Cell-coupled field-effect transistor (FET) biosensorshave attractedconsiderable attention because of their high sensitivity to biomolecules.The use of insect cells (Sf21) as a core sensor element is advantageousdue to their stable adhesion to sensors at room temperature. Althoughvisualization of the insect cell–substrate interface leadsto logical amplification of signals, the spatiotemporal processesat the interfaces have not yet been elucidated. We quantitativelymonitored the adhesion dynamics of Sf21 using interference reflectionmicroscopy (IRM). Specific adhesion signatures with ring-like patchesalong the cellular periphery were detected. A combination of zetapotential measurements and lectin staining identified specific glycoconjugateswith low electrostatic potentials. The ring-like structures were disruptedafter cholesterol depletion, suggesting a raft domain along the cellperiphery. Our results indicate dynamic and asymmetric cell adhesionis due to low electrostatic repulsion with fluidic sugar rafts. Weenvision the logical design of cell–sensor interfaces withan electrical model that accounts for actual adhesion interfaces. For example, fluorescence interference contrastmicroscopy (FLIC) has investigated the neuronal cell–substrateinterface by taking advantage of its high axial resolution.8 FLIC requires the reconstruction of silica steps with differentthicknesses on the sensor surface, and the topological structuresthemselves denature the native cell adhesion.9 Moreover, by transmission electron microscopy (TEM), Wrobel et al.reported that the distribution of interfacial gaps in human embryonickidney-293T (HEK) cells and sensors ranged from areas of strong adhesion(≤10 nm) to areas of weak adhesion (10–100 nm).10 Repeating the cryo-dehydration process for thefixation is likely to affect the dynamics of insect cell adhesion.Therefore, microscopic techniques to visualize the interfaces between“living” cells and sensors without altering the sensorsurface are necessary for the rational development of sensor devices.Cell-coupled FET biosensorsbased on complementary metal-oxide semiconductors (CMOS) have attractedmuch attention owing to their high sensitivity to biomolecules andportability.z ∼ 2 nm.11 Moreover, such precise visualization of thesample–substrate interface has the advantage of assessing biologicalinteractions such as focal adhesion assembly13 and physical interaction15 such as repulsion ofglycoconjugates16 . Recently, we have developed“insect” cell-coupled FET devices with high selectivityfor odorant molecules through the strong expression of odorant receptors(OR).18 Minor structural differences in similarsilk moth pheromone components  weresensitively discriminated based on drain current modulations of thesensors at room temperature. Previously, a scanning electron microscope(SEM) has clearly visualized dehydrated Spodoptera frugiperda (Sf21) cells in close contact with a sensor surface.19 However, spatiotemporal insights into the adhesiondynamics of intact insect cells and their origins are still unknown.The combination of IRM with FET-device sensors should therefore providespatiotemporal information about the cell-sensor interfaces.In contrast, IRM exploits interference between light reflectedfrom sample–liquid and liquid–substrate interfaces.In the case of the glass substrate used, IRM can be a prominent label-freetechnique for measuring the distance between cells and substrateswith a high axial resolution of Spodoptera frugiperda (Sf21) cells to a substrateby using IRM at room temperature (20 °C) in phosphate bufferedsaline  , whereas Sf21 cells remainedspherical (t ∼ 0 min), which thenspread radially and formed visible dark ring structures at the cellperiphery  was optimized for evaluation of the peripheral strong adhesionzone and small patch-like structures (∼1 μm) (t = 30 min). Statistical analysisshowed that the contact area of Sf21 exhibited a sigmoidal-like drasticincrease and reached a plateau at t ∼ 10 min,whereas that of HEK remained constant (t ∼ 10 min).The difference in dynamics between insect and mammalian cells is shownschematically  . The conconstant 1c. This atically 1c inset.16 Although the suspension medium did not contain any adhesion molecules, we confirmed that Sf21 cells didnot express focal adhesion kinase in the absence of serum  the presence of a specific sialicglycoconjugate with low electrical potential or (2) low amounts ofsialic glycoconjugate. To test this hypothesis, cells were treatedwith enzymes  that specifically degradesialic acid. Pretreatment with the enzyme reduced the zeta potentialof HEK cells to that of Sf21 cells, whereas the surface potentialof Sf21 cells was intact  (t ∼ 10 min). Notably, the strong adhesion with ring structures 2e.This 2) leads to symmetry breaking of adhesion with a large spreadingarea,26 possibly via glycoconjugates with negativepotential. Interestingly, the positively charged substrate did notincrease the contact area of Sf21 cells and the presence of localpatch-like structures at the cell periphery (∼1 μm) butsuppressed ring-like structures (o-methyl-β- cyclodextrin (dMβCD)and methyl-β-cyclodextrin (MβCD) for the cells. Treatmentwith dMβCD and MβCD decreased the contact area of Sf21cells due to the loss of segregation of glycoconjugates at the cellularperiphery  also confirmed a decreasein the contact area and induced the disappearance of adhesion rings were also influenced by the depletionof cholesterol, but the positively charged substrate did not influencethe presence at the cell periphery. The results indicate that smallpatch-like structures at the cell periphery are supported by the cholesterolwith a negatively charged surface. To date, the raft structures ofmammalian cells are generally invisible to conventional microscopybecause their size is below the diffraction limit.32 Therefore, fluorescence lifetime imaging is commonly usedto resolve small lipid raft structures at focal adhesion patches33 even though the spatial resolution is lower.To the best of our knowledge, the large local raft structure of thesugar moiety visible in this study’s experiment is the firstto be visualized by conventional microscopy, except for phase separationwith membrane models. These results clearly indicate that glycoconjugateswith low surface potential at the cell periphery are supported bycholesterol and serve as a graft for a glycoconjugate. To elucidatethe physical properties of the glycoconjugate raft, we examined membranefluidity.34 The tight contact region (h ≤ 40 nm) has lower generalized polarization values(GP) (h = 40–80), suggestinga “fluidic sugar raft” at the cell periphery. A previousreport revealed that ordered domains of human monocyte cell lineswere suppressed by the cholesterol depletion;35 however, the contribution of local domains to cell–substrateinterfaces was not quantitatively assessed. In contrast, IRM accuratelyfocuses on the cell–substrate interfaces and simultaneous imagingby Laurdan microscopy detected disordered tight contract areas. Treatmentof MβCD suppressed the formation of adhesion ring with decreasingGP values , and phase behavior is determined by headgroupchemistry and by the length and saturation of the tails.37 Although further efforts need to be made todetermine the lipid structures, lipid content, and physical propertiesof the cell membrane using various techniques39 and model systems to assess the fluidity of dynamical adhesion ofgiant unilamellar vesicle (GUV),40 a fingerprintof dynamic insect cell adhesion is supported by the raft of glycoconjugates.To characterize the spatial potential distributionof insect celladhesion, positively and/or negatively charged substrates were preparedby silane coupling (see method details in the ructures 3b red. Iructures 3b blue. eriphery 3d. Previon rings. The coeues(GP) 3e thantP values  indicatiN-acetyl glucosamine and sialic acidby WGA clearly showed a higher amount of Sf21 glycoconjugates thanin HEK cells, but the electrostatic charge was weaker. To elucidatethe molecular mechanism of ring-like glycoconjugate formation at thecell periphery with small patch-like structures, cholesterol depletionand inhibition of cholesterol synthesis (6 thus we believe that the electrical theory that accounts for realadhesion interfaces opens the possibility of rational design of cell-coupledFET biosensors.In conclusion, cells adhered more strongly to thesubstrate immediatelyafter the cells were placed on the substrate than HEK cells at ambienttemperature. Zeta potential measurements identified a weak surfacepotential for Sf21 cells compared to HEK cells, which was not affectedby the enzymatic digestion of surface sialic blushes. Fluorescentstaining of ynthesis  were useynthesis  which maynthesis 3c. Sf21cynthesis . Therefoynthesis  Visualiz


Planar inverted lead halide photovoltaics demonstrate remarkable photoconversion properties when employing poly(triarylamine) (PTAA) as a hole transporting layer. Herein, we elucidate the effect of ambient ultraviolet (UV) degradation on the structural and operational stability of the PTAA hole transporter through a series of rigorous optoelectrical characterization protocols. Due attention was given to the interplay between the polymer and perovskite absorber, both within the framework of a bilayer structure and fully assembled solar cells. The obtained results imply that UV degradation exerts a major influence on the structural integrity of PTAA, rather than on the interface with the perovskite light harvester. Moreover, UV exposure induced more adverse effects on tested samples than environmental humidity and oxygen, contributing more to the overall reduction of charge extraction properties of PTAA, as well as increased defect population upon prolonged UV exposure. Operational stability and structural integrity of a poly(triarylamine) hole transporter and methylammonium lead halide absorber are investigated upon exposure to UV stress. Charge transport can be further improved by using HTL materials containing cyclic hydrocarbon scaffolds built around silicone cores8 or via substitution of carbon atoms within the hydrocarbon ring with sulphur. Subdivisions formed in such a way provide additional electron pairs, aiding charge extraction through π–π stacking.9 However, when keeping in mind more simplistic and energy efficient fabrication processes suitable for low temperature processing conditions, an inverted planar structure (HTL/perovskite/ETL) is suggested as a promising alternative to mesoporous cells. In essence, adoption of inverted device architectures opens the door to implementation of cheaper flexible substrates and further reduction of overall manufacturing costs. Unlike their inorganic counterparts, organic materials have an intrinsic benefit of lower processing temperatures and PTAA stands out as the suitable and the most popular candidate for the HTL material in inverted planar perovskite solar cells. The structure of PTAA is built upon successive repetition of triarylamine units forming a high molecular weight polymer network and providing good substrate coverage, which in turn promotes nucleation of perovskite crystals, resulting in larger