 37 °C followed by additional 8 min incubation with six concentrations of mixed [3H]DA as described above. To determine the effect of amphetamine (AMPH) on Y88F/H547A-mediated transporter conformational transitions, Vmax or Km of [3H]DA uptake in WT and Y88F/H547A was determined in the presence or absence of 1 µM AMPH  as reported previously51.To confirm whether Y88F/H547A-induced increase in V2+-regulation of DA transport, we performed the competitive inhibition of DA uptake in intact PC12 cells transfected with WT or mutant, which were preincubated with a series of final concentrations of DA (1 nM-1 mM), GBR12909 (1 nM-10 µM), cocaine (1 nM-1 mM), AMPH (1 nM-1 mM) or ZnCl2  at room temperature for 10 min followed by additional 8 min incubation with a fixed concentration of [3H]DA .To determine whether these mutations alter the affinity of DAT substrate or inhibitors as well as Zn3H]DA uptake in PC12 cells transfected with WT or mutant in the presence or absence of Tat protein. In brief, cells were dissociated with trypsin/EDTA  and resuspended in culture medium and incubated at room temperature for 10 min. The dissociated cells were collected by centrifugation at 400 × g for 5 min at 4 °C and washed once with phosphate-buffered saline followed by additional 5 min centrifugation . Finally, the resulted cell pellets were resuspended in KRH assay buffer. The cell suspensions from WT hDAT or Y88F/H547A mutant were then preincubated with or without recombinant Tat1–86  at room temperature for 20 min followed by additional 8 min incubation with a mixed [3H]DA uptake . Non-specific [3H]DA uptake was determined in the presence of 10 µM nomifensine. The update reaction was terminated by immediate filtration through Whatman GF/B glass filters (presoaked with 1 mM pyrocatechol for 3 h) followed by three washes with 3 mL of ice-cold KRH buffer containing pyrocatechol using a Brandel cell harvester . Radioactivity was determined as described above.In order to determine whether double mutation of Y88 and H547 disrupts Tat and DAT interaction, we performed the specific [max or Kd) of [3H]WIN 35,428 binding in intact PC12 cells transfected with WT hDAT or mutant. Cells were washed with sucrose-phosphate buffer twice  and then incubated with one of the six concentrations of [3H]WIN 35,428  in a final volume of 500 μL on ice for 2 h. Nonspecific binding at each concentration of [3H]WIN 35,428 was determined by adding in the presence of 30 µM cocaine  and subtracted from total binding to obtain the specific binding. For competitive inhibition experiments, assays were performed in a final volume of 250 μL. Intact PC12 cells transfected with WT or mutated hDATs were incubated in buffer containing 25 μL of [3H]WIN 35,428  and one of ten concentrations of DA (1 nM- 100 μM), cocaine (1 nM- 100 μM), GBR12909 (0.01 nM- 1 μM) or ZnCl2  on ice for 2 h. Assays were terminated by removing reaction reagents in each well followed by two washes with ice-cold assay buffer. Cells were lysed with 1% SDS for an hour and subjected to the radioactivity measurement as described above.Binding assays were used to determine the kinetic parameters  were lysed rapidly in 1% SDS after preloading with [3H]DA. To collect factional efflux samples, buffer (500 μL) was added into a separate set of cell wells and transferred to scintillation vials after 1 min as an initial fractional efflux, and another 500 μl buffer was added to the same wells and collected after 10 min as second fractional efflux. Additional fractional efflux at 20, 30, 40, 50 min, respectively, was repeated under the same procedure. After last fractional efflux, cells were lysed and counted as total amount of [3H]DA remaining in the cells from each well.Basal DA efflux was performed at room temperature as described previously51. PC12 cells transfected with WT hDAT and its mutants growing in 24-well plates were preloaded with [3H]DA  for 20 min at room temperature. Subsequently, cells in all plate wells were washed with KRH for three times, cells were then incubated with KRH buffer containing AMPH (5–250 µM) or vehicle  for 15 min at room temperature. After incubation, the buffer solution incubated with AMPH or vehicle was collected as AMPH-stimulated efflux or baseline efflux. Cells were lysed in 1% SDS and subject to count for obtaining the initial amount of DA  in the cells.To further determine DAT mutation-mediated transporter confirmation transitions, basal efflux and AMPH-stimulated DA efflux in WT hDAT and its mutants were measured as reported previouslyn as the number of independent experiments for each experiment group. Pharmacological parameter values  and IC50 from [3H]DA uptake and [3H]WIN 35,428 binding were calculated by nonlinear regression analysis using a one-site model with variable slope. To determine the significant difference in the pharmacological parameters between unpaired sample groups, such as WT hDAT and its mutants, unpaired Student’s t test was used for the statistical comparisons. Significant differences between sample groups were analyzed with separate ANOVAs followed by post-hoc, Bonferroni multiple comparison test as indicated in the results Section of each experiment. All statistical analyses were performed using IBM SPSS Statistics version 25, and differences were considered significant at p < 0.05.In the current study, all data are presented as mean ± SEM with


