A significant decrease in mixture viscosity wasalso observed with increasing water content from ∼104 to ∼102 Pa s when increasing the water contentfrom 30 to 60 wt %. Time-resolved SAXS experiments on levitated dropletsof this proxy confirm the phase changes observed in bulk phase mixturesand demonstrate that coexistent nanostructures can form in droplets.Aerosol compositional and subsequent nanostructural changes couldaffect aerosol processes, leading to an impact on the climate andurban air pollution.The composition of atmospheric aerosols varies with time,season,location, and environment. This affects key aerosol properties suchas hygroscopicity and reactivity, influencing the aerosol’simpact on the climate and air quality. The organic fraction of atmosphericaerosol emissions often contains surfactant material, such as fattyacids. These molecules are known to form three-dimensional nanostructuresin contact with water. Different nanostructures have marked differencesin viscosity and diffusivity that are properties whose understandingis essential when considering an aerosol’s atmospheric impact.We have explored a range of nanostructures accessible to the organicsurfactant oleic acid (commonly found in cooking emissions), simulatingvariation that is likely to happen in the atmosphere. This was achievedby changing the amount of water, aqueous phase salinity and by additionof other commonly coemitted compounds: sugars and stearic acid . The nanostructure was observedby both synchrotron and laboratory small/wide angle X-ray scattering(SAXS/WAXS) and found to be sensitive to the proxy composition. Additionally,the spacing between repeat units in these nanostructures was watercontent dependent (i.e., an increase from 41 to 54 Å in inversehexagonal phase  An increased organic mass fraction has been linked to poorair quality.8 Additionally, some organicemissions are surface active and can decrease droplet surface tensionand influence water uptake, affecting the ability of a particle toform a cloud droplet and therefore impacting on cloud formation andthe climate.12Aerosols are emitted into the atmosphereand affect the climateand human health.14 and urban emissions.17 This has made it the compound of choice as a reactive organic surfactantaerosol proxy, e.g., see refs and lamellar phase, the latter with lateral diffusion coefficientsorders of magnitude higher than in the orthogonal direction.31 This has implications for the diffusion throughthese nanostructures of small molecules such as water and atmosphericoxidants.Oleic acid is a commonly emitted organic surfactant, withsourcesincluding marinesee refs −26. Its 37 The phase state of atmospheric aerosols can range from liquid tosemisolid and solid and is dependent on location, season, compositionand meteorology.41 Phase state can also vary in indoor particles, suggesting aerosolviscosity and diffusivity could impact on indoor air quality.42 Knowing that atmospheric aerosol compositionand phase state can vary in the atmosphere, there is potential forsurfactant molecules (such as oleic acid) to form LLC nanostructures.The feasibility of LLC and organic crystalline phase formation inaerosol particles have been demonstrated in previous studies on alevitated oleic acid–sodium oleate proxy43 and the effect on reaction kinetics of theformation of one of these semisolid nanostructures (lamellar) wasrecently quantified; lamellar phase formation reduced reactivity byabout an order of magnitude,21 potentiallyincreasing the lifetime of oleic acid by days.22 These studies have contributed to explaining why oleicacid persists longer in the atmosphere compared with laboratory experiments,a long-standing issue in this field.45Aerosol viscosity and the diffusivity of small moleculeswithinaerosol particles have a profound effect on their reactivity and abilityto take up water.46 and it has been studied qualitativelyin an atmospheric context, demonstrating that nanostructure formationcan occur in levitated droplets24 alongwith a crystalline organic phase.20 However,there is a lack of data concerning the systematic addition of atmosphericallyrelevant molecules to the mixture. We achieve this by employing smallangle X-ray scattering (SAXS), a powerful technique used previouslyto probe aggregate structures on the nanometer scale.43 A combination of laboratory andsynchrotron SAXS experiments were carried out with the latter allowingfor simultaneous wide-angle X-ray scattering (WAXS) and levitationof LLC phase mixtures. We observe and rationalize LLC phase changesand link them to variations in aerosol composition that can occurin the atmosphere, thus establishing potential atmospheric implications.This research also provides a resource for subsequent studies on theeffect of surfactant (oleic acid) nanostructure on aerosol physicalproperties and in particular, but not exclusively, in an atmosphericcontext.Thisstudy seeks a better understanding of the nanostructures accessibleto the oleic acid–sodium oleate–water organic surfactantaerosol proxy with the addition of other common atmospheric emissions(sugars and a saturated fatty acid) and by changing the amount andnature of the aqueous phase . The oleic acid–sodiumoleate system has been explored before in a biological context,43 We define a “base” oleic acid–sodium oleate–aqueousphase bulk mixture where the organic fraction was made by mixing oleicacid  and sodium oleate  in a 1:1 wt ratio. Aqueous phase (deionized water or NaClsolution) was added to afford a final organic-aqueous ratio of 1:1wt for this base mixture, which was used for all mixtures except whenthe oleic acid–sodium oleate weight ratio was specificallyvaried. Additional organic molecules (sugars and stearic acid) wereadded to the base mixture in the desired ratio (relative to the amountof oleic acid and sodium oleate) and the amount of aqueous phase wasunchanged, except when this was the variable studied.  plus the additional fructose, representing33 wt % of the organic fraction.) In this study, the wt % quoted whenreferring to a compound in a sample means the weight percentage ofthe organic fraction of the organic-aqueous mixture .The starting point for all self-assembled surfactantmixtures studiedhere are mixtures of oleic acid and sodium oleate, building on previousstudies on this self-assembled atmospheric aerosol proxy systems.After adding the organic and aqueous components together, the sampleswere vortex mixed, heated to ∼50 °C for 20 min and vortexmixed again before being frozen in a conventional freezer. Samplesappeared more homogeneous once frozen and thawed slowly at room temperature.Before the SAXS measurement, samples were allowed to reach room temperatureand were once again vortex mixed before being sealed inside thin-walledglass X-ray capillary tubes of 1.5 mm internal diameter for the SAXSexperiment.47 Self-assembled LLCphases scatter X-rays tosmall angles and give scattering patterns with characteristic Braggpeaks.48 This scattered intensity is measuredas a function of the scattering vector (q), which isinversely proportional to the spacing between equivalent scatteringplanes (d)d-spacing can changewith changing water content as water is gained or lost from the nanostructure.49 The relative position of each scattering peakfor a given nanostructure is known and can be used to determine whichnanostructure is present (see q positionratios).50SAXS is a technique used to probe aggregate materialson the nanoscale.A combination of synchrotron and laboratory SAXSinstrumentationwas used to carry out the SAXS experiments at three different facilitiesas outlined below. Simultaneous WAXS was possible with the synchrotronsetup, probing shorter repeat distances.q-range for these SAXS and WAXSmeasurements were 0.008–0.46 and 0.4–0.6 Å–1, respectively. Samples were mounted in a sample rackand secured with Scotch tape.Synchrotron SAXS-WAXSexperiments on bulk mixtures were carriedout on the I22 beamline at the Diamond Light Source (U.K.). SAXS andWAXS patterns were measured by X-rays of 12.4 keV energy with 0.1s collection times. The 24 Here, a microfocused X-ray beamof ∼14 μm (fwhm) diameter was used to acquire SAXS patternsfrom levitated droplets at 1 s acquisition time. The total SAXS-WAXS q-range for these experiments was 0.03–1.50 Å–1. Samples of oleic acid–sodium oleate (1:1wt) in an excess NaCl (1 wt %) solution were injected into a pressurenode of the acoustic levitator. Safety procedures at the beamlinemeant that SAXS experiments could not be started until ∼3 minafter droplet injection.Acoustic levitation-SAXS-WAXSexperiments were also carried outon the I22 beamline. The levitation setup is described in detail inprevious publications.An Anton Parr SAXSpoint 2.0 instrumentwas used to carry out thelaboratory SAXS experiments. A Cu source produced X-rays of 1.54 Åwavelength. The sample-to-detector distance was 0.360 m and patternswere collected for ∼3–5 min for each sample. Sampleswere enclosed in Kapton capillaries of 1.5 mm internal diameter andmounted on a sample rack.Further SAXS-WAXS experiments on mixtureswith multiple additiveswere carried out on a Xenocs Xeuss 2.0 with a Cu X-ray source anda sample-to-detector distance of 1.185 m. A sample-to-detector distanceof 0.162 m was used for WAXS measurements taken simultaneously withthe SAXS measurements. Samples were mounted between two Kapton windows.Rheological measurements were carried out using the HR-3 DiscoveryHybrid Rheometer (TA Instruments) with a 40.0 mm 1.00694° coneplate geometry (Peltier plate Steel) and used to perform an oscillatoryfrequency sweep from 0.1 to 100 rad/s on the samples with a 0.2% strainapplied at 25 °C. Dynamic viscosity of the samples was calculatedby