grains.10 The reasons for the selection of PTAA are numerous, but the most important ones are resilience to negative effects of oxygen and moisture and remarkable intrinsic hole mobility (∼4 × 10−3 cm2 V s−1),11 which can be further improved by doping, thanks to available nitrogen electron pairs located at triphenylamine bonding sites.12,13 Numerous attempts were made to improve the stability of devices employing PTAA, mainly via doping or application of interfacial barriers to prevent ion migration from the light harvester and extend operational lifetime.14–18 Although satisfactory improvements were suggested by many groups, there is surprisingly a low number of studies concerning the stability of PTAA itself. Namely, literature reports on this topic are usually related to PTAA stability after heat and moisture induced oxidation.19–21 To the best of our knowledge, there are no reports elucidating the behaviour of PTAA upon prolonged exposure to UV irradiation, which is of crucial importance for estimation of long-term stability for perovskite based photovoltaics. The effect of ultraviolet (UV) exposure on degradation of devices with mesoporous structures is associated with photocatalytic behaviour of TiO2 and the resulting interplay between accumulated defects at and disruption of charge transfer across the interface with the perovskite.22,23 Despite the aforementioned research progress related to inverted planar perovskite cells and remarkable PCEs achieved with this structure,1 there is a notable lack of information regarding the impact caused by UV induced degradation on the operational and structural stability of devices utilizing PTAA as a HTL. Our work aims to bridge this gap, by presenting readers with comprehensive and detailed studies on the effect of short wavelength photons on the stability and charge transport of perovskite solar cells containing PTAA as a hole transporter, while taking into consideration the interactions at the PTAA/MAPI interface, as well as the overall photophysics of fully assembled devices. We conducted a series of optoelectrical spectroscopy and space-charge limited experiments to monitor the effect of UV degradation on neat PTAA films, bilayers with methylammonium lead iodide light harvester (MAPI), and finally, on fully assembled solar cells with an inverted planar structure (ITO/PTAA/MAPI/PC60BM/PFN/Ag). Since UV radiation accounts for approximately 5% (4.6 mW cm−2)23 of the AM1.5G spectrum, we opted for even harsher experimental conditions by exposing our samples to more than a double of that intensity, totalling to 10 mW cm−2. In this way, we ensured that there was no underestimation of the UV impact encountered during the operation of solar cells under “real-life” conditions. In principle, the presence of encapsulant foil prevents transmission of high energy photons and provides a sufficient level of protection from UV irradiation during outdoor operation. However, the ageing process may lead towards undesirable effects such as yellowing and delamination of the protective barrier, resulting in device exposure to UV stress accompanied by oxygen and moisture ingress. In order to replicate such a scenario as close as possible, we conducted all of the experiments under ambient conditions, ensuring that all three adverse factors are accounted for. In such a way, we exclude any possible underestimation of the degradation degree arising from the isolated effect of UV, since such a degradation process would also involve the presence of oxygen/moisture under realistic operational circumstances. The effect of UV degradation on prepared samples was observed at various exposure steps  and the results at each stage of degradation were used to quantify the divergence from the behaviour of a control device stored under ambient dark conditions. Interfacial behaviour was probed with ultrafast transient absorption spectroscopy (TAS)24 and small/large perturbation transient photovoltage/photocurrent (TPV/TPC) measurements.25,26 Population of defect states and interplay with drift mobility of charge carriers were observed using space-charge-limited-current (SCLC)27 and photoinduced charge extraction by linearly increasing voltage (photo-CELIV).28 In this way, reliable assessment of charge transport properties recorded at various steps of device assembly and stages of degradation allows elucidation of device behaviour covering both short (femtosecond TAS) and longer timescales . At all stages of experiments, the structural integrity of both MAPI and PTAA was monitored and appropriate connection with charge transport properties was drawn.With the ever-increasing interest of the scientific community in the prospective applications of large area perovskite photovoltaics pertaining to industry-scale fabrication, recent research trends have been focused on increment of device operational stability. Resolution of such an important issue would further expedite the adoption of lead halide solar cells for large-scale applications, which is further supported by energy conserving, cost effective and high through-output solution processing. Most of such efforts were aimed towards application of devices built upon mesoporous architectures, as they exhibited the highest power conversion efficiency (PCE) and operational stability. Such a structure, referred to as regular or normal, commonly utilizes TiO−1 were cleaned in a three-step process consisting of ultrasonication with deionized water, acetone and isopropanol for 10 minutes each. After drying, the substrates were transferred to a nitrogen filled glovebox for ozone treatment, in order to remove any organic residues. A hole transporting layer was spin-coated from a solution of PTAA  in toluene at 6000 rpm, followed by annealing at 110 °C for 10 minutes. Afterwards, MAPI was fabricated according to the Lewis acid/base adduct protocol described by Lee et al.29 from the precursor solution 1 mmol of PbI2 (Sigma-Aldrich), 1 mmol of methylammonium iodide , 0.9 mmol of dimethyl sulfoxide  and addition of thiourea  in 636 μl of anhydrous N,N-dimethylformamide (Sigma-Aldrich). Perovskite layers were spun over the previously prepared polymer film at 6000 rpm for a total of 30 seconds and anhydrous diethyl ether antisolvent was added 10 seconds into the spinning step. Thereafter, the MAPI films were annealed at 100 °C for 10 minutes and the bilayer samples obtained in such a way (ITO/PTAA/MAPI) were used for TAS measurements. On the other hand, devices intended for transient photovoltage/photocurrent characterization were fabricated by the addition of a 100 nm thick silver electrode over the bilayer, resulting in an ITO/PTAA/MAPI/Ag structure. Next, hole-only devices used during SCLC experiments were formed by evaporation of an 80 nm thick Au electrode over the previously described bilayer to yield a symmetric ITO/PTAA/MAPI/Au structure. Finally, fully assembled solar cells employed in steady state J–V and drift mobility investigations (photo-CELIV) were fabricated after PC60BM (Solenne BV) was spin-coated over the bilayer (20 mg ml−1 in anhydrous chlorobenzene) at 1500 rpm followed by deposition of poly (PFN)  in anhydrous methanol. Afterwards, 100 nm of Ag was thermally evaporated over a shadow mask to outline an active area of 4 mm2 for a completely assembled planar inverted cell architecture (ITO/PTAA/MAPI/PC60BM/PFN/Ag).Indium tin oxide (ITO) prepatterned substrates  with a sheet resistance of ∼15 Ω sq−2. The degradation process was monitored under the ambient atmosphere in the absence of external light and relative humidity was kept close to 45%. The power conversion efficiency (PCE) of the fabricated solar cells was measured under a N2 atmosphere and 100 mW cm−2 illumination generated by a Xenon lamp calibrated with an Oriel 91150v silicon solar cell (Newport). Steady-state J–V curves were recorded at a constant scan rate of 10 mV s−1 using a multiplexer test board (Ossila) and accompanying software suite. External quantum efficiency (EQE) spectra were recorded using an integrated  system and a chopper operating at a frequency of 146 Hz. Scanning electron microscopy images were taken using a Jeol JSM-7000F operating at 15 kV. The topography of samples was examined with an atomic force microscope (AFM)  operating in tapping mode, with the addition of an external lock-in amplifier  for a Kelvin probe force microscopy (KPFM) configuration. Calibration of the cantilever  for work function measurements was done using a standard HOPG sample. X-ray diffraction (XRD) patterns were obtained using a D/MAX-2000 X-ray diffractometer with monochromatic Cu Kα irradiation (λ = 1.5418 Å) at a scan rate of 4° min−1. Absorbance of PTAA/MAPI bilayers was measured in the 250–850 nm range, using a UV-vis spectrophotometer . Attenuated Total Reflection (ATR) IR spectra were recorded on a Bruker Vertex 70v spectrometer. Transient absorption spectroscopy measurements were performed on a Newport (TAS-1) transient absorption spectrometer. The excitation source was a pulsed Yb:KGW-based laser  with the central wavelength at 1026 nm, a pulse duration of 170 fs and a repetition rate of 1 KHz. During the study, the pump fluence was kept constant at 1.5 mJ cm−2. Optoelectrical characterization was performed on a transient module of a commercially available measurement platform, ARKEO (Cicci Research s.r.l.). Transient photovoltage was operated in the small-perturbation regime by confining the amplitude of the LED pulse to <10% of the equilibrium voltage established at the background field. This approach ensured that the transient voltage signal followed mono-exponential decay and the resulting fits mirrored the lifetime of charge carriers. On the other hand, the transient photocurrent response was monitored under large-perturbation conditions and 50 μm extraction to an external circuit, while the duty cycle was set at 1/2 in order to allow sufficient time for device loading and relaxation of decay tails. Open circuit voltage and short circuit current conditions were controlled via response from transimpedance and differential voltage amplifiers. Charge injection during photo-CELIV experiments was performed using a fast LED (470 nm) with a Lambertian radiation pattern and 120° viewing angle operating at 100 mA of driving current. The offset voltage was set to prevent premature extraction of the generated charge carriers between the injection pulse and extraction ramp. The selected range (0.4–0.8 V) was conditioned with the degradation stage of measured solar cells. The data were collected using a high-speed waveform generator driving one electrode with a ramp of 1.25 × 105 V s−1 applied after 3 μs delay, while the other electrode was connected to a transimpedance amplifier and monitored with a 100 MHz single-shot bandwidth digitizer.UV degradation was conducted within a custom-built chamber illuminated with a series of PHILIPS UV-A lamps emitting at 350–400 nm (distribution peak at 370 nm) totalling to 10 mW cm60BM/PFN/Ag) incorporating a fullerene ETL. In all cases, MAPI films were coated over 40 nm thick layers of PTAA. Bilayers formed in this way were exposed to UV radiation (illuminated from the polymer