Primary pleural synovial sarcoma is a malignant tumour and thought to be more aggressive than synovial sarcoma which occurs in extremities. Its treatment strategy has not been fully established because of its rarity. We report a primary pleural synovial sarcoma case which achieved a long‐term survival with repeated surgery of recurrent pleural tumour. A 39‐year‐old man presented with a gradually enlarged tumour in the left hemithorax. The tumour was resected and diagnosed as primary pleural synovial sarcoma. The tumour was slowly growing and repeatedly recurrent in the left pleura. The surgical resections for the recurrent tumours were performed 6 years and 11 years after the initial surgery. Intriguingly, recurrent tumour which developed after second surgery exhibited temporally spontaneous regression. Our patient remains alive 12 years after the initial surgery. Repeated resection of metastatic lesion can achieve long survival in primary pleural synovial sarcoma. Synovial sarcoma is a soft tissue malignant tumour and the majority of that occur in extremities. Primary pleural synovial sarcoma is extremely uncommon and its therapeutic strategy has not been clearly established SYT‐SSX1) fusion gene was detected by reverse transcriptase‐polymerase chain reaction, so a diagnosis of primary pleural synovial sarcoma was made.A 39‐year‐old man was referred to our hospital for investigation of a mass detected by chest X‐ray. A chest computed tomography (CT) scan revealed pleura‐based opacity in the left hemithorax. The patient had no history of any serious illnesses. His laboratory findings were normal. We performed an ultrasound‐guided biopsy twice, but no definitive diagnosis was obtained. Follow‐up CT at 8 months after the second biopsy showed progressive enlargement of the lesion Fig. A. We perTwo years after the initial surgery, a CT scan revealed a tiny nodule adjacent to the mediastinum in the left upper hemithorax. The tumour gradually enlarged Fig. B, and 6 We herein described a case of primary pleural synovial sarcoma that grew slowly and exhibited late recurrence and temporally spontaneous regression of the recurrent tumour. Repeated surgical resection of the recurrent tumours contributed to long‐term survival.Primary pleuropulmonary synovial sarcoma is thought to be more aggressive than synovial sarcoma arising from the extremities. According to a previous study, the median time to local recurrence or metastasis among patients with localized primary pleuropulmonary synovial sarcoma was 8.5 months The recurrent tumour that occurred after the second surgery spontaneously regressed. Spontaneous regression is a rare phenomenon that involves complete or partial disappearance of a tumour without treatment The standard treatment of synovial sarcoma is wide resection combined with radiation, as appropriate. Complete tumour resection improves survival In conclusion, repeated surgical resection of metastatic lesions can lead to long‐term survival in patients with primary pleural synovial sarcoma.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


Tumour budding has been described as an independent prognostic feature in several tumour types. We report for the first time the relationship between tumour budding and survival evaluated in patients with muscle invasive bladder cancer. A machine learning-based methodology was applied to accurately quantify tumour buds across immunofluorescence labelled whole slide images from 100 muscle invasive bladder cancer patients. Furthermore, tumour budding was found to be correlated to TNM (p = 0.00089) and pT (p = 0.0078) staging. A novel classification and regression tree model was constructed to stratify all stage II, III, and IV patients into three new staging criteria based on disease specific survival. For the stratification of non-metastatic patients into high or low risk of disease specific death, our decision tree model reported that tumour budding was the most significant feature , and no clinical feature was utilised to categorise these patients. Our findings demonstrate that tumour budding, quantified using automated image analysis provides prognostic value for muscle invasive bladder cancer patients and a better model fit than TNM staging. Current clinical guidelines for both prognosis and treatment strategies are based on Tumour Node Metastasis (TNM) staging system; of which MIBC cancers are comprised of pT2–pT4 tumours, with or without nodal and distant metastasis2. Patients with MIBC are recommended for radical cystectomy with pelvic lymph node dissection. Approximately 25% of newly diagnosed bladder