dividing the storage modulus by the radial frequency.A qualitative summary of thephases observed in this study arepresented in 48 In this study, thephase with the largest negative interfacial curvature is the close-packedinverse micellar phase. The lamellar phase has zero interfacial curvature.In summary, the order from largest to smallest negative interfacialcurvature is as follows: inverse micellar; close-packed inverse micellar;and inverse heagonal and lamellar.The following discussion refersto interfacial curvature, morespecifically negative interfacial curvature for the inverse phasesstudied here. This is the curvature at the water–surfactantheadgroup interface.q-range to afforda 1D scattering pattern. The 2D SAXS patterns did not exhibit anyLLC phase orientation. Examples of scattering patterns for the phasesobserved in this study are presented in After collection of the 2DSAXS pattern from each sample, a radialintegration was performed over the 17 The relative proportionsof these molecules can vary and can be a measure of how much a samplehas aged.4SI). Note that the d-spacingfor all oleic acid-stearic acid compositions is slightly smaller (by3–4 Å) than that of the dry oleic acid–sodium oleatemixture . This suggests thatthe alkyl chain monolayers of the lamellar bilayer can pack closertogether, which is likely due to the lack of a kink in the stearicacid alkyl chain compared to the one induced by the cis double bondin oleic acid. The presence of WAXS peaks supports this since thesepeaks arise from well-packed adjacent alkyl chains.51Oleic acid istypically found in organic emissions together with its saturated analogue,stearic acid.21 a hypothesis also described by Hearn et al.52 This may also explain the trend in reactivityobserved by Katrib et al., who reacted oleic acid-stearic acid particleswith ozone. They observed that the ozonolysis reaction effectivelystopped at high stearic acid compositions.53 Though explanations were offered, no experiments on the nanostructureof those particles were carried out; our work provides experimentalevidence suggesting that the formation of these tightly packed lamellaecould play a role in determining reactivity. The difference in fattyacid “steric configuration” has also been suggestedto explain the difference in reactivity between the trans- and cis- isomers of oleic acid observed during field measurements.45The availability of the double bond to the atmosphericoxidantozone is likely to be affected by the spacing between alkyl chainsby reducing the diffusivity of ozone through the organic phase, limitingthe reaction to the surface layers ,57 Such a pH range would affect the degree to which weak acids, suchas oleic acid (pKa ∼ 5), are protonated.The surfactant headgroup charge affects the effective headgroup areawhich in turn can modify the curvature of a LLC phase.29 We simulated a change in headgroup charge byadding increasing amounts of sodium oleate to oleic acid, as wouldbe found in less acidic conditions. Fd3m symmetry), inverse hexagonalphase , and the lamellar phase.Finally, headgroup charge and oleic acid-oleateratio depend onacidity. Aerosol particle acidity can vary with a typical pH rangeof ∼1–6, though this is difficult to measure directlyand is normally inferred by applying theoretical models.Note that coexistent phases were also observed and were determinedwhere more than one SAXS peak could be indexed unambiguously to acertain phase. Coexisting phases are an important feature of thisstudy and have atmospheric implications  increases with water content  resulted in a transition between these two moleculararrangements with quite different physical properties which wouldaffect key aerosol processes such as water uptake and chemical reaction.A similar trend is observed when adding varying amounts of 1 wt %NaCl solution .Aqueous droplets containingoleic acid–sodium oleate and an excess of NaCl solution (1wt %) were levitated at an elevated humidity (∼86% RH). Theinitial SAXS pattern revealed the hexagonal phase  wascommonly observed at higher NaCl concentrations before phase separation is most probably due to a coexistent disorderedinverse micellar phase. This inverse micellar phase peak is more prominentfor the six-component mixture. In both cases the close-packing ofthese inverse micelles imply an increase in viscosity, potentiallyby orders of magnitude66 that could behighly relevant in the atmospheric context (see The dominance of close-packed inverse micellesin these multicomponentmixtures was unexpected. A broad peak at low-text see .4 Pas at 10 wt % water to ∼102 Pa s at 60 wt % water(67 and are consistentwith what was predicted from kinetic modeling of self-organized oleicacid (∼102 – 103 Pa s).22 Although the mixtures used for rheology weredominated by the inverse hexagonal phase, they remained as unidentifiedcoexisting phases. Therefore, we cannot ascribe these viscositiesto a specific phase. This is, however, a more realistic representationof what might occur in the atmosphere.The dynamic viscosity of the model1:1 oleic acid–sodium oleate mixture decreased significantlywith increasing water content, going from ∼10 % water7. These d-spacing clearly have a plasticizingeffect on the mixture, allowing the organic phase to flow more freely.The increased water content and subsequent increasein inversehexagonal phase 68 In light of this, our results suggest that a variation in aerosolcomposition could have a marked impact on the resulting moleculararrangements of organic surfactants found in these aerosols; a majorclass of which are fatty acids, such as oleic acid studied here  phase.66 Additionally, diffusion becomesanisotropic for the lamellar and inverse hexagonal phases; there isa significant difference between lateral and orthogonal diffusionin the lamellar phase.31 These variationsin viscosity and diffusivity have implications for the uptake of tracegases  into aerosol particlesand can determine the time scale of bulk diffusion within them, impactingon aerosol persistence and atmospheric lifetime.70The viscosity of atmospheric aerosol particlesaffects water uptakeand chemical reactivity, and these are two determiners of aerosolpersistence and subsequent influence on the atmosphere.71 For example,organic coatings are present on marine aerosols,13 occasionally resulting in an inorganic core-organic shellmorphology.62 Phase separation also happensin atmospheric aerosols73 and heterogeneitiesin particle viscosity have been observed to develop during the ozonolysisand humidification of aerosol proxies.74 During thepresent study, we observed an appreciable amount of nanostructuralheterogeneity in the bulk mixtures. The most common coexisting phaseswere the inverse hexagonal and ordered inverse micellar phases. Thereare significant differences in physical characteristics between thesetwo phases due to their different geometries. For example, the inversehexagonal phase has directionally dependent water diffusion and ismore viscous than the inverse micellar phase, observed by us qualitatively.We have shown that composition influences fatty acid nanostructure.If aerosol composition varies within a single aerosol particle, therecould be a similar spatial heterogeneity in fatty acid nanostructure,impacting aerosol processes and subsequent effects on urban air pollutionand climate through variations in the time scales of aerosol tracegas uptake due to the implied viscosity differences.33Heterogeneityis a feature of many atmospheric aerosols. A rangeof imaging techniques have demonstrated that atmospheric aerosolsare not always well-mixed.5 with aerosol light scatteringand absorptioncontributing to radiative forcing.2 Wequalitatively found visual differences between the optically isotropicphases with cubic symmetry (close-packed inverse micellar) and opticallyanisotropic phases  ;75 the former was translucentand the latter was opaque. This observation suggests that aerosollight scattering could be affected by the optical characteristicsof the fatty acid nanostructures, resulting in an additional indirecteffect on the climate.Aerosol optical properties can vary with environmentand over timeWe have explored the molecular arrangementsaccessible to the oleicacid–sodium oleate fatty acid aerosol proxy in levitated dropletsand bulk mixtures. This was achieved by addition of other common atmosphericemissions (sugars and saturated fatty acids) and by adjusting theamount of water and salinity of the aqueous phase. Notably, the compositionof the proxy heavily influenced the resulting nanostructure. Overa small range of aqueous phase salinity, a phase transition was observedbetween two nanostructures known to have different physical characteristicsand phase changes were also observed in levitated droplets. The viscositywas observed to decrease by orders of magnitude with increasing watercontent.76 However, this study is a step toward linking the laboratory withthe real world when considering the relatively novel proposition ofLLC phase formation in the atmosphere.Bridging the gap between simple aerosol proxy systemsand realatmospheric measurements continues to be a challenge .The potential importanceof fatty acid self-organization in theatmosphere is added to by the demonstration of the range of (oftencoexisting) nanostructures observed in this study both in bulk mixturesand levitated droplets, and even in the more complex mixtures withup to six components we tested here. We now have better knowledgeof the phase space accessible to these molecules, providing a basisfor future, more quantitative investigations into the physical characteristicsof the nanostructures discussed in this study and likely encounteredin the atmosphere.