side) in the presence of oxygen and ambient humidity, spanning over a total duration of 24 hours, with sampling intervals at 2, 5, 8, 12, 16 and 24 h. Doping of MAPI solution with thiourea has been previously demonstrated as a highly reproducible fabrication method yielding large crystals, due to the Lewis acid/base interactions between electron pairs from S-donors and acceptors from lead halides.29,30 Consequently, we obtained perovskite grains of remarkable size, often exceeding 3 μm, as clearly demonstrated by SEM micrographs shown in In this study, the effect of UV degradation was monitored within the operational framework of a bilayer structure (ITO/PTAA/MAPI) with or without a top electrode and fully assembled inverted perovskite solar cells . The resulting steady-state current–voltage curves shown in 41,42 This process can be identified by the emergence of an additional peak in the XRD spectrum at 12.7° originating from the presence of the PbI2 phase.42–44 Indeed, the corresponding peak marked with an asterisk appears in the pattern of the UV treated sample. It should be kept in mind that the perovskite retains its structural integrity even upon exposure to the harshest degradation conditions, as all diffraction peaks characteristic of the 110, 220 and 330 planes of MAPI remained detectable and retained high intensity.45–47 The degradation of PTAA films was monitored using ATR-IR spectroscopy performed under vacuum conditions in order to exclude the appearance of unwanted peaks originating from the environment. The resulting spectra recorded for each period of UV exposure are shown in −1 region corresponding to the stretching of C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O bonds resulting from oxidative degradation.19 Therefore, deterioration of PTAA is limited to the interplay between hydrocarbon rings and polymer chain length. The declining intensity of the peak at 1493 cm−1 ascribed to the stretching of double carbon bonds within the aromatic ring suggests partial decomposition of the cyclic structure over time.48 This trend is accompanied by the other CC peak located at 1602 cm−1, mirroring the same reduction trend as the period of UV degradation increases.Moreover, distortion of the surface potential seems to be governed by the effect of UV exposure, since the sample exposed to only oxygen and moisture yielded more uniform contrast maps, as shown in Fig. S3.−1. This region nests a well-defined peak population corresponding to the C–N stretching 49 and further intensity drop of the amine bond fingerprint becomes noticeable after the 5–16 h window. The stretching of C–N bonds characteristic of aliphatic amines gives a weak signal at 1170 cm−1, seemingly unresponsive to UV illumination. Alternatively, this occurrence could be assigned to C–O etheric stretching caused by oxygen inclusion, in which case it would have been defined by a strong peak.50 Hence, we can dismiss this possibility, since peak intensity in our case is close to the background noise. The signal of the peak at 817 cm−1 arising from aromatic C–H bending and the corresponding shoulder assigned to the same bond within a meta xylene configuration become indistinguishable after the 12 h mark. It should be pointed out that the impact of UV degradation was insufficient to induce any morphological changes of PTAA films, as clearly indicated in AFM topography images  summarized in After PTAA was subjected to UV light for a duration exceeding 2 h, another visible drop of signal intensity was observed in the area spanning from 1200 to 1380 cms Fig. S2. So far,τ1) is attributed to charge carrier trapping at the perovskite grain boundaries and perovskite/HTL interfaces.52,53 It represents the transit time for excited carriers to move from the conduction band to the trap states. In principle, faster τ1 indicates quicker trap-filling, leading to larger splitting in the quasi-Fermi energy levels and consequently to more efficient free charge carrier injection, resulting in enhanced electrical characteristics of MAPI films. It should be mentioned that the density of trap states and their depth exert a noticeable effect on τ1 constant.41In particular, the first time component (τ1 compared with the corresponding time components of UV degraded bilayers. The second time constant (τ2) represents the time required for hole injection from the perovskite layer into the PTAA polymer, with faster hole injection being indicative of better electrical characteristics of the devices. Inspection of values listed in τ2 in comparison to UV exposed samples. Interestingly the τ2 value doesn't vary considerably between different UV exposure times, implying that the PTAA/perovskite interface is stable and its charge transfer properties do not deteriorate beyond the initial degradation stage. Moreover, the third slow time component (τ3) which is representative of the exciton recombination time is the slowest in fresh films, meaning that the free carriers have longer availability for injection to the hole transporter. We performed additional fitting according to the polynomial model based on the following relation:n is the charge carrier density and k1, k2 and k3 are trap-assisted, bimolecular and Auger trimolecular recombination rates, respectively. All recombination rates for the studied samples are summarized in et al.54 highlights how slower bimolecular recombination rates (k2) are indicative of longer free-carrier diffusion length in the perovskite layer, which favours the efficiency of the planar heterojunction perovskite solar cells (PSCs). Notably, it becomes apparent that the pristine sample exhibits a slightly slower bimolecular recombination rate which agrees with the better electrical characteristics of the corresponding device. The charge carrier lifetime trend was probed using the TPV technique on devices having the previously defined photoactive bilayer structure, with the addition of a top electrode necessary for the formation of electrical contact (ITO/PTAA/MAPI/Ag). Experiments were performed under open circuit conditions and perturbations of small amplitude in order to ensure that the density of photogenerated charge carriers didn't interfere with the established electrical field. In this fashion, voltage transients followed single exponential decay and we were able to directly observe the lifetime of charge carriers. The resulting trend is depicted in Notably, the fresh sample exhibited slightly faster 55 Thus, we can correlate the sharp drop of carrier lifetime after the 5 h mark with the obstruction of charge flow through the sample arising from structural disordering, as well as capacitive effects introduced by the optoelectrical measurement protocol.56,57 Moreover, we note the absence of any apparent charge reservoir, as this occurrence would enhance the random-walk mechanism during the carrier transport and cause a decay trend of higher orders.58,59 As mentioned earlier, all transient decays in our case exhibited a mono-exponential trend which is not indicative of any effectual accumulation of charge carriers.60 The full set of TPV transients taken at each step of UV degradation is given in the ESI , according to the following relation:69q stands for elementary charge, d for thickness, while ε and ε0 are the dielectric constants of MAPI and vacuum permittivity, respectively. VTFL represents the transition point between ohmic and trap-filled regimes and is defined as the trap filling limit voltage. ntraps distribution in each of the tested devices. The initial increase of trapping states occurs after 2 h of UV treatment when the defect population saturates and stays levelled until the degradation time extends beyond 16 h, resulting in a further increase of trap concentration. The lowest population of traps was detected in the reference device, which is consistent with the results obtained from TAS, where the fresh specimen exhibited the fastest trap filling features. The increase of ntraps is accompanied by the shift of onset defining VTFL, which stems from the activation of additional trapping centres at higher voltages and reduction of film quality, as expected from devices subjected to prolonged degradation conditions.70 Moreover, the observed trend in the ohmic region suggests an increase of current corresponding to the leakage upturn, in accordance with UV exposure length.On the other hand, TAS was operated within the terahertz time-domain, which makes it non-sensitive to recombination phenomena occurring outside narrowly localized areas.I Fig. S7. The mec71–73 A further voltage increase leads to the space-charge-limited regime, a trap-free zone where device operation is governed only by the drift mobility of holes. The slope of the current–voltage curve in this domain is dictated by the quadratic growth (as per the Child–Langmuir law).74 In principle, it is possible to deduce hole mobility in the SCLC region according to the Mott–Gurney relation.75 However, in the case of absorbers with high intrinsic population of mobile species, like perovskites, straightforward estimation of drift mobility is convoluted, due to the unfavourable ratio between film thickness and size of the actual barrier for carrier injection.76 To that extent, we conducted photo-CELIV studies to gain insight into the charge carrier drift mobility distribution as a function of UV exposure. Since the photo-CELIV measurement protocol requires fully functional solar cells to reliably extract parameters necessary for estimation of drift carrier mobility, it was necessary to expand the active structure from a bilayer to a complete device (ITO/PTAA/MAPI/PC60BM/PFN/Ag). Thus, the existing bilayer structure was further expanded by deposition of a PCB60M layer on the reference and UV treated samples, followed by inclusion of PFN buffer and an Ag top electrode, resulting in the desired inverted planar architecture. We note that the interface established between MAPI and PC60BM formed in this way may differ from the case when they were to be formed prior to the UV exposure. Nonetheless, our aim was to monitor the capacity of PTAA for hole transport in response to UV degradation. Hence, exclusion of such an effect on the ETL and respective interface with the absorber helps prevent possible masking of the response. In principle, photo-CELIV utilizes short light pulses to generate charge carriers and extracts them afterwards, using a triangular ramp after a chosen delay time. The current response obtained in this way can be used to estimate the mobility of charge carriers according to the equation proposed by Lorrmann et al.:77j0 stands for displacement current, Δj for current superimposed over j0, d for thickness, A is the extraction ramp and tmax is the time necessary for the current to reach its maximum value. Transient responses corresponding to all degradation scenarios presented in 78 Besides visual tracking, dispersion phenomena can be quantified using the ratio between tmax and the time defined by the full width at half maximum (t1/2) of the transient superimposed over displacement current. Charge transport devoid of any dispersive features corresponds to t1/2 : tmax = 1.2.28 Dispersive