cancer patients present with muscle invasive disease. In contrast to non-muscle invasive bladder cancer (NMIBC), MIBC is highly aggressive with a high incidence of metastasis and a poor prognosis where 5-year survival rates vary between 30 and 50%4.Muscle-invasive bladder cancers (MIBC) are classified as having grown into or through the muscle layers of the bladder wall5. In addition to inter- and intra-reporter variability, TNM staging may not adequately encompass the complex and dynamic behaviour of the disease10. There is therefore a need to substantially improve patient stratification and earlier definitive treatment for high risk MIBC patients. A morphological analysis of the different patterns of tumour invasion may hold important pathological information, which could allow for a better risk stratification of patients with MIBC.Despite recent research into novel treatment and surgical strategies the mortality rates and prognosis of MIBC patients have remained immutable over the past 30 years12. Most studies have shown that a higher density of TB is typically present at the invasive front  compared to tumour core 17. It has been suggested that TB reflects an early step in progression towards metastasis due to their cell’s reported phenotypic epithelial to mesenchymal transition (EMT)20. Despite reports of observer variability, studies in other cancer types have continuously demonstrated the significant association of TB with adverse clinicopathological features and the independent significance of TB as a prognostic factor25. Information about the role of TB in bladder cancer is scarce26 and it has not yet been reported to be associated with disease specific survival (DSS) in MIBC.Tumour budding (TB) is defined as discrete clusters of up to four cancer cells that reflects an infiltrative invasive growth pattern28, is based on the automated detection of both the nuclei and the segmentation and classification of cancer cell clusters. Our automated solution enables the quantification of TB at the patient level via the computation of seven image analysis features, while eliminating inter-observer variability. Survival analysis provided evidence of the prognostic value of TB quantification in MIBC patients and showed that a combination of clinicopathological features with TB predicted patient survival more precisely than the standard TNM staging.In this study, we evaluate whether TB can more precisely predict MIBC patients at a low or medium risk of disease specific death than current clinical guidelines based on TNM staging. To this end, we introduce an image analysis solution for the detection of tumour buds, which following from our previous worksThis study comprised 100 MIBC patients with a median age at the time of surgical resection of 68 years . The follow-up time was up to 113.13 months. DSS was gathered from NHS Scotland’s patient database and was defined by the patient’s cause of death being directly from their bladder cancer. Median DSS is 23.63 months. In terms of TNM staging, 25% of patients were diagnosed with stage II, 41% with stage IIIA, 4% with stage IIIB and 30% with stage IV. No positive lymph nodes were found in 78 patients and 1–2 lymph nodes contained cancer in 22 patients. Full patient characteristics can be found in Table −11) and highest hazard ratio  than any other image analysis or clinical feature. Of all the treatment combinations, as displayed in Supplementary Table −06) and Cox-regression  than any other TB quantification method. Full survival univariate statistics for each analysed parameter are stated in Table TNM staging, returned the lowest log rank p-value (p = 4.76E2) test of independence was used to examine associations between clinicopathological data and the ‘number of TB in the core’ , and pT stage  which were also the two most significant clinicopathological features associated with patient survival and were previously demonstrated predictors of patient outcome. No other clinicopathological feature was associated with TB.The chi-squared (χ−09). The resulting sub-group of stage II and III MIBC patients without metastasis (N = 70) was further optimally separated by the ‘number of TB in core’ feature. The associated log rank p value (p = 0.0091) and hazard ratio  showed the statistical significance of this second split against other TB features and clinicopathological parameters  of tumours buds across four different methods. Methods 1 and 2 were based on work by Karamitopoulou E et al.21 and Ueno H et al.32 and the recommendations of ITBCC12. Method 3 was inspired by Lohneis P et al.24 applying ten 0.238 mm2 fields of view. Finally, Method 4–7 are novel approaches quantifying tumour buds across the whole slide or large regions of interest; methods only made feasible by the application of automated image analysis. Despite the fact that the ITBCC guidelines represent the first international effort to standardise manual TB count methodology, consistent reporting may still be affected by