Induced pluripotent stem cells (iPS cells) represent a particularly versatile stem cell type for a large array of applications in biology and medicine. Taking full advantage of iPS cell technology requires high throughput and automated iPS cell culture and differentiation. We present an automated platform for efficient and robust iPS cell culture and differentiation into blood cells. We implemented cell cluster sorting for analysis and sorting of iPS cell clusters in order to establish clonal iPS cell lines with high reproducibility and efficacy. Patient-specific iPS cells were induced to differentiate towards hematopoietic cells via embryoid body (EB) formation. EB size impacts on iPS cell differentiation and we applied cell cluster sorting to obtain EB of defined size for efficient blood cell differentiation. In summary, implementing cell cluster sorting into the workflow of iPS cell cloning, growth and differentiation represent a valuable add-on for standard and automated iPS cell handling. Clonal iPS cell lines and embryoid bodies (EB) of defined size are obtained by cell cluster sorting in an automated platform. Induced pluripotent stem cells (iPS cells) are engineered stem cells, which are readily obtained from somatic cells of patients by reprogramming . They reTransferring conventional laboratory processes into industrial manufacturing requires advanced methods and machineries, which are reliable, reproducible, scalable, and adaptable. Automation of distinct steps and integrated systems of iPS cell generation, culture and differentiation have been previously described . For exaHuman iPS cells harbor the potential to develop into all cell types of our body but iPS cell differentiation towards hematopoietic cells has been notoriously challenging . FrequenTherefore, in the present work, we embarked on the strategy to automate 1) iPS cell culture on MEF feeder, 2) EB formation from iPS cell clusters and 3) their differentiation into hematopoietic cells. Additionally, we capitalized on cell cluster flow cytometry for 1) analyzing and sorting individual iPS cell clusters to obtain clonal iPS cell lines and 2) sorting EB of defined size for hematopoietic differentiation.+ hematopoietic cells using the CytoTune reprogramming kit (Thermo Fisher Scientific) as described in our previous work (patient 1 in https://hpscreg.eu) line 1 and line 2 are referred to as UKAi004-D and UKAi004-B, respectively. The “unstable” iPS cell line used in the present work was generated as described previously (The iPS cell lines used in the present work were derived from an aggressive systemic mastocytosis (ASM) patient by reprogramming peripheral blood CD34eviously  and was eviously .2. Microscopy was performed with an automated EVOS Fluorescence Digital Inverted Microscope . Tissue culture plate handling was with a 6-axis robot  mounted on an overhead gantry and encased with a custom-made laminar flow system .MEF and iPS cell handling was performed on the automated platform Hamilton STARlet liquid handling unit (LHU)  with the following functional units: tube carrier, tilt module, plate carriers, tip carriers, shaker, liquid waste container and multiple positions for 6-well plates . Cells wThe Hamilton VENUS III software was used to develop the operating system for automated iPS cell culture and differentiation in the LHU. The operating system is composed of several task-specific methods. A number of specific commands were stringed together, forming a method. Methods were edited by the syntax-free graphical method editor. A set of files, including ‘Deck Layout’, ‘Carrier and Labware’, ‘Sequence’, ‘Liquid Class’, and ‘Library’, were linked to the output of the method editor. Every method was associated with several parameters, which can be defined by the user based on the task.MEF feeder layer generation: MEF feeder cells were seeded in 6-well tissue culture plates by LHU . MEF werFive to 7 days after passaging on MEF feeder layer, iPS cells in 6-well plates were treated in the LHU with dispase (Stem Cell Technologies) for 10–15 min or collagenase IV (Gibco) for 45–60 min. Enzyme was aspirated and 1 ml of IMDM culture medium (Gibco) supplemented with 10% FCS (PAN Biotech) and 100 U/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific) was added to each well. Colonies were harvested in LHU with custom-made pipetting positions and gently transferred to 50 ml Falcon tubes. The suspension of iPS cell clusters was homogenized by gently pipetting  and submLive-cell staining of iPS cells in culture with TRA-1-60-Vio488 (Miltenyi Biotec) was performed following manufacturer instructions. Image acquisition was performed with EVOS Fluorescence Digital Inverted Microscope. Alternatively, iPS cells were first treated with collagenase IV for 45 min. iPS cell colonies were gently detached and harvested into a 50 ml Falcon tube, washed twice with KO-DMEM (Thermo Fisher Scientific), stained with TRA-1-60-Vio488 as above and subjected to analysis with large particle sorter COPAS FP-1000.l-glutamine . Medium change was performed every second day. Immunofluorescence staining for trilineage markers α-fetoprotein (AFP), β-tubulin III (TUJ-1) and T-box transcription factor 3 (TBX3) was performed as described  and data analysis was done with FlowJo software (Tree Star). Antibodies used for FACS are listed in Analysis and sorting of cell clusters (iPS cell clusters and EB) was performed with large particle sorter COPAS FP-1000. Key parameters for optimization of cell cluster collection were “Drop Width”, “Sort Delay” and “Minimum Separation Width”. “Minimum Time of Flight (ToF)” was set as cell cluster size cut-off threshold. Cell clusters were characterized by optical density and fluorescence intensity and sorted as they exit the flow cell by an air sorting mechanism. Desired cell clusters were collected as described for each experiment.iPS cell clusters on MEF feeder were generated manually or by automation with the LHU and transferred to 6-well Clear Flat Bottom Ultra-Low Attachment plates (Corning) for EB formation. Hematopoietic differentiation was performed as previously described . BrieflyiPS cell-derived hematopoietic cells were centrifuged on glass slides with Shandon Cytospin 4 cytocentrifuge (Thermo Fisher Scientific) followed by fixation with methanol at room temperature. Cells were stained with Diff Quik (Medion Diagnostics) followed by mounting with Entellan (Merck). Image acquisition and analysis was performed with Leica DMRX microscope (Leica) and Leica Application Suite software (Leica Microsystems), respectively. ImageJ was used for image handling.RNA isolation was performed with NucleoSpin RNA Kit  following the manufacturer´s instructions. cDNA was synthesized using MultiScribe High Capacity cDNA Reverse Transcriptase Kit (Thermo Fisher Scientific). RT-qPCR was performed on StepOnePlus Real Time cycler with FAST SYBR Green master mix (Thermo Fisher Scientific). Sequences of primers used in the present study are listed in EB generation from feeder-free iPS cell cultures was performed with micro-contact printed (µprinted) vitronectin arrays and automated by LHU. Briefly, polydimethylsiloxane stamps with circular features of 600 µm in diameter were used to pattern the surface of 6-well tissue culture plates with vitronectin arrays. iPS cells were seeded on patterned plates by LHU and cultured in StemMACS iPS-Brew XF (Miltenyi Biotech) . iPS celConfluency of MEF feeder was calculated from phase contrast image scans of the automated EVOS Fluorescence Digital Inverted Microscope with modified PHANTAST algorithm . The algp-values are indicated in the respective figure legends.Statistical analyses were performed in Prism 7 (GraphPad). The statistic test used, and The reliable and robust automation of iPS cell culture is key to high throughput application of iPS cells in disease modeling and drug screening. This involves 1) iPS cell culture, the 2) isolation of individual clonal iPS cell lines and 3) iPS cell differentiation into the desired cell types  protocol for automated culture of iPS cells on MEF feeder layer or on feeder-free vitronectin-coated plates . We developed preprogrammed modules to automate all steps of MEF feeder layer seeding, generating confluent and homogenous MEF feeder-coated wells with high reproducibility . HomogenStep 2iPS cell cloning is readily performed with cell cluster sorterThe establishment of iPS cell lines after reprogramming or genetic engineering (i.e. by CRISPR-Cas9 technology) relies on the isolation of individual iPS cell colonies, which develop from a single cell, thus generating a colony where all cells are clonal and share the same genetic information . FrequeniPS cells were seeded as single cells on MEF feeder at low cell density and expanded to colonies, and iPS cell colonies were harvested by collagenase IV treatment. iPS cell clusters were obtained by gentle mechanical agitation and individual iPS cell clusters were sorted into 24 well plates, precoated with MEF feeder layer, based on particle time of flight (TOF) and extinction coefficient . The sorNext, we proceeded to demonstrate that iPS cell clonality is preserved during sorting. To this end, iPS cell clusters were labeled with PKH26 (red) or CFSE (green), mixed and then subjected to cell cluster sorting. Individual iPS cell clusters bearing either PKH26 (red) or CFSE (green) color code, but not both, were obtained and sorted . Thus, oReprogramming of somatic cells into iPS cells is a complex process and frequently incompletely reprogrammed iPS cells are obtained. Such incompletely reprogrammed iPS cells are unstable and exhibit reduced staining for pluripotency markers . Thus, wStep 3Automated differentiation of iPS cells into hematopoietic cellsiPS cells are valuable tools for disease modeling and drug discovery since they represent an essentially unlimited cell source and are able to differentiate into virtually any cell type of the human body . FrequenTherefore, we applied the same modular preprogramming approach with the LHU for automation of EB formation . No morp5 μm2 in gate 1 (G1) or 3.36–8.35 × 105 μm2 in gate 2 (G2)  . Particloduction .Additionally, a novel and innovative approach to control EB size is to use micro-contact printing (µCP) . iPS celIn summary, we show that our automated platform stands as an efficient and robust system for iPS cell culture and differentiation. Cell cluster sorting allowed for analysis and sorting of iPS cell clusters and establishment of clonal iPS cell lines from bulk populations. In addition, EB formation is efficiently automated in our platform and combined with cell cluster sorting to enrich for EB with specific size. The EB generated by automation were successfully differentiated towards hematopoietic cells with efficiency and quality indistinguishable from manual processing. Finally, our automated platform output has high upscaling potential, as seven 6-well plates can be handled simultaneously by the LHU and several rounds of automated iPS cell culture and cell cluster sorting can be performed daily .Automation of iPS cell generation, culture, genetic manipulation, and differentiation into cells/tissues of interest is key to the large scale application of patient and disease specific iPS cells in disease modeling and drug screening . Human iHere, we report on a user-friendly, modular preprogrammed, automated platform for iPS cell culture on a feeder-free or MEF based culture system. We developed a library of preprogrammed automation steps that can be easily selected and combined by the user to create customized protocols. This approach allowed us to use the same platform also for blood cell differentiation. Importantly, our modular system requires only basic training to operate the software and almost no programming skills from the users. Furthermore, our pipeline relies on two main operational blocks: the liquid handling unit (LHU) and the cell cluster sorter, thereby reducing establishment and operational costs. The flexibility of our platform did not compromise its robustness and sterility, as no contamination occurred throughout the entire study.Several studies reported on the automation of iPS cell culture, addressing automation of iPS cell reprogramming, clonal isolation, induced clonal stability, and differentiation towards several tissues . Our worAnother attractive approach is the combination of our automated pipeline for iPS cell culture and EB generation with bioreactor-based hematopoietic differentiation strategies, which have shown high yields of functional hematopoietic cells with therapeutic value . In addiOur study paves the way for the future development of specific automation protocols aiming the large-scale establishment and CRISPR/Cas9-mediated genetic engineering of iPS cell lines. This includes iPS cell differentiation towards desired cells and tissues, which are most suitable for disease modeling and drug screening studies.