distribution of UV treated devices outlined in 79Such behaviour is in line with that previously noted by TPC analysis and described reduction of PTAA charge extraction capabilities, since the formation of shunt pathways imposes a barrier for collection of photogenerated carriers.60BM layer. Nevertheless, since pristine PC60BM was used during fabrication of all devices, any potential trend disturbance introduced by the ETL would be equally exerted on all samples and the resulting distribution should remain preserved. Finally, readers should keep in mind that photo-CELIV detects collection of the fastest carriers at respective electrodes regardless of charge species sign, hence the difference between electrons and holes is imperceptible to this technique.It is evident from the inset of In summary, we evaluated the effect of UV stress on the stability and charge transfer properties of PTAA and the interface formation with the MAPI absorber, through a series of rigorous optoelectrical characterization experiments. Each experimental procedure was conducted at different stages of UV exposure conjoined with the oxygen and humidity impact, to estimate the degree of degradation encountered under realistic operational circumstances. The resulting morphological degradation fingerprint points towards the significance of grain boundaries and ion migration, while the impact of UV exposure on the structural integrity of PTAA/MAPI bilayers exceeds that of oxygen and humidity. Furthermore, both the polymer and perovskite show only partial signs of degradation, lacking any indications of severe structural decomposition. Transient absorption spectroscopy revealed a stable PTAA/MAPI interface, resilient to UV intensity and extended periods of illumination. These findings coupled with TPC tracked charge extraction impose the conclusion that deterioration of PTAA film quality hinders efficient hole collection. The charge recombination decay indicates a non-uniform distribution of carrier lifetimes arising from impeded charge flow due to the structural disarrangement originating from the bulk of the bilayer, rather than the interface between the absorber and PTAA. Trap state distribution revealed by SCLC measurements demonstrates nearly monotonic UV induced dependency of defect population until the degradation threshold reaches the point of further trap generation at longer degradation times. This trend is accompanied by the formation of shut channels, allowing leakage of the current and appearance of a visible hysteretic response during the photoconversion process. The carrier drift mobility investigated using photo-CELIV revealed a noticeable initial drop upon UV exposure, followed by the relaxation to uniform values, suggesting partial dependence of the mobility on degradation time of PTAA. Additionally, tested samples exhibited signs of dispersive transport and increased dependence of drift mobility on the electric field, once the cells were subjected to extended periods of UV illumination. Finally, we can infer that PTAA degradation caused by UV light interferes with the mechanism of charge transport and appropriate actions should be taken to improve its operational resilience, such as doping with suitable materials or shielding from the adverse effects of ultraviolet irradiation by inclusion of an additional layer.The authors declare no conflicts of interest.NA-001-C9NA00246D-s001


Since its first description 25 years ago, color Doppler twinkling has been a compelling ultrasound feature in diagnosing urinary stones. While the fundamental cause of twinkling remains elusive, the distinctive twinkling signature is diagnostically valuable in clinical practice. It can be inferred that if an entity twinkles, it empirically has certain physical features. This work investigates a manipulable polymeric material, polymethyl methacrylate (PMMA), which twinkles and has measurable surface roughness and porosity that likely contribute to twinkling. Comparative investigation of these structural properties and of the twinkling signatures of breast biopsy markers made from PMMA and selected commercially available markers showed how twinkling can improve ultrasound detection of devices intentionally designed to twinkle. While this specific application of detecting breast biopsy markers by twinkling may provide a way to approach an unmet need in the care of patients with breast cancer, this work ultimately provides a platform from which the keys to unlocking the fundamental physics of twinkling can be rigorously explored.The online version contains supplementary material available at 10.1186/s41747-022-00283-z. Surface roughness is associated with the color Doppler ultrasound twinkling phenomenon.Polymethyl methacrylate is a manipulable material with measurable surface roughness and porosity.Polymethyl methacrylate markers twinkle with multiple transducers using a range of ultrasound frequencies.The twinkling artifact, described as a radiological sign, on color Doppler ultrasound , 2, is cConsequently, color Doppler ultrasound, which is generally not used in breast radiology could play a significant role  for examThis work investigates a manipulable polymeric material, polymethyl methacrylate (PMMA)  that twiPMMA  mixed according to specifications on the package insert was made into a 1.3-mm diameter, 8-mm long cylindrical construct comparable in size to conventional biopsy markers by extruding the PMMA from a 15-gauge hole punched into the hub of a needle attached to a 3-cc syringe.a) of four commercial metallic breast biopsy markers: TriMark® cork , Tumark® Q (Hologic®), UltraClip™ ribbon , and SenoMark™ O clip . Optical surface characterization to measure Sa was performed using a consistent magnification of × 20 for all markers. Overall shape and curvature of the markers were removed from the surface characterization using 4th order polynomial curve fitting of the optical measurement data [Based on earlier developed techniques , a non-cent data .Scanning electron microscopy (SEM)  images captured the surface irregularities of PMMA and the metallic biopsy markers. Porosity was determined using micro-computed tomography  using provided computed tomography analyzer software  based on thresholding and regions of interest on 15-μm slice thicknesses .ex vivo in pork belly meat using a clinical system  with 9-L and ML6-15 linear array transducers, both generally used in breast ultrasound, and a C1-6 curvilinear transducer typically used in abdominal ultrasound . To minimize experimental bias, the markers were placed at roughly the same depth between 1 and 2 cm deep and spaced minimally apart so that they could be scanned simultaneously. For the phantom study, two gel phantoms were stacked on top of each other to minimize backscatter from the tabletop.Ultrasound of the four commercial markers and the PMMA marker was performed in a gel phantom and 125I seed next to it for localization without definite visualization of the marker on B-mode imaging. A twinkling score of 2 and below would require additional imaging features or information before an 125I seed would be used to localize it.Scanning parameters such as ultrasound transmit frequency, color scale, and gain were adjusted to optimize twinkling. For radiological assessment, a twinkling score was defined from 0 (least twinkling and least confident detection) to 4 (most twinkling and most confident detection) . In geneex vivo pork belly meat by B-mode imaging using the ML6-15 transducer. With color Doppler, a distinct twinkling signature (twinkling score ≥ 3) was noted for the cork, PMMA, and Q markers. Relative to the transducers, the twinkling signature in both the gel phantom and the pork belly meat was most pronounced (highest twinkling scores) with the C1-6 transducer (color transmit frequency 3.1 MHz in the gel phantom and 3.1 MHz in pork belly meat) followed by the 9L transducer (color transmit frequency 5.0 MHz in the gel phantom and 3.1 MHz in pork belly meat), and least with the ML6-15 transducer (color transmit frequency 6.3 MHz in both the gel phantom and pork belly meat). This relationship was particularly evident in the pork belly study. Twinkling signatures that scored a 4 were evident over a range of color frequency settings towards the lower end of the spectrum for each transducer  compared to the ribbon and the O markers (< 1 μm), consistent with previously published results [Sa of the PMMA marker (5−6 μm) was between that of the cork and Q markers. SEM images capturing the surface irregularities of PMMA and the four metallic biopsy markers used in this study supported the Sa measurements. The color Doppler ultrasound twinkling signature was both present and comparable for the markers with high Sa  and essentially absent for markers with low Sa (ribbon and O markers) as shown in Figs. The  results . The Sa was high −10 μm coSa) was 60.6%. The porosities of essentially solid wire-based constructs such as the ribbon (low Sa) and the O (low Sa) markers were not measurable. The exception was the Q marker (high Sa) which had non-detectable porosity. Unlike Sa, the association between porosity and twinkling appears weaker, favoring surface roughness as a stronger contributor to twinkling . The porosity of the cork marker  and microscopic (μm) surface roughness features, once the association between surface roughness and twinkling is better understood. Adding an ultrasound twinkling dimension to biopsy marker detection in the care of patients with breast cancer addresses a clinical need and is readily translatable to practice. By building a platform for defining what surface characteristics create and optimize twinkling, commercially available markers could be optimized to twinkle, and 3D printed makers could be made from material readily available and less expensive, such as PMMA.Current hypotheses on the causes of twinkling have highlighted the presence of air bubbles that vibrate in response to ultrasound insonification and rough surfaces that cause rapid phase changes in the backscattered ultrasound –19. ThisA twinkling signature associated with a biopsy marker has the potential to improve challenges breast radiologists face during preoperative localization of targets that have responded well to neoadjuvant therapy and are now radiologically normal or occult. While color Doppler ultrasound for detection of twinkling is not a standard part of breast radiology, it is readily available on nearly all cart-based and portable ultrasound vendor platforms. This technological development demonstrates how PMMA with measurable surface roughness features can provide a promising medium to better understand the underlying causes of the twinkling phenomenon on color Doppler ultrasound.The limitations of using twinkling to detect breast biopsy markers include false-positive entities that twinkle. Sources of false-positive twinkling related to an application in breast radiology include blood flow, microcalcifications , calcifiFuture work will involve creating biopsy markers that are “super twinklers” by refining their surface roughness. In so doing, the underlying causes of ultrasound twinkling may be better understood.