subjectivity, and inter- and intra-observer variability. Furthermore, manual quantification can be a time consuming process. To overcome these issues and standardise the quantification of TB we designed a fully automated ML based methodology capable of quantifying both the number and density of TB across specific regions of interest within the whole slide image.Despite TB being reported as an independent predictor of patient outcome in several disease areas, it has not been accepted as a core item in the clinical guidelines17, our results link intra-tumoural budding to a worse prognosis in MIBC. Interestingly, in this study, and in contrast with the majority of work in other cancer types, the number or density of TB within the invasive front of MIBC was not significantly associated with DSS. Additionally, we assessed the association between TB and a number of clinicopathological features  in MIBC.It is interesting to note that in this study, TB quantified across the tumour core outperformed “field of view” methodologies, based on the ITBCC or published recommendations, when assessed against DSS prediction. Together with previous studies36 and immunofluorescence (IF)38. The use of cytokeratin-based immuno-labelling as a tumour mask clearly identifies TB even in cases that display a high density of peri-tumoural inflammatory infiltrate or reactive stromal cells. This fluorescence cytokeratin-aided distinction of TB allows for a more accurate recognition of TB when applying automated image analysis. However, the automated quantification of histopathological features may still be confounded by tissue artefact and necrotic debris. This is particularly true for methodology that employs object segmentation based on signal intensity thresholds or sole colour information, which can result in false positive and false negative detections. A further obstacle to overcome, when employing threshold based image analysis across patient cohorts, is inter- and intra-heterogeneity. The large inter- and intra-sample heterogeneity of the signal utilised to automatically segment tissue, for example tumour from stroma, may lead to inaccurate segmentation when run across large cohorts. Machine learning, and in particular convolutional neural network and context based random forest, was used to overcome the above limitations. By enabling the automatic definition of high-dimensional decisions based on colour and hierarchical texture information, the employed algorithms yield a precise quantification of true tumour bud objects against other false positive objects while ensuring robustness against variability in the visual appearance of tumour regions , recent reports have provided meaningful information about TB and disease progression using both immunohistochemistry (IHC)28, Weis et al. recently proposed an application specific approach to recognise TB in IHC images of colorectal carcinoma based on classification convolution neural networks41. We believe that the two approaches differ in both their aim and their methodology. Regarding the methodology, Weis et al. performed segmentation of the tumour mask utilising mainly colour information: colour deconvolution was employed followed by k-mean clustering and heuristic post-processing rules. Tumour buds were then selected among the resulting tumour objects using a classification convolutional neural network. In contrast, we employ a semantic segmentation neural network for the segmentation of the tumour objects and detect all nuclei in the image. The selection of the tumour buds among the detected tumour objects then relies on the heuristic definition of a tumour bud based on the number of nuclei within a tumour cell cluster  provided that corresponding manual annotations are available for training. As an example, we have first developed the regression random forest model used for cell detection for the analysis of H&E slidesUpon analysing the full MIBC cohort, no TB parameter was as significantly associated with patient survival as clinical TNM staging even though the parameter ‘number of TB in core’ was shown to be strongly prognostic for survival. By excluding the metastatic patients from the analysis, the objectively reported ‘number of TB in core’  outperformed all clinical features for predicting disease specific death . Due to the unavailability of patient material containing cancer tissue for some cases, the study number was reduced to 110. The archived formalin fixed paraffin embedded (FFPE) tissue block that contained the deepest invasion of cancer was selected for the 110 patients after review of haematoxylin and eosin (H&E) labelled slides by both a pathologist (DJH) and research scientist (PDC)  was performed on de-paraffinised 3 μm FFPE tissue sections using a Dako link 48 instrument . The primary antibody against Pan-cytokeratin  was utilised to visualise urothelial cells and nuclei were counterstained with Hoechst . A Carl Zeiss AxioScan.Z1 whole slide scanner  was used to capture and digitise whole