Regions of interest (ROI) included the frontal, parietal, lateral temporal, and occipital cortices, the posterior cingulate/precuneus, and the striatum. The most frequent occurrence of Aβ deposition was in the posterior cingulate/precuneus . The second most frequent site was the lateral temporal cortex , followed by the lateral parietal cortex  and other lesions, such as the frontal and occipital cortices. The striatum was the least frequently affected. Our study found that the posterior cingulate/precuneus and the lateral temporal and parietal cortices may be the earliest areas to be affected by Aβ accumulation. Longitudinal follow-up of focal brain amyloid deposition may help elucidate the evolutionary pattern of Aβ accumulation in the brain of people with AD continuum.Accumulation of aggregated amyloid-β (Aβ) in the brain is considered the first pathological event within the pathogenesis of Alzheimer’s disease (AD). It is difficult to accurately identify the initial brain regions of Aβ accumulation due to the time-lag between the start of the pathophysiology and symptom onset. However, focal regional amyloid uptake on amyloid PET scans may provide insights into this. Hence, we aimed to evaluate the topographic distribution of amyloid deposition in patients with cognitive impairment and to identify the starting order of amyloid accumulation in the brain using conditional probability. We enrolled 58 patients composed of 9 normal cognition (NC), 32 mild cognitive impairment (MCI), and 17 dementia showing focal regional amyloid deposition corresponding to a brain amyloid plaque load (BAPL) score of 2 among those who visited the Memory Clinic of Asan Medical Center and underwent an  Amyloid-β (Aβ) and hyperphosphorylated tau deposition are two major pathologic components of Alzheimer’s disease (AD) pathogenesis ,2. The a18F]-florbetaben is an 18F-labeled polyethylene glycol stilbene derivative with high in vitro affinity and specificity for β-amyloid plaques [18F]-florbetaben PET is conducted through a triple scale system, termed β-amyloid plaque loading (BAPL) [18F]-florbetaben was found to exhibit high sensitivity and specificity for pathologically confirmed amyloid plaque detection ; moreover, these studies detected a marked and statistically significant association between the results of visual-based qualitative assessments and quantitative analyses -flutemetamol amyloid PET study reported that focal amyloid deposition refers to a transitional state  -florbetaben amyloid PET scans as well as ApoE (apolipoprotein E) genotyping. ApoE genotyping was conducted by extracting genomic DNA with polymerase chain reaction (PCR) from peripheral venous blood -florbetaben amyloid PET images were acquired over the course of 20 min at the dynamic mode (4 × 5 min frames). Scanning was performed 90 to 110 min after injection of a bolus  into an antecubital vein.All PET images were acquired using Discovery 690, 710, and 690 Elite PET/CT scanners . [18F]-florbetaben PET images were assessed by consensus among three physicians . Interpretation was conducted visually by comparing the activity in the cortical gray matter with activity in the adjacent cortical white matter. In general, only four regions of interest  were interpreted in a visual rating of [18F]-florbetaben PET findings. We included the occipital cortex and striatum in these evaluations in order to clearly identify spatial patterns of amyloid deposition. Each of these brain regions was scored according to the RCTU and BAPL systems, described below.-florbetaben PET images.We were interested in assessing the evidence that one brain region would tend to have earlier β-amyloid involvement than another brain region by evaluating -florbetaben amyloid PET scanning, though there have been some studies that analyzed amyloid deposition patterns based on post-mortem observations [18F]-florbetaben amyloid in vivo PET readings [To our knowledge, this is the first study to reveal the sequence of amyloid deposition as measured by [rvations ,12,30,31readings . This paThe risk of developing AD is known to increase by approximately 1.8 times in amyloid-positive individuals as compared with amyloid-negative individuals of the same age . With thInterestingly, the BAPL2 patients enrolled in our dataset included those with MCI or even those in the dementia (AD) state. According to the amyloid hypothesis, amyloid deposition occurs 15–20 years before dementia symptom onset. As such, if BAPL2 status represents the early stage of amyloid deposition, most BAPL2 patients should be in the asymptomatic or mild symptomatic stage. In this regard, it could be assumed that subthreshold levels of amyloid deposition may be responsible for cognitive impairment in this patient group . PreviouWe acknowledge some limitations to this study. For example, the cross-sectional nature of this study is an important limitation. Future longitudinal studies will be necessary in order to better understand how amyloid accumulation occurs and spreads over time. Since the rate of amyloid deposition is an important factor in evaluating the clinical manifestations and prognosis of AD patients, a follow-up study needs to be conducted in order to better understand these associations. Second, tests for other core AD biomarkers, such as tau protein, were not conducted in the current study. This opens the door for the possibility of BAPL2 being non-specific for AD in etiology. However, we tried to exclude other causes of cognitive decline through appropriate diagnostic testing, including with regard to MRI scans, blood laboratory analyses, and other clinical findings. Finally, we evaluated visual rating results rather than quantitative analysis of PET data, such as standardized uptake value ratio (SUVR) values. The concordance rate between cortical SUVR and visual read categorization is known to be excellent and the degree of agreement between visual and quantitative analyses of florebetaben PET in our center is also very high. Moreover, either visual read or quantitative analysis would not matter in determining whether there is regional amyloid uptake in the brain.Our study showed that patterns of amyloid deposition in BAPL2 patients analyzed by CP calculations were similar to those found in AD patients. We conclude that BAPL2 amyloid status is more likely to represent the early stage of amyloid deposition of Alzheimer’s disease. However, further longitudinal studies incorporating not only amyloid but also tau imaging findings will be warranted in order to reveal the natural history and the temporospatial spread of amyloid within the BAPL2 patient group.


Acquisition of dense, three-dimensional, force fieldswith intramolecularresolution via noncontact atomic force microscopy (NC-AFM) has yieldedenormous progress in our ability to characterize molecular and two-dimensionalmaterials at the atomic scale. To date, intramolecular force mappinghas been performed exclusively at cryogenic temperatures, due to thestability afforded by low temperature operation, and as the carbonmonoxide functionalization of the metallic scanning probe tip, normallyrequired for submolecular resolution, is only stable at low temperature.In this paper we show that high-resolution, three-dimensional forcemapping of a single organic molecule is possible even at room temperature.The physical limitations of room temperature operation are overcomeusing semiconducting materials to inhibit molecular diffusion andcreate robust tip apexes, while challenges due to thermal drift areovercome with atom tracking based feedforward correction. Three-dimensionalforce maps comparable in spatial and force resolution to those acquiredat low temperature are demonstrated, permitting a quantitative analysisof the adsorption induced changes in the geometry of the moleculeat the picometer level. Since then, the technique has produceda wealth of impressive results, including the ability to observe bondorder in organic molecules,2 localizationof charge within a molecule,3 imaging thestages of a chemical reaction,4 molecularmanipulation at the single bond level,5 and detailed investigation of on-surface synthesized 2D materialssuch as graphene nanoribbons,6 among manyothers.9 In particular, many of these results make use ofhigh-resolution spectroscopic measurements, such as high spatial density“force mapping” of single molecules,12 to elucidatekey information about the sample, beyond that obtained via conventionalimaging.The demonstration of submolecular resolutionby Gross et al.13 and the need for an extremely stableenvironment with negligible thermal drift (as the tip must approachextremely close to the adsorbed molecule in constant height in orderto probe the Pauli repulsion regime).1 Thesepractical considerations drastically limit the accessibility of thetechnique and its use in studying systems under conditions relevantto real-world applications.Although demonstrating unparalleled resolution andsensitivity,these results have been almost exclusively limited to operation onplanar metallic substrates at low temperature . This is due to the twin requirements of bothcreating and maintaining a functionalized probe by controlled pickupof a single CO molecule (unstable at temperatures above ∼10K),16 including demonstrationof single images showing submolecular resolution at room temperature.17 In each of these works, the use of semiconductingmaterials for both tip and substrate inhibits diffusion of the moleculesand can result in tip apexes that are stable for many hours even atroom temperature. When repeated observations of a small area (suchas a single molecule) are required at elevated temperatures, it isnecessary to use feedforward drift correction19 to compensate for the instabilities caused by small temperaturevariations. This technique in principle allows the imaging of thesame nanometer-sized area for prolonged periods and has previouslybeen used to acquire high density force maps of atomically clean substratesat room temperature.21It has previously been demonstratedthat intramolecular contrastcan be obtained on nonmetallic substrates at elevated cryogenic temperatureswithout deliberate tip functionaliztion,In this paper we show itis possible to reproducibly acquire densetwo-dimensional (2D) and three-dimensional (3D) force fields overa small organic molecule adsorbed on the Si(111)-(7 × 7) surfaceat room temperature. This is accomplished using standard silicon cantileversand a commercial NC-AFM instrument, without special tip treatmentor deliberate tip functionalization. The significant challenges ofroom temperature operation are overcome via a combination of modifiedscan protocols and scripted drift correction. The data is shown