The intrinsic high heterozygosity of cassava makes conventional breeding ineffective for rapid genetic improvement. However, recent advances in next generation sequencing technologies have enabled the use of high-density markers for genome-wide association studies, aimed at identifying single nucleotide polymorphisms (SNPs) linked to major traits such as cassava mosaic disease (CMD) resistance, dry matter content (DMC) and total carotenoids content (TCC). A number of these trait-linked SNPs have been converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers for downstream application of marker assisted selection.We assayed 13 KASP markers to evaluate their effectiveness in selecting for CMD, DMC and TCC in 1,677 diverse cassava genotypes representing two independent breeding populations in Uganda.Five KASP markers had significant co-segregation with phenotypes; CMD resistance (2), DMC (1) and TCC (2), with each marker accounting for at least 30% of the phenotypic variation. Markers located within the chromosomal regions for which strong marker-trait association loci have been characterised  had consistently superior ability to discriminate the respective phenotypes.The results indicate varying discriminatory abilities of the KASP markers assayed and the need for their context-based use for MAS, with PSY2_572 particularly effective in selecting for high TCC. Availing the effective KASP markers on cost-effective genotyping platforms could facilitate practical implementation of marker-assisted cassava breeding for accelerated genetic gains for CMD, DMC and provitamin A carotenoids. Manihot esculenta Crantz), a climate-resilient crop grown on approximately 18 million hectares in Africa, offers great potential to end extreme hunger, achieve food security, improve nutrition and eradicate poverty if ideal varieties are deployed  elimination of genotypes with unfavorable alleles at the early stage of the selection scheme, thus allowing for field testing of a reduced number of genotypes, (ii) rapid introgression of resistant genes into existing cassava clones, in places where the disease has recently spread to, for example in Southeast Asian countries , (iii) eThe advent of next generation sequencing technologies has renewed the hope for MAS in plant breeding by enabling the identification and use of trait-linked single nucleotide polymorphism (SNP) markers in selection. With sequencing and annotation of the cassava genome nearly complete , it is nIn the case of cassava, the use of KASP markers in selection decisions is yet to be fully integrated into breeding programs. Effective deployment of markers for routine MAS requires an assessment of their predictive ability through technical and biological validations in independent populations  . TechniTwo diverse cassava populations from Uganda’s breeding program at National Crops Resources Research Institute (NaCRRI) were used as independent genetic resource for the KASP marker validation. One population was constituted by 653 genotypes segregating for DMC and TCC in the provitamin A cassava breeding pipeline (pVAC population) while the second trial had 1,024 genotypes (white-fleshed population) segregating for CMD resistance. The two populations were phenotyped concurrently at Namulonge, central Uganda, during 2018/2019 cropping season. Namulonge is a known hotspot for CMD due to the high prevalence of cassava geminiviruses and super abundance of whitefly vector population in the area. Each trial was laid down in an augmented design using 10 plants per plot and two checks replicated within each block, all planted at spacing of 1 x 1 m. Furthermore, rows of CMD infected plants of cultivar BAO (highly susceptible to CMD) were planted as source of inoculum within and around the trial for white-fleshed population to increase disease pressure. Plants were allowed to grow in field under natural conditions for 12 months, with weeding done regularly when needed.CMD severity was scored on plant basis at three, six and nine months after planting using the 1-5 scale , where 1where DSW = dry sample weight and FSW = fresh sample weight.https://www.nextgencassava.org/). The markers were a product of GWAS using a West African cassava germplasm (Thirteen KASP markers associated with CMD (3), DMC (4) and TCC (6) were selected from genomic resource under the Next Generation Cassava Breeding project . Briefly, the genotyping used the high throughput PCR SNPline workflow using 1 μL reaction volume in 96-well plates for PCR. The KASP genotyping reaction mix comprised three components: (i) sample DNA (~10 ng); (ii) marker assay mix consisting of target-specific primers; and (iii) KASP-TFTM Master Mix containing two universal FRET (fluorescence resonant energy transfer) cassettes (FAM and HEX), passive reference dye (ROX™), Taq polymerase, free nucleotides, and MgCl2 in an optimized buffer solution. The SNP assay mix was specific to each marker and consisted of two Kompetitive allele-specific forward primers and one common reverse primer. Finally, the PCR products were fluorescently read, and allele calls made using KRAKENTM software.Tissues were collected from young newly expanded leaves of plants growing under natural field conditions. Four leaf discs of 6 mm diameter were punched into wells of sample collection plates and desiccated using silica gel for 48 hours. Plates containing dry leaf tissues were shipped to Intertek, Australia. The KASP marker details are available through the Excellence in Breeding repository here: lme4 package for R statistical software  such that ZblockB represented the random effect for blocks and ϵ was the residual such that 2H) as:Where ranef function in lme4 package.where ggpubr package in R were used to visualize segregation of marker genotypes for each phenotype, and statistical differences among the genotypes were compared using the Kruskal-Wallis test. Marker effects were further evaluated by regressing the marker genotypes onto respective phenotypes for estimation of phenotypic variance accounted to by each marker. In this case, linear regression was performed using the lm function in R such that marker genotypes and the corresponding phenotypes were treated as independent and response variables, respectively.Boxplots drawn with the The KASP markers for CMD resistance targeted the CMD2 locus , which h= false positive, TN = true negative, FN = false negative, TP = true positive  allele (TG or TT) significantly co-segregated with low CMD severity compared to the unfavorable allele for which the associated genotypes had mean CMD severity >3 Figure 3Three markers tested for TCC  also had segregation patterns likened to dominant gene effect. For marker PSY2_572, genotypes with at least one copy of the favorable allele (AA or CA) co-segregated with high levels of TCC Figure 4For DMC, two markers (S1_24197219 and S6_20589894) exhibited dominant segregation patterns Figure 5An inverse relationship was noted between TCC and DMC Figure 62R = 0.45). Only one marker (S1_24197219) accounted for up to 30% of variation in DMC, with the remaining three explaining ≤5% of the phenotypic variance. Meanwhile, markers PSY2_572 and S1_24636113 accounted for the 42% and 30%, respectively. All other TCC markers had low 2R values, with S1_30543962 accounting for 15% of the phenotypic variation and S15_7659426 explaining none of the observable variation in the trait.When we regressed the marker genotypes onto the respective phenotypes, all markers showed significant association with traits, except two markers (S8_25598183 and S15_7659426) for TCC Table 3.Principal component analysis further revealed similarities between markers located within the same chromosomal positions. For example, CMD markers S12_7926132 and S12_7926163 had identical contribution to the total variation in the principal components; the same observation was true for TCC markers PSY2_572, S1_24636113  and relatively low false positive rate (~38%) Table 4.https://www.nextgencassava.org/) have yielded important genomic resources, such as well annotated genome sequence  and industry. Dry matter content, provitamin A carotenoids content and virus disease resistance are must-have traits for cassava product profile in Uganda . The twoThe robust and high call rates for the marker genotypes demonstrated their usefulness in screening cassava germplasm of broad genetic background. However, the wide variability in the predictive ability of the markers suggests the need for their case-by-case deployment in cassava breeding. For example, the consistent and significant co-segregation of allelic states of markers S12_7926132 and S12_7926163 with CMD severity indicates their reliability for MAS. In fact, markers S12_7926132 and S12_7926163 may be tightly linked to the functional gene in conferring CMD resistance in cassava given their close proximity to CMD2 resistance locus previously identified on chromosome 12 . Given, DNA polymerase δ subunit 1 (MePOLD1) as the functional gene on chromosome 12 responsible for CMD2 resistance. That study generated eight novel KASP markers that, when incorporate into the existing genomic resource, could reinforce prospects of MAS for CMD2 resistance in cassava breeding.The allelic segregation of markers S12_7926132 and S12_7926163 points to two important aspects. Foremost, the consistent dominant allelic effects exhibited in the marker segregation underpin CMD2 is a qualitative trait under additive genetic effect. Despite the classical qualitative segregation exhibited for CMDs, there was a considerable variation in severity levels for susceptible clones, which could be attributed to differences in plot-based viral loads or other genetic factors controlling plant fitness. Secondly, the large proportion of variation in CMDs unexplained by the marker effects indicates the need for continuous improvement for effective operationalization of MAS platforms in cassava. Through whole genome sequencing and genetic variant analysis,  fine-mapManes.01G124200.1, which is a phytoene synthase (PSY) gene known to increase accumulation of provitamin A carotenoids in cassava roots  can be found below: WE conceptualized the study, guided field trials and wrote the original manuscript. OE phenotyped trial for CMD and reviewed manuscript. FG phenotyped trial for DMC and TCC. SM guided field trials and edited manuscript. TA guided field trials and edited manuscript. EN guided laboratory analyses for DMC/TCC and edited the manuscript. AO guided data analyses and reviewed manuscript. IR guided marker data analyses and edited manuscript. RK guided the study conceptualization and provided funds. All authors contributed to the article and approved the submitted version.www.nextgencassava.org/) funded by the Bill and Melinda Gates Foundation through Cornell University (grant number OPP1048542).This work was supported through the Next Generation Cassava Breeding project  for providing detailed technical information on the 13 KASP markers.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 reviewer XZ is currently organizing a Research Topic with one of the authors RK.