slide fluorescence images with a 20x objective .45. The automated image analysis algorithms were developed for the detection of nuclei and the segmentation of urothelial-specific PanCK positive regions from both stroma and artefacts prior to the classification and quantification of tumour buds. The following paragraphs provide more details on the image analysis methods.The quantitative image analysis of tumour buds was performed using machine learning and deep learning approaches based on Definiens Tissue Phenomics® technology 46 modified to include transposed convolution layers47 and skip connections48. As detailed in our previous work27, the second model combined a layer normalisation step, a fully CNN and a classification visual context Random Forest (RF)49. In comparison to our previous work27, the second model’s PanCK normalisation step relied on the detection of the PanCK reference objects from the output of the first network and not from heuristic rules. The outputs of the two networks were then ensemble by merging the two tumour masks detected by either of the two networks. The networks were trained based on the manual delineation of tumour and non-tumour regions (including tissue and staining artefacts as well as necrosis) on a subset of images prior to their application across the entire cohort of images. Examples of detected tumour masks are provided in Fig. The tumour cell clusters are defined as the connected components of the tumour mask (PanCK positive labelled cells). The tumour mask is segmented by ensemble prediction of two deep-learning models, trained on manual annotations. The two models read as follow. First, a semantic segmentation convolutional neural network (CNN) was developed based on the Inception architecture28, these two resulting prediction maps are inputs to a “local maxima” algorithm that outputs the position of the nuclei. This is illustrated in Fig. Nuclei were detected based on the training and application of two regression visual context RF models that respectively predict the pixel-wise spatial proximity to the closest nucleus as well as the size of the corresponding nucleus. As detailed in our previous workWe performed a quantitative evaluation of the tumour segmentation and the nuclei detection algorithms on a set of 10 and 5 validation fields of view respectively, selected on whole slide images unused for training  4–7: Quantification of the total number of tumour buds and their density in the detected tumour core and in the detected invasive front.Given the detected tumour buds and the estimated tumour core and invasive front, we quantified the number and density of both peri-tumoural (tumour buds within the invasive front) and intra-tumoural (tumour buds within the tumour core) detected budding. More precisely, the four following automated quantification methods were employed:et al.21. The size of the field of views of Method 1 and Method 2 was defined as recommended by the International Tumour Budding Consensus Conference (ITBCC) for the reporting of TB in colorectal cancer12. The third method was proposed by Lohneis P. et al.24. The fourth method explored other quantification approaches than the ones previously described in the literature and leads to an additional set of four tumour bud related features. The resultant (m = 7) TB related features are set out in Table et al.50 and restricted in this study the number of tested tumour bud related predictors. This aimed at limiting the impact of multiple hypothesis testing.The first and third methods are based on the quantification methods previously described by Karamitopoulou E. https://keras.io/) (Version 2.0.9) running on top of TensorFlow51 (Version 1.4.0). Other operations, such as the training and prediction of the classification and regression RF models as well as the computation and export of the tumour bud related features, were performed in an extended version of the Definiens Developer XD software52.The convolutional neural networks were trained and predicted using the open source Python deep learning library Keras  using the survival package (Version 2.39–5) and the survminer package (Version 0.4.3). When meaningful, we performed p values correction to account for the m = 7 hypotheses resulting from the definition of the TB related features. Corrected p values, or q values, were computed using the R package BioconductorOur survival decision tree see Fig.  analysisOur survival decision tree differs from the standard classification tree: while the standard classification tree finds at each node the decision that minimises the misclassification rate between the two resulting branches, we instead return the decision that minimises the log rank test p value between the survival characteristics of the two resulting branches. By bypassing the need of an arbitrary set of classes, the proposed survival decision tree has the advantages of keeping both the time and event information and of maintaining the continuous characteristic of survival