tohave similar spatial and force resolution to data acquired in lowtemperature environments, and we highlight how acquisition of 3D forcedata allows a quantitative analysis of the forces responsible forintramolecular contrast, the quality of the probe, and even minordistortions of the molecular structure induced during surface adsorption.22 In Assessing the feasibilityof room temperature intramolecular force mapping required a prototypicalmolecule–substrate system that was known to be stable at roomtemperature, and which had already demonstrated amenability to submolecularresolution imaging at cryogenic temperatures. Naphthalene tetracarboxylicdiimide (NTCDI) adsorbed onto the Si(111)-(7 × 7) surface hasbeen shown to have a commensurability between the reactive oxygenend groups of the molecule, and the silicon adatom spacing, whichallows it to adsorb in an almost planar configuration in a numberof sites in the Si(111)-(7 × 7) unit cell, as demonstrated inprevious combined STM/NC-AFM studies at cryogenic temperatures.z position of adjacentspectra.16 Acquisition of constant height(CH) data for force mapping is conceptually much simpler, but it requiresthat the residual vertical drift over the period of a single scanis ≲10 pm. Because fluctuations in ambient temperature resultin a continuously shifting thermal drift direction and magnitude,even under optimized conditions this practically limits the maximumacquisition time of a single frame to ∼1–2 min  simulations, that showthat both Si-OH-terminated and Si-terminated probes do not react stronglywith adsorbed organic molecules,2 the absolute bondlengths within the molecule are nonetheless exaggerated by ∼10%compared to those calculated via DFT. By comparing the measured bondlengths from 26 images, we estimate that the tip apex in this instance has a lateralstiffness kxy ∼1 N/m  and the stiffer CuOx tip termination(kxy ∼ 10 N/m).27Aquantitative analysis of the high-resolution images in 11 or deflection of the tip apex,12 it isnecessary to acquire grid spectroscopy data in order to allow thedirect visualization of the complex force field arising between thetip and adsorbed molecule.Although imaging issufficient for simple characterization of molecular structure forplanar molecular systems, in order to identify more complex quantitiessuch as the adsorption angle, distortion in the molecular configurationdue to surface–molecule interaction,F(z) curves extracted from both grids over key sites on the molecule.The relatively weak minimum (∼100 pN) in the F(z) curves is indicative of a nonreactive probeinteracting primarily via van der Waals and Pauli repulsive interactions.Because the oxygen atoms of the molecule are bound to the surface,there are no strongly reactive sites present, and consequently weonly detect a small variation in the force minima over the differentareas of the molecule on the order of ∼5–15 pN  images extracted from the 3D dataset at decreasing tip–sample heights show a clear evolutionin contrast as previously described for similar systems at low-temperature.15 Representative F(z) curves extractedfrom the same data set at key sites over the molecule show a similarvariation in minima and force resolution as those extracted from the2D grids, confirming the correspondence between the two methodologies.Two-dimensional force maps, in concert with conventionalconstantheight imaging as demonstrated in 17 In our measurements we detecta ∼ 10 pN difference between those sites in both the 2D griddata shown in F(z) turning pointsbetween subsequent grids on the same molecular site could vary onthe order of ∼30 pN  curve occurs) from each curve to create 2D and3D z* maps, which have previously been used to extractquantities such as the adsorption angle11 and combined topographic/chemical information28 of molecular systems. The high sensitivity of these mapsreveals additional information about the system, both in terms ofthe adsorption geometry of the molecule, and the nature of the tip,which must be carefully decomposed.Another key advantage of acquiring higher dimensional forcemapsas opposed to standard CH imaging is the ability to perform more sophisticatedanalysis on the spectroscopic data to extract detailed quantitativeinformation about the studied system. In 22 In the data sets shownin 26 We note that thisis flat within our experimental and computational error and intuitivelycorrect as the molecule and adsorption geometry are symmetric withrespect to the surface along this axis.Due to the symmetry of the molecule we simplifydiscussion of thedata by considering the maps acquired across the long and short axesof the molecule. Turning first to the short axis, it is known thatNTCDI adsorbed on Si(111)-(7 × 7) can adopt both planar and tiltedadsorption configurations, although from combined experimental andDFT studies it is expected that the planar configuration will be morecommonly observed.z* map,with the end nitrogen atoms being pulled down belowthe central aromatic rings. In the 3D data set the nitrogen atomswere determined to be 11 ± 7 pm below the central bond of themolecule, whereas for the 2D data set a similar analysis resultedin an estimate of 18 ± 9 pm. This is very similar to the resultsof the same analysis applied to the simulated PPM data set, whichshows a height difference of 18 ± 4 pm  to remove the electrostatic force dueto the contact potential difference (CPD). Due to the semiconductingnature of the tips, no tunnel current was observed throughout theexperiments, nor did we use any scanning tunnelling microscopy (STM)based techniques to prepare the tip apex. Atom tracking was used toapply feedforward correction for imaging and for 3D data acquisition.All z heights are given relative to the tip heightover the molecule at the frequency shift (Δf) feedback value used for feedback during atom tracking. Site-specificforces were extracted from Δf(z) spectroscopy curves using the Sader–Jarvis algorithm31 after subtracting the non-site specific forcesby measurement of the tip–sample interaction over a cornerhole site.33 All NC-AFM imaging was performedin constant height (CH) mode, except for large overview images, whichwere performed using a multipass scan mode introduced by Moreno etal.16 (referred to as adaptive height (AH)mode in this manuscript), which uses an intermittent disengagementof the feedback loop to obtain pseudoconstant height images in therepulsive regime on samples with topographically different regions.Data were acquired usinga commercial Omicron Nanotechnology VT-STM/NC-AFM,operated using a Nanonis controller. All experiments were performedin ultrahigh vacuum (UHV) at room temperature. The Si(111)-(7 ×7) surface was prepared by standard flash annealing and a low coverageof NTCDI was prepared by depositing the molecules from a homemadeKnudsen cell onto the room temperature substrate. Commercial siliconcantilevers (Nanoworld) were treated by argon sputtering to removethe native oxide layer and subsequently prepared on clean Si(111)surfaces via gentle indentation to the surface until good NC-AFM resolutionwas achieved. Cantilever deflection was measured using a standardfour quadrant photodetector, rather than an optical interferometeras described in previous cantilever studies,


The objective of this study was to determine if locally delivered FK506 could prevent allogeneic nerve graft rejection long enough to allow axon regeneration to pass through the nerve graft. An 8mm mouse sciatic nerve gap injury repaired with a nerve allograft was used to assess the effectiveness of local FK506 immunosuppressive therapy. FK506-loaded poly(lactide-co-caprolactone) nerve conduits were used to provide sustained local FK506 delivery to nerve allografts. Continuous and temporary systemic FK506 therapy to nerve allografts, and autograft repair were used as control groups. Serial assessment of inflammatory cell and CD4+ cell infiltration into the nerve graft tissue was performed to characterize the immune response over time. Nerve regeneration and functional recovery was serially assessed by nerve histomorphometry, gastrocnemius muscle mass recovery, and the ladder rung skilled locomotion assay. At the end of the study, week 16, all the groups had similar levels of inflammatory cell infiltration. The local FK506 and continuous systemic FK506 groups had similar levels of CD4+ cell infiltration, however, it was significantly greater than the autograft control. In terms of nerve histmorphometry, the local FK506 and continunous systemic FK506 groups had similar amounts of myelinated axons, although they were significantly lower than the autograft and temporary systemic FK506 group. The autograft had significantly greater muscle mass recovery than all the other groups. In the ladder rung assay, the autograft, local FK506, and continuous systemic FK506 had similar levels of skilled locomotion performance, whereas the temporary systemic FK506 group had significanty better performance than all the other groups. The results of this study suggest that local delivery of FK506 can provide comparable immunosuppression and nerve regeneration outcomes as systemically delivered FK506. Peripheral nerve injuries can lead to permanent loss of motor and sensory function and debilitating chronic pain with less than 50% of patients achieving adequate functional recovery after peripheral nerve injury and repair –3. PeripClinical reports of nerve allograft treatment have been scarce, even after the advent of microsurgical techniques and immunosuppressant drugs in the 1980s . AllogenSeveral studies have shown in both animal models and limited clinical case reports that temporary systemic immunosuppression (e.g. FK506) can prevent allogeneic nerve graft rejection and that nerve regeneration can reach similar levels to the autograft –27. ThesWe hypothesized that temporary local delivery of FK506 to an allogeneic nerve graft could prevent allogeneic graft rejection and allow nerve regeneration to proceed through the allogeneic nerve graft. In previous work, we developed an FK506-delivering nerve conduit that provides sustained local delivery of FK506 for at least 2 months , 37. In A depot FK506 delivery system in the shape of a nerve conduit was created by embedding FK506 into poly(L-lactide-ε-caprolactone) (PLC) following a previously described protocol . 