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Spatial transcriptomic technologies and spatially annotated single-cell RNA sequencing datasets provide unprecedented opportunities to dissect cell–cell communication (CCC). However, incorporation of the spatial information and complex biochemical processes required in the reconstruction of CCC remains a major challenge. Here, we present COMMOT  to infer CCC in spatial transcriptomics, which accounts for the competition between different ligand and receptor species as well as spatial distances between cells. A collective optimal transport method is developed to handle complex molecular interactions and spatial constraints. Furthermore, we introduce downstream analysis tools to infer spatial signaling directionality and genes regulated by signaling using machine learning models. We apply COMMOT to simulation data and eight spatial datasets acquired with five different technologies to show its effectiveness and robustness in identifying spatial CCC in data with varying spatial resolutions and gene coverages. Finally, COMMOT identifies new CCCs during skin morphogenesis in a case study of human epidermal development. This work presents a computational framework, COMMOT, to spatially infer cell–cell communication from transcriptomics data based on a variant of optimal transport (OT). The complex structures and functions of multicellularity are achieved through the coordinated activities of various cells. Cells make decisions and accomplish their goals by interacting with an environment consisting of external stimuli and other cells. A major form of cell–cell interaction is cell–cell communication (CCC), mainly mediated by biochemical signaling through ligand–receptor binding that induces downstream responses that shape development, structure and function.1. Computational tools have been developed to estimate CCC activities from scRNA-seq data3 using signaling databases6. Most of these methods rely on the expression levels of ligand and receptor pairs and explicitly defined functions. For example, the products of ligand and receptor levels7 or non-linear Hill function-based models6 are used. In addition, these methods emphasize different aspects of CCC. For example, CellPhoneDB5, ICELLNET7 and CellChat6 account for the multi-subunit composition of protein complexes; SoptSC8, NicheNet9 and CytoTalk10 utilize downstream intracellular gene–gene interactions; and scTensor11 examines higher-order CCC represented as hypergraphs. These inference methods designed for scRNA-seq data have provided biological insights based on non-spatial transcriptomic data13. However, these non-spatial studies often contain significant false positives given that CCC takes place only within limited spatial distances that are not measured in scRNA-seq datasets. Improvements can be made by filtering the inferred CCC using spatial annotations14.Traditionally, CCC studies were restricted to a few cell types and a small number of selected genes at the resolution of cell groups. Recently, the emergence of single-cell transcriptomics  has enabled the examination of tissues at single-cell resolution at unprecedented genomic coverage20 provides information on the distance between cells or spots containing multiple or fractions of cells. At various cellular resolutions these technologies measure the spatial expression of hundreds to tens of thousands of genes in 2-dimensional (2D) or 3-dimensional tissue (3D) samples21. Methods and software24 developed for non-spatial data analysis have been applied to spatial data, with a small number of methods designed specifically for spatial data. Giotto builds a spatial proximity graph to identify interactions through membrane-bound ligand–receptor pairs23; CellPhoneDB v3 restricts interactions to cell clusters in the same microenvironment defined based on spatial information25; stLearn relates the co-expression of ligand and receptor genes to the spatial diversity of cell types24; SVCA26 and MISTy27 use probabilistic and machine learning models, respectively, to identify the spatially constrained intercellular gene–gene interactions; and NCEM fits a function to relate cell type and spatial context to gene expression28. However, current methods examine CCC locally and on cell pairs independently, and focus on information between cells or in the neighborhoods of individual cells. As a result, collective or global information in CCC, such as competition between cells, is neglected.Spatial transcriptomics29, developmental trajectory reconstruction30 and spatial annotation of scRNA-seq data32. Naturally, one can form an optimal transport problem by viewing ligand and receptor expression as two distributions to be coupled with a cost based on spatial distance34. However, when using classical optimal transport, different molecule species with significantly different expression levels are normalized to ensure the same total mass, which renders the units of distributions unable to be compared. Furthermore, multiple ligand species can bind to multiple receptor species, resulting in competition. Of the 1,735 (secreted) ligand–receptor pairs in the Fantom5 database35, 72% of ligands (372 of 516) and 60% of receptors (309 of 512) bind to multiple species. Such competition between multiple molecule species is ubiquitous and a critical biophysical process but it is ignored in existing methods. Although recent optimal transport variants such as unbalanced optimal transport and partial optimal transport can deal with unnormalized distributions and avoid certain coupling due to signaling spatial range and simultaneous consideration of multiple species38, they introduce other issues. Specifically, unbalanced optimal transport38 in its common form uses Kullback–Leibler divergence as a soft constraint on marginal distribution preservation. This approach may result in the total amount of coupled signaling molecule species significantly exceeding the total amount of either ligand or receptor initially available. By contrast, partial optimal transport36 requires an additional parameter, the total coupled mass, which is usually difficult to estimate in the context of CCC inference.Optimal transport has recently been used for transcriptomic data analysis, including batch effect correction38. An efficient algorithm is developed specifically for solving the collective optimal transport problem.To adapt optimal transport theory for the application of CCC inference, we present a method called collective optimal transport, which is capable of preserving the comparability between distributions, ensuring that the total signal does not exceed the individual species amounts (ligand or receptor), enforcing spatial range limits of signaling, and handling multiple competing species. The collective optimal transport method achieves this by optimizing the total transported mass and the ligand–receptor coupling simultaneously, unlike existing optimal transport methods. By introducing an entropy regularization to enforce the inequalities for marginal distributions, the collective optimal transport can be reformulated as a special case of the general unbalanced optimal transport frameworkBased on collective optimal transport, we develop COMMunication analysis by Optimal Transport (COMMOT), a package that infers CCC by simultaneously considering numerous ligand–receptor pairs for either spatial transcriptomics data or spatially annotated scRNA-seq data equipped with spatial distances between cells estimated from paired spatial imaging data; summarizes and compares directions of spatial signaling; identifies downstream effects of CCC on gene expressions using ensemble of trees models; and provides visualization utilities for the various analyses.We show that COMMOT accurately reconstructs CCC on simulated data generated by partial differential equation (PDE) models and outperforms three related optimal transport methods. We then apply COMMOT to analyze scRNA-seq data that have been spatially annotated using paired spatial datasets and five types of spatial transcriptomics data that differ with respect to spatial resolution or gene coverage. Finally, we examine a specific system of human epidermal development and elucidate connections between CCC and skin development.Ligands and receptors often interact with multiple species and within limited spatial ranges Fig. . Considens cells or spots and nl ligand species and nr receptor species, the collective optimal transport determines an optimal multi-species coupling k to receiver cell l through ligand i and receptor j. This is achieved by solving a minimization problem, I is the index set for ligand and receptor species that can bind together, and i on spot k. The species-specific cost matrix i by infinity. The competitions between molecule species and cells are considered by assuming that a given receptor species or cell has limited capacity for interactions, such that a stronger inferred interaction with one ligand species or cell reduces the potential of interaction with other ligand species or cells  found in different regions of the innermost basal layer of the epidermis, a differentiating spinous cell cluster in the intermediate layer, and a granular cell cluster in the outermost living layersers Fig. . This reers Fig. .Fig. 2RoThe spatial signaling between epidermal cells was inferred in the integrated dataset by considering ligand–receptor pairs annotated in the database CellChatDB. For example, our computational analysis predicted that molecular interactions between the ligands GAS6 and PROS1 with their receptor TYRO3 (GAS6-TYRO3 and PROS1-TYRO3) are significant in granular cells and moderately present in basal cells Fig. . This pr39 , NOTCH and JAK/STAT  pathways39 Fig. –16. For 39 Fig. . WNT sigKRT15 and KRT5, as well as lower expression of the known terminally differentiated granular cell markers LOR and FLG, reinforcing the WNT pathway’s known role in stem cell proliferation40. The analysis also predicted that higher WNT signaling would increase the expression of BCAM, POSTN and STMN1, the expression localization of which we confirmed by immunostaining on human epidermis  data of the mouse hypothalamic preoptic region with 161 genes and 73,655 cells across 12 slices along the anterior–posterior axis43  data of mouse placenta with 903 genes and 7,203 cells46 Fig. . Midkine46 Fig. , suggest18  CCC profile matrix for the ns cells and nlr ligand–receptor pairs. Differential expression analysis of the cell types and CCC profile found neuron cells to be most active through various ligand–receptor pairs, and distinct CCC activities for relatively rare cell types  data of mouse secondary somatosensory cortex with 10,000 genes measured in 523 individual cells18 Fig. . Using tpes Fig. . Predictpes Fig.  correlatWnt5b, Wnt10a and Wnt2b, while the differentially expressed genes in cluster 3  included known target genes of the WNT signaling pathway such as Gja1 and Acsf2 and the known corresponding intracellular signaling transductors Lrp5 and Lrp6  are often neglected. To prioritize the genes that are more likely to be regulated by CCC, we used a random forest modelirs Fig. . Such anirs Fig. .16 dataset of mouse brain tissue with 3,355 spots and 32,285 genes  by solving a global optimization problem that accounts for potential higher-order interactions between the multiple ligand and receptor species. To this end, we introduce collective optimal transport that determines a collection of optimal transport plans for all pairs of species that can be coupled simultaneously. As a result, the coupling between a species pair will affect other couplings and vice versa, which cannot be realized in traditional optimal transportns spatial locations and a set of nl ligand species and nr receptor species, a collective optimal transport problem is formulated as follows:i on spot k, j on spot l and F penalizes the untransported mass μi and vj. The coupling matrix k to spot l through the pair consisting of the ligand i and receptor j for I is the index set of ligand and receptor species that can bind. The cost matrix Ci,j) if Dk,l ≤ Ti,j) of the cluster-level CCC is determined by performing n independent permutations of the cluster labels and computing the percentile of the original signaling strength in the signaling strengths resulting from these label permutations. Permuting cluster labels after computing the spot-level CCC matrices may neglect communications between different clusters. To address this limitation, we provide an option that randomly permutes the locations of all spots or the spots within each cluster and then computes the spot-level CCC matrices.To elucidate CCC among cell states or local groups of spots, we aggregate the spot-by-spot CCC matrix n grid points and is represented by an array The spatial signaling direction is described by a vector field defined on a discretized tissue space consisting of P < 0.05 are kept in the edge sets To compare two cluster-level CCC networks i, and i quantified as the percentage of differentially expressed genes. The function R converts the vectors into ranks and σ is the standard deviation of the rank variables.The Spearman’s correlation coefficient is used to quantify the correlation between the inferred signaling activity and the activity of the known target genes across the cell clusters, defined as 53 is used to identify the genes that are differentially expressed with respect to After computing the CCC matrix 55 that takes a potential downstream gene as the output, and 55 is used.The identified differentially expressed CCC genes may be regulated by other genes in cells through gene regulation. To further prioritize the downstream genes, the expressions of which are affected by CCC, we train a random forest regression modelk-nearest neighbors method and k set to 100 and the maximum distance threshold of 1000 μm for Visium data and 500 μm for seqFISH+ data. The heteromeric ligand–receptor pairs in CellChatDB were converted to pairs of individual subunits. The spatCellCellcom function was then used to generate the cluster-level communication scores with the adjust_method set to fdr. For CellPhoneDB v3 analysis, the distance between clusters was quantified as the