information. In contrast, the use of a classification tree would require to arbitrarily define the classes either (1) by directly taking the event  as class, or (2) by classifying patients with time-to-event higher than an arbitrary reference time  as high survival class and patients with time-to-event lower than this reference time as low survival class.54, resulting in the decisions at each node to be independently optimised for each patient. While overfitting the decisions on all patients would result in stronger separation between the survival groups, the employed leave-one-out approach enables a better assessment of how the found results would generalise to another dataset. This is of high importance given the limited size of our current dataset (N = 100). The leave-one out analysis reads as follows: for a given patient, the decision is chosen to minimize the log rank p value of the separation of the remaining N-1 patients into low and high survival branches. Each patient is therefore left out of the selection of its optimal decision. The final grouping is obtained by applying the individual leave-one-out cross validated decisions to the corresponding individual patients, which results in the independence between the patients and the decisions.Each split’s feature and associated optimal cut-off value were validated together with the whole decision tree using leave-one-out pre-validation to prevent overfittingBecause the low number of TNM IIIB patients  as well as on the three categories  resulting from the proposed survival decision tree model, also denoted ‘TB stage model’. The hazard ratios together with their confidence intervals were computed to estimate the effect of the two respective staging methods. The respective TNM stage II and TB model stage II’ were taken as baseline for both models, leading to the following analysis: TNM III vs TNM II, TNM IV vs TNM II, TB III’ vs TB II’ and TB IV vs. TB II’. Overall significance of the two models were evaluated with the likelihood ratio test, Wald test and score log-rank test. This last analysis was performed together with the computation of the associated Kaplan Meier curves in R (Version 3.3.2) using the survival package (Version 2.39–5) and the survminer package (Version 0.4.3).Ethical approval for this study was obtained from the NHS Lothian Tissue Governance Unit , conforming to protocols approved by East of Scotland Research Ethics Service (REC). Patient material was surplus to diagnosis and no further consent was required.Supplementary InformationSupplementary Material 1Supplementary Material M2Supplementary Material M3Supplementary Material M4Supplementary Material M5Supplementary Material M6Supplementary Material M7


Arterial elastance (Ea) represents the total afterload imposed on the left ventricle, and it is largely influenced by systemic vascular resistance (SVR). Although one can expect that Ea is influenced by peripheral endothelial function, no data are available to support it in patients. The aim of this study was to investigate the relationship between Ea, SVR, and microvascular perfusion in critically ill patients undergoing the fluid challenge (FC).  A prospective study in patients receiving a fluid challenge. A pulse wave analysis system  was used to estimate Ea and an incident dark field (IDF) handheld device  to evaluate the sublingual microcirculation. Microvascular perfusion was assessed using the proportion of small-perfused vessels (PPV). Relative changes in each variable were calculated before and after FC; fluid responsiveness was defined as an increase in the cardiac index by at least 10% from baseline. n=10 for hypotension; n=5 for oliguria; n=3 for lactate values greater than 2 mmol/l; n=2 for tachycardia), including 12 fluid responders. There was a strong correlation between Ea and SVR  and only a weak correlation between Ea and PPV at baseline . Ea decreased from 1.4 [1.2–1.6] to 1.2 [1.1–1.4] mmHg/mL (p=0.01), SVR from 1207 [1006–1373] to 1073 [997–1202] dyn ∗ s/cm5 (p=0.06), and PPV from 56 [51–64] % to 59 [47–73] % (p=0.25) after fluid challenge. Changes in Ea were significantly correlated with changes in SVR, but not with changes in PPV.  We studied 20 patients requiring a fluid challenge ( The correlation between Ea and indexes of microvascular perfusion in the sublingual region is weak. The impact of microcirculatory perfusion on the arterial load is probably limited. Cardiovascular alterations in the critically ill, which are frequently characterized by arterial hypotension, require prompt and adequate correction to prevent organ failure , 2. ArteFluid therapy is one of the most commonly used therapeutic intervention, which aims at correcting hypovolemia, increasing cardiac preload and cardiac output, and potentially improving microcirculatory blood flow , 5, 6. FThe aim of this study was to investigate the relationship between Ea, SVR, and PPV in critically ill patients who received a fluid challenge in an attempt to correct signs of