10% w/vThe in vivo study protocols were executed as approved by the Institutional Animal Care and Use Committee of the University of Utah. 94 adult male Tg(Thy1-YFP)16Jrs  mice were used in the experimental groups and 33 adult male BALB/c mice were used as allogeneic sciatic nerve donors . These two strains of mice were chosen because they have differences in major histocompatibility complex class I and class II molecules. The experimental mice were divided into experimental groups: Autograft (n = 29), Allograft + Local FK506 (n = 29), Allograft + Temporary Systemic FK506 (n = 7), and Allograft + Continuous Systemic FK506 (n = 29). In all animals, an 8mm sciatic nerve gap was created and repaired by either an 8mm autologous or allogeneic sciatic nerve graft. The Autograft group, received a nerve autografts without any additional drug or device treatment. The Allograft + Local FK506, received a nerve allograft that was placed through the inner lumen of the FK506-loaded nerve conduit. The Allograft + Continuous FK506 and Allograft + Temporary Systemic FK506 groups received a nerve allograft and daily subcutaneous injections of FK506; however, daily injections were discontinued after 6 weeks for the Temporary Systemic FK506 group. FK506 solutions for injection were created by diluting clinical grade intravenous FK506 solution  into 0.9% sodium chloride to a final concentration of 1.667 mg/ml. For the serial assessment of immune response and nerve regeneration, n = 5 mice from each group were sacrificed at weeks 1, 2, and 4, and n = 7 mice from each group were sacrificed at weeks 6 and 16. The Temporary Systemic FK506 group only has n = 7 animals, which were sacrificed at week 16. The mice sacrificed at weeks 1, 2, and 4 underwent nerve tissue harvesting for histologic assessment of the immune response to the nerve graft, and mice sacrificed at weeks 6 and 16 also underwent functional ladder rung testing and gastrocnemius muscle mass recovery evaluation. Nerve histomorphometry was performed only at weeks 4, 6, and 16 since nerve regeneration prior to 4 weeks is minimal.The rodent study protocols were executed as approved by the Institutional Animal Care and Use Committee of the University of Utah. The mice were housed according to the IACUC and University guidelines at the University of Utah. Mice were anesthetized with isoflurane. A longitudinal incision was made in the posterior distal thigh of the hind limb, separating the natural muscle planes. In the autograft group, the sciatic nerve was isolated and transected immediately distal to the sciatic notch and proximal to the peroneal and tibial bifurcation, for a total length of 8mm. The proximal and distal ends of the isograft was flipped and it was sutured back into the transected ends of the intact sciatic nerve were then repaired using 9–0 nylon epineural sutures. For the allograft group, the sciatic nerve was transected immediately distal to the sciatic notch and an 8mm portion of the nerve was removed. An 8 mm sciatic nerve allograft from a donor BALB/c obtained in a similar fashion was sutured to the transected ends of the host animal using 9–0 nylon epineural sutures. In the Allograft + Local FK506 group, the allograft was placed through the inner lumen of the FK506-loaded nerve conduit. Animals were sacrificed according to the timeline mentioned above for tissue harvest. Animals were provided daily carprofen postoperatively according to IACUC guidelines to alleviate pain. Animals were sacrificed via cervical disolocation under isoflurane anesthesia.The ladder rung assay evaluates an animal’s ability to perform skilled locomotion, which is correlated with sensorimotor functional recovery . The aniThe sciatic nerve grafts were harvested from the animals immediately upon sacrifice based upon the timeline in 5 μm sections were taken from the paraffin embedded nerve graft tissue described above and immunohistochemically (IHC) stained for CD4+ lymphocytes. Slides were deparaffinized and an antigen retrieval step was performed with 6.0 pH sodium citrate for 20 minutes. The slides were then stained with a rabbit monoclonal anti-mouse CD4+ primary antibody ab183685 at a 1:500 dilution  and then a polyclonal goat anti-rabbit HRP antibody  was used for secondary staining. The slides were counterstained with hematoxylin. The slides were imaged with light microscopy using an Axio Scan.Z1  and were scored for CD4+ cell infiltration severity based on a 0–3 scale: 0 = normal, 1 = mild, 2 = moderate, 3 = severe .A 5mm portion of the sciatic nerve immediately distal to the distal suture line of the nerve graft was harvested at the time of sacrifice. The nerve tissues were fixed in formalin for 24 h and then postfixed in 2% osmium tetroxide solution  for 60 min, dehydrated, and embedded into Epon 812 resin . 0.5 μm sections were stained with toluidine blue and imaged via light microscopy with a ZEISS AxioScan.Z1 . Analysis was performed using ImageJ to determine nerve fascicle area, axon density, and total number of myelinated axons. Stereological techniques were used to obtain unbiased representations of the total number of myelinated axons and axon diameter per cross section , 42.Experimental/MassContralateral) x 100.The gastrocnemius muscle of both hind legs was harvested immediately after sacrifice by careful dissection at the tendinous origin and insertion points. The muscles were weighed and the relative muscle mass of the experimental leg was calculated by comparing the weight to the contralateral side: Relative % Gastrocnemius Muscle Mass =  was used for all statistical analysis. The inflammatory cell and CD4+ T-cell infiltrate severity score data was analyzed with a Kruskal-Wallis test with a pairwise comparison post-hoc test. The gastrocnemius muscle mass recovery, nerve histomorphometry, and ladder rung assay data was analyzed with a one-way analysis of variance with a Student’s t-test post-hoc test for group comparisons.Serial histological assessment of inflammatory and CD4+ cell infiltration was performed to determine the immune response time course to allogeneic nerve grafts with either systemic or local delivery of FK506 immunosuppression. Since inflammatory cells and CD4+ lymphocytes have been implicated as critical role players in Wallerian degeneration and nerve regeneration, the autograft control group was used to determine the native inflammatory and CD4+ cell baselines in a nerve injury scenario , 44. See2) had significantly (p<0.05) greater average myelinated axon density than both the Local FK506  and Continuous Systemic FK506 groups , and the Continuous Systemic FK506 group also had significantly (p<0.05) greater myelinated axon density than the Local FK506 group. At week 16, no significant differences of average myelinated axon density were found, indicating that local delivery of FK506 had similar levels of immunosuppressive effects as the systemic delivery of FK506 in this study in regards to nerve histomorphometry. However, nerve histomorphometry outcomes were significantly lower than both the Autograft and Temporary Systemic FK506 group, suggesting that sustained FK506 treatment produces an inhibitory effect to nerve regeneration outcomes. Also, at week 16, the Autograft and Temporary Systemic FK506 group had no significant differences in nerve histomorphometry outcomes, suggesting that discontinuing FK506 immunosuppression after nerve regeneration has passed through the allogeneic nerve graft will produce nerve regeneration outcomes similar to the Autograft.Nerve histomorphometric analysis was performed to assess nerve regeneration across the various nerve grafts and treatments. Total number of myelinated axons, axon diameter, and axon density was quantified for each animal sacrificed at weeks 4, 6, and 16 . At weekMuscle mass recovery after denervation is a gross measure of motor neuron end-target regeneration . The relThe ladder rung assay was used to assess functional recovery after nerve injury. Skilled locomotion was assessed by measuring the slip ratio  of the animals crossing a horizontal ladder. The ladder rung assay data can be found in Local delivery of immunosuppressant drugs is a novel field of research with only a limited number of studies being published to date –32, 35. We serially assessed the immune response to the allogeneic nerve grafts with varying immunosuppressive treatments to determine if local delivery of FK506 could produce similar levels of immunosuppression as systemically delivered FK506 . An autoIn regards to the nerve regeneration outcomes at the end of the study, the Local FK506 and Continuous Systemic FK506 group had no statistical differences in the nerve histomorphometry, gastrocnemius muscle mass recovery, and ladder rung assay outcomes. This result indicates that similar levels of nerve regeneration were achieved between the groups receiving either local or systemic delivery of FK506. However, the Local FK506 and Continuous Systemic FK506 groups had significantly lower total number of myelinated axons than the Autograft and Temporary Systemic FK506 groups, and they also had lower gastrocnemius muscle mass recovery than the Autograft group. For the ladder rung assay at week 16, the Local FK506, Continuous FK506, and Autograft groups had no significant differences between groups, therefore there was no difference in functional recovery from the autograft. The observed elevated presence of CD4+ cells in the Continuous FK506 and Local FK506 group may be to the presence of acute rejection, which led to lower histomorphometry and muscle mass recovery outcomes than the autograft. It is also common for solid-organ transplants  in the clinic to experience sub-clinical and clinical acute rejection even with immunosuppressive therapy, which is histologically described by increased mononuclear immune cell infiltration . It can Another interesting takeaway from the study is that the Temporary Systemic FK506 group achieved similar levels of nerve regeneration and significantly better skilled locomotion functional recovery than the Autograft group . At the In recent years, there have been several studies investigating the efficacy of locally delivered FK506 immunosuppressive therapy to allogeneic tissue to prevent tissue rejection; however, they have primarily focused on delivering local immunosuppressive therapy to vascularized composite allografts –34. To dIn conclusion, results from this study suggest that local delivery of FK506 to nerve allografts can provide similar immunosuppression and nerve regeneration outcomes as systemic FK506 delivery, and that temporary FK506 administration to a nerve allograft can produce similar nerve regeneration end-results as an autograft repair. Additionally, temporary FK506 delivery may provide an added benefit of enhanced sensorimotor functional recovery, which may be attributed to the added neurotrophic effects of FK506 early on in the regeneration process. Further studies are needed to determine the optimal local FK506 concentrations and duration of release for local immunosuppression in nerve allograft repair strategies.