average distance between cells from the pair of clusters. The command ‘cellphonedb method statistical_analysis’ was used to generate CellPhoneDB results with the threshold parameter set to 0.1.For the CellChat analysis the spatial data were treated as non-spatial scRNA-seq data, and the count matrix was first normalized using the normalizeData function. The data were then filtered using the functions identifyOverExpressedGenes and identifyOverExpressedInteractions with the default parameters. The cluster-level communication scores in CellChat were computed using the computeCommunProb function with default parameters, and the results were further filtered using the filterCommunication function with min.cells set to 10. The ligand–receptor pairs categorized under ‘Secreted Signaling’ in the CellChatDB were examined. For Giotto analysis, the count data were first normalized using the normalizeGiotto function with default parameters. A spatial network was then created using the createSpatialNetwork function with the Frozen tissue sections (10 μm) were fixed with 4% paraformaldehyde in PBS for 15 min. Ten percent BSA in PBS was used for blocking. Following blocking, 5% BSA and 0.1% Triton X-100 in PBS was used for permeabilization. The following antibodies were used: mouse anti-KRT5 , mouse anti-KRT15 , mouse anti-BCAM , mouse anti-FGF7 , mouse anti-STMN1 ; mouse anti-IGFBP6 ; mouse anti-PMAIP1 , mouse anti-POSTN ; mouse anti-FLG ; rabbit anti-LOR ; mouse anti-TYRO3 ; rabbit anti-GAS6 ; and rabbit anti-PROS1 . Secondary antibodies include Alexa Fluor 488  and Cy3 AffiniPure . Slides were mounted with Prolong Diamond Antifade Mountant containing DAPI (Molecular Probes). Confocal images were acquired at room temperature (22.2 ºC) on a Zeiss LSM700 laser scanning microscope with a Plan-Apochromat ×20 objective or ×40 and ×63 oil immersion objectives.Homo sapiens probes from Advanced Cell Diagnostics were used: Tyro3 probe (429611), Gas6 (427811-C2) and Pros1 (506991-C2). Confocal images were acquired at room temperature on an Olympus FV3000 confocal microscope with a Plan-Apochromat ×20 objective or ×40 and ×60 oil immersion objectives.Frozen neonatal human foreskin tissue sections were used for RNA in situ hybridization using RNAscope kit v2  as per the manufacturer’s instructions. The following Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41592-022-01728-4.Supplementary InformationSupplementary Figs. 1–36, Supplementary NoteReporting SummaryPeer Review File


Calc, a mechanistically linked (but independent) biomarker for distant metastasis, is prognostic in some cohorts of triple-negative patients. Here, we develop and compare several digital pathology-based machine vision algorithms to investigate if a combined TMEM-MenaCalc biomarker could provide improved prognostic information over and above that of either biomarker alone.90% of breast cancer mortality is caused by distant metastasis, a process that involves both dissemination of cancer cells to distant sites as well as their proliferation after arrival. However, prognostic assays currently used in the clinic are based on proliferation and do not measure tumor cell dissemination potential. We previously reported that the density of Tumor Microenvironment of Metastasis (TMEM) doorways  is a prognostic biomarker for the development of distant metastasis in hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) patients. We have shown further that MenaCalc scores and determine if a combination of these biomarkers improves the ability to predict development of distant metastasis over and above that of either biomarker alone. Methods: This retrospective study included a subset of 130 patients (65 patients with no recurrence and 65 patients with a recurrence at 5 years) from the Calgary Tamoxifen cohort of breast cancer patients. Patients had confirmed invasive breast cancer and received adjuvant tamoxifen therapy. Of the 130 patients, 86 cases were suitable for analysis in this study. Sequential sections of formalin-fixed paraffin-embedded patient samples were stained for TMEM doorways (immunohistochemistry triple staining) and MenaCalc (immunofluorescence staining). Stained sections were imaged, aligned, and then scored for TMEM doorways and MenaCalc. Different ways of combining TMEM doorway and MenaCalc scores were evaluated and compared to identify the best performing combined marker by using the restricted mean survival time (RMST) difference method. Results: the best performing combined marker gave an RMST difference of 5.27 years (95% CI: 1.71–8.37), compared to 3.56 years (95% CI: 0.95–6.1) for the associated standalone TMEM doorway analysis and 2.94 years (95% CI: 0.25–5.87) for the associated standalone MenaCalc analysis. Conclusions: combining TMEM doorway and MenaCalc scores as a new biomarker improves prognostication over that observed with TMEM doorway or MenaCalc Score alone in this cohort of 86 patients.Purpose: to develop several digital pathology-based machine vision algorithms for combining TMEM and Mena Distant metastasis is the primary cause (~90%) of death from breast cancer . HoweverPreviously, we reported the discovery of the “Tumor Microenvironment of Metastasis” (TMEM) doorway, a portal into the blood vasculature composed of a tumor cell overexpressing the actin regulatory protein Mena, a perivascular macrophage, and an endothelial cell, all in direct contact . TMEM doCalc, we cannot rule out its existence.While these studies clinically validated the use of TMEM Score for prognosticating metastatic outcome in HR+/HER2− patients (the largest subgroup of breast cancer patients), a statistically significant association between TMEM Score and distant recurrence outcome was not observed in the triple-negative or HER2+ breast cancer subpopulations, perhaps due to the smaller number of these subjects available for analysis. While there is currently no evidence of a connection between the HER2 receptor status and TMEM doorways or MenaTo identify those cancer cells within the tumor that are capable of intravasation, we developed the in vivo invasion assay, a technique capable of isolating the motile fraction of cancer cells from the rest of the immobile bulk of the primary tumor ,12,13. UINV, have been shown to confer a pro-metastatic motile phenotype and are found to be expressed exclusively in invasive and disseminating tumor cells .,22.Calc ll cells A or restll cells B. This rfication .(1)MenaAll statistical analyses were performed and automated using R 4.0.4 in RStudio .In each of the four TMEM doorway tests , patientCalc analyses were performed in a similar way to the four TMEM doorway analyses.The eight standalone MenaCalc test were paired to form a combined test pair , expanding the analysis to include other breast cancer subtypes and, importantly, accounting for other variables with a multivariate analysis.In conclusion, we have developed and evaluated a potential new biomarker for prognosticating metastatic progression in ER+/HER2− breast cancer patients that combines the previously published TMEM doorway and Mena


Ulva armoricana extracts on MDV infection in vitro.Marek’s disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by an alphaherpesvirus, Marek’s disease virus (MDV). MD is presently controlled by systematic vaccination of animals, which protects efficiently against the development of clinical disease. However, MDV vaccines do not prevent the multiplication and spread of MDV field strains and may favor the emergence of strains with increased virulence. Therefore, MDV persists to be a major problem for the poultry industry and the development of new alternative strategies to control MDV is needed. Seaweed extracts have previously been shown to exert immunomodulatory and antiviral activities, especially against herpesviruses. The objective of the present study was to explore the effect of We could demonstrate that the ulvan extract as well as its vitamin-enriched formulation reduce the viral load by about 80% at 24 h post-infection in infected chicken fibroblasts at concentrations that are innocuous for the cells. We also observed a substantial decrease in MDV plaque size suggesting that ulvans impede MDV cell-to-cell spread in vitro. Moreover, we showed that ulvan extract could promote MDV reactivation in lymphoid cells.Our data provide the first evidence that the use of the ulvan extract could be a good alternative to limit MDV infection in poultry. Marek’s disease virus (MDV) is an alphaherpesvirus inducing lethal lymphoma in chickens. MDV infection progresses in 3 major phases: (i) an early cytolytic infection corresponding to the entry of the virus through the respiratory tract and its transport to the lymphoid organs where it infects B-cells and activated T-lymphocytes; (ii) a latent phase (between 7 to 10 days post-infection (pi)) in CD4 + T lymphocytes, which is characterized by the integration of the viral genome into the genome of the host cells; (iii) the tumor phase occurring in CD4 + T lymphocytes leading to malignant lymphoma formation and the death of animals from the 3rd week of infection . ConcomiMDV is endemic worldwide and, despite the availability of vaccines, MDV continues to threaten the poultry industry with economic losses estimated at 1–2 billion dollars per year , 4. VaccNumerous scientific publications have reported that algae extracts or algae-derived compounds can exhibit inhibitory activities against viral infections . ThUlva armoricana, is able to activate chicken heterophils and monocytes in vitro and in vivo [.While research on the antiviral and immunomodulatory properties of algae is sparking great interest in human medicine, studies related to animal health are still scarce, particularly with regard to herpesvirus infections. We recently demonstrated that ulvans, the sulfated polysaccharides (SPs) fraction purified from  in vivo . This suPrior to determining the effect of ulvans and Searup® on MDV replication, we evaluated the cytotoxicity of the extracts on avian cells. Chicken embryo fibroblasts (CEFs) and the lymphoid cell line 3867 K were treated with four increasing doses  of the vitamin complex (VitComp), Searup® or the ulvans extract. Untreated cells were maintained in culture and used as negative control. In order to exclude any side effects induced by degradation products of the compounds and/or cellular metabolites, the culture media was replaced after 48 h by fresh media supplemented (or not for the control condition) with the defined treatment doses. At 24, 48, 72 and 96 h post-treatment, the viability of CEFs was monitored by evaluating the level of ATP produced by the cells (CellTiter Glo assay) and the viability of 3867 K was assessed by flow cytometry using the Viability Dye eFluor™ 780.We could show that the purified ulvans extract and the vitamin complex had virtually no cytotoxic effect on primary chicken fibroblasts Fig. A. TreatmThe MDV transformed T-lymphocytes (3867 K) treated with the different formulations showed a level of viability similar to untreated cells (of about 80%), independently of the doses used Fig. B. A sligUlva armoricana as well as the commercialized Searup® formulation can impede MDV dissemination in vitro in the concentration range recommended in poultry farms (1 ml/l/24 h).To determine whether ulvans treatment can impact MDV lytic replication, we infected CEFs and treated the infected cells with increasing doses of the vitamin complex, Searup® and the ulvans extract. The viral load was quantified at 24, 48, 72 and 96 hpi by qPCR. Our results showed that all treatments significantly decreased MDV lytic replication in CEFs  in vitro. To this end, we used the 3867 K cells, a lymphoblastoid cell line that expresses the green fluorescent protein (GFP) fused to the tegument protein UL47 upon reactivation, as described previously , 28. TheThe intensification of chicken farms around the world, the incomplete protection of anti-MDV vaccines and the emergence of new hypervirulent pathotypes of MDV suggest that MDV could become a major problem in the poultry industry if new therapies or vaccines are not developed. Most efforts to control MDV are focused on developing next-generation vaccines. Although many natural products or chemicals have been shown to be effective against many viral infections affecting humans and animals, only a few studies have reported antiviral activity of compounds against MDV infection –32. HereUlva armoricana SPs also limit MDV replication in chicken fibroblasts since the viral load was strongly reduced while the number of MDV plaques was similar to that of untreated cells. In addition, the significant reduction of MDV plaque size suggests