tissue hypoperfusion.This is a retrospective analysis of prospectively collected data conducted in a 35-bed mixed Department of Intensive Care between January and May 2018. The study was approved by the ethical committee (P2018/128), which waived the need for an informed consent, considering the retrospective nature of the study. We studied adult (>18 years old) patients, treated by mechanical ventilation, who received a fluid challenge (FC), while they were equipped with a radial arterial catheter for routine arterial pressure (AP) and cardiac output monitoring using an uncalibrated pulse-contour method device. Exclusion criteria were as follows: diseases that could affect the quality and reliability of the arterial signal, such as aortic valve disease, aortic aneurysms, and cardiac arrhythmias , and poor quality of the arterial pressure signal due to excessive over- or underdamping of the catheter-transducer system checked using the fast flush test.A fluid challenge was performed because of clinical or biological signs of impaired organ perfusion and according to the clinical judgment of the attending physician based on hemodynamic monitoring variables, as indicated by one or more of the following: tachycardia (>100 beats/min); mean arterial blood pressure (MAP) lower than 65 mmHg; oliguria (<0.5 ml/kg/h); blood lactate level greater than 2 mEq/L.2 between 35 and 45 mmHg. FC was performed over 20 minutes using a minimal volume of 500 mL of crystalloid or colloid solutions. Unfortunately, there is no agreement on which specific increase of CO after FC should be considered to define a patient as “responder”  days.Twenty patients , and CVP increased from 7 [7–11] to 10 [8–12] (p < 0.01) after the fluid challenge, while SVR, PPV, MAP, and CO did not significantly change. Twelve patients (60%) were responders to fluid challenge (p=0.05) and SVR from 1266 [1082–1751] to 1073 [1002–1185] dyn ∗ s/cm5, (p=0.03) after FC. CVP increased from 7 [6–7] to 9 [7–10] mmHg (p < 0.01) and CO from 4.4 [3.4–5.0] to 5.0 [4.6–5.5] L/min (p=0.05) after FC. PPV and MAP did not change significantly. In the nonresponders, MAP increased significantly after FC , while Ea, SVR, PPV, CVP, and CO did not significantly change. In particular, MAP increased in seven nonresponders. Five of these patients had a history of cardiomyopathy, one had a sepsis at the time of FC, and the last one was treated with V-V ECMO.Overall, Ea decreased significantly from 1.4 [1.2–1.6] to 1.2 [1.1–1.4] mmHg/mL  was observed. A weak correlation between Ea and PPV was found at T0 and for all data, but not at T1. ΔEa was significantly correlated with ΔSVR but not with ΔPPV  in septic patients , 21. It Finally, we studied the microcirculation network in a specific organ , while Ea assessed by the MostCare is the reflection of the vascular tone of the cannulated artery , which would have a different response to therapeutic interventions and a different microvascular flow than the sublingual area. Our findings also suggest that potential surrogates of sublingual microvascular perfusion, such as SVR, cannot estimate microvascular function at the bedside and that handheld computer-controlled devices with a high-resolution image sensor are the only adequate tool to quantify capillary perfusion.dyn), which could potentially be associated with changes in volume load and arterial tone more than the static one we measured.This study had some limitations. First, the relatively small number of patients enrolled limited the analysis between responders and nonresponders to fluid challenge; however, the aim of this study was to compare Ea to microcirculatory parameters before and after fluid challenge, and this association would remain weak even by adding more patients. Second, the majority of our patients received a crystalloid solution, and colloids or red blood cells transfusion may have different effects on the microcirculation. Also, the patient population was still heterogeneous, with a potentially different basal microcirculatory perfusion condition and different response to fluid therapy. Moreover, we were not able to stratify the severity of the patients at the time of fluid challenge and the evolution of the diseases after fluid administration, and we could not fully understand the effect of the fluid on microcirculation. We cannot exclude that the results of the present study could be only the consequence of the population studied and the timing on the evolution of the disease. Third, we assessed Ea using an uncalibrated pulse-contour waveform analysis device, while the “gold standard” is still considered the ventricular pressure-volume with invasive aortic pressure –24. In aArterial elastance significantly decreases after the fluid challenge, and this change is correlated with systemic vascular resistance. We observed only a weak correlation between arterial elastance and microvascular perfusion, implying that the impact of microcirculatory perfusion on the arterial load is probably limited.