P < 0.001), and less likely to have a family history of ADPKD , a detectable PKD1 or PKD2 mutation , or progression to CKD stage 3 or stage 5 (P < 0.001). Patients with atypical polycystic kidney disease by imaging represent a distinct prognostic group with a low likelihood of progression to CKD.Using age- and height-adjusted total kidney volume, the Mayo Clinic Imaging Classification provides a validated approach to assess the risk of chronic kidney disease (CKD) progression in autosomal dominant polycystic kidney disease (ADPKD), but requires excluding patients with atypical imaging patterns, whose clinical characteristics have been poorly defined. We report an analysis of the prevalence, clinical and genetic characteristics of patients with atypical polycystic kidney disease by imaging. Patients from the extended Toronto Genetic Epidemiology Study of Polycystic Kidney Disease recruited between 2016 and 2018 completed a standardized clinical questionnaire, kidney function assessment, genetic testing, and kidney imaging by magnetic resonance or computed tomography. We compared the prevalence, clinical features, genetics, and renal prognosis of atypical versus typical polycystic kidney disease by imaging. Forty-six of the 523 (8.8%) patients displayed atypical polycystic kidney disease by imaging; they were older (55 vs. 43 years;  Progressive cyst expansion distorts the kidney architecture and ultimately leads to ESKD in a large proportion of patients4. With the recent approval of Tolvaptan as the first disease-modifier drug for ADPKD6, identifying high-risk patients who may benefit from this treatment is a clinical priority8. The Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease has shown that total kidney volume (TKV) expands quasi-exponentially during adult life at ~ 5% per year and is a sensitive marker for predicting chronic kidney disease (CKD) progression in ADPKD9.Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder with a prevalence of at least 1/1000, and is an important cause of end-stage kidney disease (ESKD)11, for enrichment of high-risk patients in clinical trials12, and is now commonly used in clinical practice13. However, it requires visual inspection of MR images to exclude cases with atypical imaging patterns (class 2) which were present in 8.8% (52/590) of patients from the Mayo Clinic derivation cohort but excluded from subsequent analyses10. The typical imaging (class 1) pattern for the Mayo Clinic Imaging Classification is defined as bilateral and diffuse cyst distribution, where all cysts similarly contribute to TKV. By contrast, atypical polycystic kidney disease is defined by one of the following imaging patterns: (i) unilateral, (ii) asymmetric, (iii) segmental, (iv) lopsided, or bilateral cystic disease with (v) unilateral or (vi) bilateral kidney atrophy , the Mayo Clinic Imaging Classification (MCIC) provides a validated approach for CKD risk stratificationCOL4A1 mutation (n = 1); complex diagnoses of ADPKD with a second kidney disease (n = 2); and no PKD1 and PKD2 mutation results available (n = 3) ; non-ADPKD diagnoses (n = 8) including simple cysts (n = 4), peri-pelvic cysts (n = 2), congenital anomalies of the kidney and urinary tract n = 1), and cystic disease related to a , and cysP < 0.001) with a male predominance ; they were less likely to have a family history of ADPKD  or a detectable PKD1 or PKD2 mutation , suggesting milder CKD in the former group. Consistent with this notion, patients with atypical kidney imaging patterns showed excellent kidney survival as defined by the absence of CKD stage 3 or stage 5 .The clinical characteristics of patients with typical versus atypical kidney imaging patterns are detailed in Table PKD1 (n = 3 for non-truncating and n = 1 for truncating) or PKD2 (n = 5) mutation. Only one patient in this cohort displayed the pattern of acquired unilateral atrophy, without an identifiable PKD1 or PKD2 mutation.Of the 46 patients with atypical polycystic kidney disease  of our patients with atypical kidney imaging patterns would have been misclassified as being at high risk for progression to ESKD .Significant variability in cystic liver disease severity was observed in patients with atypical polycystic kidney disease by imaging: 29 (63.0%) had fewer than two liver cysts, 11 (23.9%) had between two and ten cysts, and 6 (13.0%) had more than ten cysts. Mild liver disease (≤ 10 cysts) was more frequent in patients with atypical imaging patterns compared to patients with typical imaging patterns  which is very similar to that reported in the discovery cohort used for the Mayo Clinic Imaging ClassificationTo fully capture all observed atypical kidney imaging patterns, we added two additional patterns to the Mayo Clinic Imaging Classification. Firstly, a "segmental sparing” pattern, characterized by bilateral and diffuse cystic disease with sparing of one pole of one or both kidneys, which is relatively common  in this study. Secondly, a “mild lopsided” pattern, where 15–49% of TKV is attributable to 2–5 cysts (similar to the “lopsided” pattern where ≥ 50% of TKV is attributable to 2–5 cysts); it is the most common atypical imaging pattern in our cohort . Both of these patterns were strongly associated with the clinical and genetic features of atypical polycystic kidney disease, namely low probability of a positive family history, low probability of a detectable mutation, or slowly progressive kidney disease.15. Mosaicism refers to the occurrence of two genetically distinct cell populations within an individual, due to the somatic mutation of a single pluripotent stem cell during embryogenesis or development17. Due to dilution and variable involvement of the affected cells, a mosaic individual with ADPKD often presents with de novo cystic kidney disease with atypical imaging  patterns, as exemplified by the proband of TOR135 we previously reported18. However, the diagnosis of mosaicism is technically challenging and frequently missed by Sanger sequencing, which is frequently used for ADPKD, due to dilution of the mutation signal from the admixture of normal and mutant cells—a difficulty that can be overcome by next-generation sequencing with high-read depth19. Indeed, by using next-generation sequencing, a recent study of 387 PKD1 and PKD2 mutation-negative patients with ADPKD identified 20 PKD1 somatic mosaics, with at least 6 of them displaying atypical kidney imaging patterns15. In addition to somatic mosaicism, PKD2 (n = 5) and PKD1 non-truncating (n = 3) mutations, which are typically associated with mild cystic kidney disease, were found in 8 of 9 (88.9%) mutation-positive patients with atypical imaging patterns in our study. Next-generation sequencing for mutation screening of additional genes, such as PRKCSH, GANAB, ALG8, ALG9, SEC61B, SEC63, DNAJB11, and HNF1B, which may be associated with mild or atypical polycystic kidney disease, may allow for further elucidation of the genetic causes of atypical polycystic kidney disease by imaging14.Forty-five of the 46 patients with atypical polycystic kidney disease  showed complete sparing of cystic disease with normal parenchyma in one or more parts of the kidneys. Sparing of more than one region of the kidney is a common finding (40/46 or 87.0% in this cohort) and is suspicious of somatic mosaicismPKD1 and PKD2, or progression to advanced CKD. The causes of atypical polycystic kidney disease by imaging are heterogeneous and may include somatic mosaicism, mild disease associated with PKD1 non-truncating or PKD2 mutations, as well as mutations in other cystic disease genes. Incorrect identification of atypical imaging patterns can lead to inappropriate risk stratification. Elucidating the genetic causes of atypical polycystic kidney disease by imaging with next-generation sequencing, including broad cystic gene panels, will advance our understanding of this clinical syndrome and help clinicians counsel patients on the most appropriate management strategy.In conclusion, approximately 9% of a large cohort of patients with ADPKD displayed atypical polycystic kidney disease by imaging. Compared to patients with typical kidney imaging patterns, they were older, and less likely to have a family history of ADPKD, a detectable mutation in https://www.cimpkd.ca/) and 22 patients seen at St. Joseph’s Healthcare in Hamilton, between March 1, 2016 and September 30, 2018. All study patients fulfilled the ultrasound or magnetic resonance imaging (MRI) based diagnostic criteria for ADPKD; none had an eGFR less than 15 mL/min/1.73 m2 at the time of recruitment21. They were referred by more than 100 academic and community nephrologists in the Greater Toronto Area for risk stratification by kidney MRI and genetic testing, and potentially, novel therapeutic interventions. All study patients provided informed consent to a pre-specified research protocol approved by the Research Ethics Board at the University Health Network in Toronto and St. Joseph’s Healthcare in Hamilton, both in Ontario, Canada. Research was performed in accordance with the Declaration of Helsinki.Study patients (n = 543) were recruited from the extended Toronto Genetic Epidemiology Study of PKD (eTGESP), which enrolled 521 patients seen at the Centre for Innovative Management of Polycystic Kidney Disease  images were collected and used for the analyses. Their estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation from the last clinic follow-up22.All study patients completed a standardized clinical questionnaire that included their demographics, detailed family history (with an annotated pedigree available for every proband in the study), and potential complications of ADPKD. In addition, their serum creatinine, 10. Two additional categories  were included in our classification to cover atypical presentations not accounted for in the original paper; they were seen relatively frequently in our clinical experience to warrant their inclusion in this study. Segmental sparing is defined by generalized cystic disease with sparing of one pole of one or both kidneys. Mild lopsided was introduced to describe patterns suggestive of lopsided but with dominant cysts amounting to 15–49%, as opposed to equal to or greater than 50%, of TKV. A systematic analysis of cyst patterns was conducted on MRI or CT scan independently by two different investigators (IAI and AZW), including one radiologist (AZW). Whenever the investigators differed as to the interpretation and could not come to an agreement, a senior abdominal radiologist (KK) was brought in to settle ambiguous cases.TKV was assessed by an experienced radiologist (MP) using the ellipsoid formula . All images were visually inspected for polycystic kidneys with atypical imaging patterns as outlined in the Mayo Clinic Imaging ClassificationPKD1 and PKD2 was performed by targeted exome sequencing as per the published protocol23. All pathogenic mutations identified through targeted exome sequencing were confirmed by Sanger sequencing using a validated PCR protocol18. All nonsense, frameshift, and canonical splice-site mutations were grouped as protein-truncating mutations, and non-synonymous missense or atypical splice site mutations were grouped as non-truncating mutations. In-frame insertions/deletions (in-frame indel) were classified separately. Non-truncating mutations were evaluated for their pathogenicity using bioinformatics prediction algorithms , review of the PKD mutation database (http://pkdb.mayo.edu), and evaluation of familial co-segregation whenever possible18. All mutation-negative patients were re-screened by multiplex ligation-dependent probe amplification to detect large gene rearrangements24.Mutation screening for Biochemical, genetic, and volumetric parameters were compared between the participants with polycystic kidneys and typical or atypical imaging patterns. Statistical analysis was performed in GraphPad Prism and R. Categorical variables were reported as frequency (percentage) and normally distributed continuous variables were reported as mean ± standard deviation, while non-normal continuous variables were reported as median . Patient characteristics were compared using the Mann–Whitney test and Fisher’s exact test. Kaplan–Meier curves were plotted to compare kidney survival  for patients with typical versus atypical polycystic kidney disease by imaging, and tested for statistical significance using the log-rank test. Censoring was done at death, development of CKD stage 3 or 5, or age at the most recent follow-up.Supplementary Information.