that the ulvans extract prevents MDV spread. Algae SPs have also been shown to be able to hinder the early stages of viral infection (adsorption/attachment and penetration of the virus) by binding directly to viruses or to viral receptors present on the surface of cells [Ulva armoricana can modulate the immune response notably by triggering the expression of a number of pro-inflammatory cytokines  [Ulva armoricana SPs extract can also control MDV infection/dissemination through the secretion of factors such as IFN-α which was shown to strongly impede the infectivity of MDV [Several studies reported that algae SPs are able to directly inhibit transcription/replication of alpha and gammaherpesviruses after viral internalization in the host cell , 24, 33.of cells , 34–37. of cells –40. Alsod IFN-γ) . We thusy of MDV .Aside the effect of the ulvan extract, we showed that the vitamin complex alone also reduced the replication of MDV. Nutrients, notably vitamins A and D, are known to exert antiviral activities and to stimulate immunity (for review ). LikewiUlva and especially its formulated version Searup® could promote the reactivation of MDV. We have not currently identified the mechanisms/factors triggered by ulvans that may contribute to MDV reactivation. However, among the many mechanisms identified to induce herpesvirus reactivation, we suspect that ulvans may promote MDV reactivation through the induction of pro-inflammatory cytokines expression, oxidative stress and/or the modulation of key cellular pathways , SPs extract from r review ). Interetivation , 44. Alstivation . In additivation , 47. Fintivation , 48.Ulva armoricana SPs extract ulvans and its commercialized Searup® formulation considerably reduce the dissemination of MDV in vitro with limited to no cytotoxicity for avian cells. Clearly the anti-MDV activities of ulvans warrant further investigation in vivo, however, based on our data it can be speculated that the ulvans SPs extract could limit MDV infection at the early stages of infection. Ulvans could indeed exert an antiviral activity by limiting replication/dissemination of MDV and/or by stimulating the innate immune system of chickens [In conclusion, we demonstrated that the chickens . In addiAltogether, our results identify ulvans as a natural seaweed extract with potent antiviral activity against MDV, and potentially other avian viruses, that could be readily exploited in alternative treatment strategies for controlling viral diseases in poultry farming.Chicken embryonic fibroblasts (CEFs) were prepared from 12-day-old specific pathogen-free White Leghorn (LD1) embryos and maintained in culture as previously described . CEFs inUlva armoricana SPs fraction (ulvans) (ii) Searup® (Olmix SA) composed of the ulvans fraction formulated with vitamins A and D3 and (iii) the vitamin solution without seaweed extracts (VitComp) containing vitamins A and D3 according to the European legislation (EU 2015/724 and EU 2017/1492). The range of concentration  of SPs extract from Ulva corresponds to 11,5 mg/L to 46 mg/L of active seaweed ingredient. In the formulated version Searup®, the dose of SPs extract range between 8 mg/l to 32 mg/L. Of note, the concentrations used in the study was defined according to the recommended dose of Searup® (1 ml/l) used in poultry farms. Treatments were added to the culture media of infected CEFs from 1h post-infection (hpi). The 3867 K cells were cultured at a density of 5.105 cells/P6-well plates in media containing the different treatments. All media supplemented with the different treatments doses were renewed after 48 h of culture. In a second set of experiment, the 3867 K cells were treated with 2 ml/l of VitComp, Searup® or ulvans extract in combination with 0.5 mM of sodium butyrate , a histone deacetylase inhibitor known to trigger herpesvirus reactivation [Cells were treated with increasing doses of (i) the tivation , 51, 52.3) were grown in opaque-walled 96-well plates (Thermo Scientific) in 100 μl complete media containing the different doses of VitComp, Searup® or ulvan extract. At 24, 48, 72 and 96 h post-treatment (hpt), cells were washed in PBS1X and incubated with 100 μl of ATP detection reagent for 10 min at room temperature in the dark. Luminescence was measured using a GloMax-Multi Detection System (Promega). Relative ATP production values for treated cells were calculated as fold changes with reference to mean values obtained for non-treated cells (set to 1). Three independent experiments were performed in triplicate.The viability of CEFs incubated with the different concentrations of VitComp, Searup® and ulvans was assessed by quantification of ATP levels using the CellTiter-Glo® kit (Promega) according to the manufacturer’s instructions. Cells (5 × 10The viability of 3867 K lymphoid cells was assessed at indicated time points using the Fixable Viability Dye eFluor™ 780 (eBioscience) according to standard procedures. Viable cells were detected by flow cytometry using a MoFlo AstriosEQ high-speed cell sorter . A first gating was based on morphological criteria (FSC/SSC) in order to eliminate cellular debris and damaged cells. A second gating was then performed based on the eFluor 780 staining which allowed us to discriminate between dead (eFluor positive) and viable (eFluor negative) cells.6 copies of the cellular genome quantified by detection of the Gallus gallus inducible nitric oxide synthase gene (iNOS).CEFs infected with the recEGFPVP22 virus were treated at 1 hpi with increasing doses  of the different treatments. Infected CEFs left untreated were used as negative control. MDV replication was assessed by real-time quantitative PCR (qPCR) as previously described –55. At 2In 3867 K cells, MDV reactivation  was detected by flow cytometry based on the expression of the late antigen UL47-GFP. First, viable cells were selected on morphological criteria (FSC/SSC) and the eFluor 780 staining. The eFluor 780-negative population (viable cells) was then further gated for UL47-GFP expression to determine the percentage of reactivated cells (eFluor 780 negative/UL47-GFP positive).At 96 hpi, CEFs infected with MDV and treated with the vitamin complex, Searup® and ulvans were fixed using 4% paraformaldehyde and MDV plaque numbers were counted. MDV dissemination was evaluated by measuring the size of plaques visualized by GFP-expression, as previously described . At leasp values < 0.05 were considered statistically significant as indicated in the figure legends.All graphs and statistics were performed using the GraphPad Prism software version 5.02 . Data are presented as means and standard error of the mean (±SEM) from at least 3 independent experiments. The Kruskal-Wallis test was used to compare differences in multiple groups and the Mann-Whitney (two-tailed) test was used to compare non-parametric variables between two groups.


Takotsubo cardiomyopathy is a form of non-ischemic cardiomyopathy characterized by transient systolic dysfunction. The prevalence of Takotsubo cardiomyopathy has been estimated to be about 2% overall but about 10% amongst women presenting with clinical manifestations of acute coronary syndrome. The overall mechanism of the disease still remains unclear. However, treatment of Takotsubo cardiomyopathy appears to be similar to congestive heart failure (CHF) medical management. This case highlights the classic presentation exhibited very similar to acute coronary syndrome and diagnostic criteria for Takotsubo (stress-induced) cardiomyopathy. Takotsubo cardiomyopathy, also known as Broken-Heart Syndrome, started being reported in the 1990s by Japanese authors . They foA 60-year-old woman with a past medical history of anxiety and stomach ulcers presented to the hospital for an acute onset of 10/10 chest pain radiating to her back. She had witnessed her husband fall flat on his face. She immediately called for emergency medical services (EMS) who advised her to initiate cardiopulmonary resuscitation (CPR) because he likely had a cardiac arrest. However, due to her husband’s morbid obesity, she was unable to flip her husband over in order to start CPR. As EMS arrived and attempted to revive her husband, the patient started to have severe 10/10 chest pain in addition to being tearful with worsening anxiety as a result of experiencing the ordeal. Despite EMS’s efforts, they were unable to revive her husband and he was pronounced dead at the scene. Due to the patient’s new onset of chest pain, EMS decided to treat her with aspirin and nitroglycerin and transported her to the hospital. The patient’s initial electrocardiogram (ECG) at the hospital showed normal sinus rhythm, heart rate 75/min, normal axis, QTc of 428ms, inverted T-waves in leads V4-V5 and flattened T-waves in leads III and V6 Figure . High sensitivity troponins had an initial result of 584 that trended up to 4192 before down trending to 1650. She was hemodynamically stable while in the emergency room. Her chest X-ray during the initial evaluation in the emergency department was unremarkable. Computed tomography angiography (CTA) chest had ruled out a pulmonary embolism and aortic dissection. Her chest pain improved without receiving additional medications while in the emergency room and resolved when she was moved up to the medical/surgical floor. On collecting additional history from the patient, she endorsed an extensive family cardiac history including her mother who had a myocardial infarction at the age of 48 years followed by a quadruple coronary artery bypass graft at the age of 50 years, and her father who had multiple episodes of myocardial infarction and coronary artery disease also treated with a quadruple coronary artery bypass graft. Her paternal grandfather passed away from a myocardial infarction. Her paternal aunt and uncle had a history of myocardial infarction. Her maternal grandfather had a stroke.A transthoracic echocardiogram (TTE) showed a left ventricular ejection fraction of 40-45% with basal hyperkinesis and apical akinesis suggesting stress-induced cardiomyopathy Figure . Due to the increased risk factors, lab results, ECG findings, and TTE findings, cardiac catheterization was discussed with the patient who agreed to have the procedure done. Cardiac catheterization showed notable apical hypokinesis with normal left main coronary artery; left anterior descending had 30% stenotic lesion; left circumflex had 50-60% stenotic lesion; right coronary artery had 40% stenotic lesion  transient akinesia or dyskinesia of left ventricular apex and mid-ventricular segments, 2) absence of angiographic evidence of acute plaque rupture or obstructed coronary arteries, 3) ECG abnormalities of either ST-segment elevation and/or T-wave inversion, and 4) absence of cranial trauma, intracranial bleeding, pheochromocytoma, myocarditis, and hypertrophic cardiomyopathy . The disease frequently presents with chest pain and dyspnea resembling acute myocardial infarction but some patients may present with symptoms of acute decompensated heart failure like dyspnea on exertion, fatigue, weakness, and gravity-dependent pitting/non-pitting edema . ECG mayMany suggest beta blockers for the blockade of the elevated catecholamines on the myocardium in Takotsuto cardiomyopathy. However, beta blockers do not always provide protection against recurrent Takotsubo cardiomyopathy ,10. AngiOur patient met all the criteria for the diagnosis of Takotsubo cardiomyopathy because she had ECG alteration (T-wave inversion), troponin level elevation, apical akinesia on transthoracic echocardiogram and no flow-limiting lesion on cardiac catheterization, with no known disease associated with elevated catecholamine or documented cranial pathologies. Although symptoms had resolved for our patient and she did not develop symptoms of acute decompensated heart failure, precautions and risk discussions were provided along with guideline-directed medical therapy and follow-up arrangements for psychiatric therapy for her late husband and for repeating the transthoracic echocardiogram in three months.Takotsubo cardiomyopathy or stress-induced cardiomyopathy results from left ventricular dysfunction that can present similarly to acute coronary syndrome or heart failure. Diagnostic criteria have been developed to better diagnose this form of cardiomyopathy. The prognosis is favorable with most patients recovering within weeks to months and few experiencing permanent dysfunctions. There is now better awareness from a physician's standpoint of Takotsubo cardiomyopathy. Patient education about the disease will create better awareness in order to improve avoidance of stressors that can result in recurrence or more harm.