Despite its crucial impact on treatment accuracy and therefore patient safety, image-guided radiation therapy (IGRT) during adjuvant breast radiotherapy widely lacks standardized recommendations and guidelines. A wide variation of different IGRT methods and schedules is used in radiotherapy centers over the world, yet reliable evidence remains sparse for most of these approaches. In this study, we took a closer look at a common approach, by which IGRT frequency is altered based on the magnitude of setup errors in the beginning of the radiotherapy treatment.The current study aims to assess the suitability of setup errors during the first three treatment fractions to determine cone-beam computed tomography (CBCT) frequency in adjuvant breast radiotherapy. For this, 45 breast cancer patients receiving non-hypofractionated radiotherapy after lumpectomy, including a simultaneous integrated boost (SIB) to the tumor bed and daily CBCT imaging, were retrospectively selected. In a first step, mean and maximum setup errors on treatment days 1–3 were correlated with the mean setup errors during subsequent treatment days. In a second step, dose distribution was estimated using a dose accumulation workflow based on deformable image registration, and setup errors on treatment days 1–3 were correlated with dose deviations in the clinical target volumes (CTV) and organs at risk (OAR). No significant correlation was found between mean and maximum setup errors on treatment days 1–3 and mean setup errors during subsequent treatment days. In addition, mean and maximum setup errors on treatment days 1–3 correlated poorly with dose coverage of the CTVs and dose to the OARs. Thus, CBCT frequency in adjuvant breast radiotherapy should not be determined solely based on the magnitude of setup errors during the first three treatment fractions. The use of increasingly complex target volumes and highly conformal irradiation techniques in breast cancer radiotherapy requires precise position verification and adjustment through image-guided radiation therapy (IGRT), now more than ever. In recent years, body-surface scanners emerged as an alternative imaging method to cone-beam computed tomography (CBCT) and planar imaging. Yet, recent studies confirmed CBCT as the gold standard for IGRT as it reduces setup errors and allows for a superior spatial match with the planning CT ,2. The bA substantial drawback of CBCT is the increased imaging dose administered to the patient, especially with daily imaging schedules . SeveralDue to the persistent lack of recommendations and guidelines, CBCT-based IGRT practice varies profoundly between different institutions. Following a thorough literature research, it can be concluded that most institutions prescind from publishing their CBCT-based IGRT protocols and that the available data originate almost entirely from individual study protocols ,8,9,10 aThe idea of an individual approach based on geometric shifts at the beginning of the treatment stands to reason, as it enables an easy standard operating procedure complying with the recommendations of a case-by-case assessment for IGRT frequency ,16,17. Yn = 18) or 63 Gy (n = 27) in 28 Fx to the tumor bed. The radiotherapy plans were generated in Varian Eclipse 15.6 using VMAT technique according to current guidelines [Population and radiotherapy: A total of 45 breast cancer patients receiving adjuvant radiotherapy with daily CBCT imaging (≥27 CBCTs in 28 fractions [Fx]) were retrospectively included in our analysis. All patients were treated in our institute between May 2016 and September 2020. Informed consent was obtained from all participating patients, and the study approval was granted by the local ethics committee. All treatments included whole breast irradiation following lumpectomy and a SIB to the tumor bed. A total of 76% of patients were additionally treated with RNI to the supra- and infraclavicular regions, and 56% of patients were additionally treated with RNI to the internal mammary region. The prescribed dose was 50.4 Gy in 28 Fx to the whole breast and either 58.8 Gy n =  or 63 Gyidelines ,20, and Positioning and IGRT: Skin markers defined during CT simulation were used for patient setup. The marker-based position was then verified and corrected through daily kilovoltage (kV) CBCT imaging using the Varian On Board Imager 1.6 . For each treatment fraction, the CBCT scans were automatically registered to the planning CT using three degrees of freedom and a gray value algorithm, followed by a manual adjustment if necessary. The primary points of interest for manual adjustments were the breast- and boost-CTV as well as the OARs. After three treatment fractions, the position of the skin markers was adjusted using the mean CBCT-based couch shifts along the X-, Y- and Z-axis in order to prevent systematic errors.Assessment of setup errors: Setup errors were defined as deviation of the marker-based position from the CBCT-adjusted position. By correlating the mean and maximum setup errors in X-, Y- and Z-direction of the first three fractions with the mean deviations of subsequent fractions, we aimed to assess the ability of an early evaluation of position accuracy to predict setup accuracy over the entire course of treatment.CBCT-corrected patient setup for all 28 fractions (“gold standard protocol”);CBCT-corrected patient setup for the first 3 fractions and optimized marker-based patient setup for all subsequent fractions (“reference protocol”).Effect of setup errors on dose distribution: In a second step, we aimed to assess whether mean and maximum setup errors in X-, Y- and Z-direction on treatment days 1–3 correlate with dose coverage in the CTVs and dose to the OARs. For this, a previously established dose accumulation workflow  based onThe accumulated dose cubes for both protocols were reimported into Varian Eclipse and compared for dose distribution in the CTVs (breast and tumor bed) and OARs , ipsilateral lung and contralateral breast). Deviations of dose parameters between the gold standard and reference protocol were assessed and correlated to the setup errors on treatment days 1–3.Statistical analysis: All statistical analyses were performed using the SPSS statistical analysis software package . Pearson product-moment correlation coefficients were used to assess the correlation between setup errors on treatment days 1–3 and setup errors on subsequent treatment days as well as dose distribution in the CTVs and OARs. Interpretation of correlation coefficients was based on the widely recognized Cohen standard .The mean setup errors [range] during the first three radiotherapy fractions (days 1–3) were 0.2 cm [0–0.9 cm] in lateral, 0.2 cm [0–1.5 cm] in cranio-caudal (CC) and 0.2 cm [0–1.3 cm] in anterior–posterior (AP) dimension. The mean setup errors [range] during subsequent fractions (days 4–28) were 0.3 cm [0–1.9 cm] in lateral, 0.4 cm [0–2.1 cm] in CC and 0.3 cm [0–3.2 cm] in AP dimension. No significant correlation was found between mean setup errors on days 1–3 and mean setup errors on days 4–28. Similarly, no significant correlation was found between maximum setup errors on days 1–3 and mean setup errors on days 4–28 in lateral, CC and any dimension, whilst a moderate correlation was found between maximum setup errors on days 1–3 and mean setup errors on days 4–28 in AP dimension. As shown in p-values are summarized in For breast-CTV, no significant correlation was found between dose coverage (V95%) and mean setup errors in lateral, AP and any dimension. A moderate correlation was found between dose coverage and mean setup errors in CC dimension. Similarly, no significant correlation was found between dose coverage and maximum setup errors in lateral and AP dimension, whilst a moderate correlation was found between dose coverage and maximum setup errors in CC and any dimension. For SIB-CTV, no significant correlation was found between dose coverage and mean setup errors. Accordingly, no significant correlation was found between dose coverage and maximum setup errors. The relation between CTV coverage and setup errors on days 1–3 is illustrated in p-values are summarized in For heart and LAD, no significant correlation was found between dose exposure (Dmean) and mean setup errors in lateral and CC dimension. A moderate correlation was found between dose exposure and mean setup errors in AP and any dimension. Accordingly, no significant correlation was found between dose exposure and maximum setup errors in lateral and CC dimension, whilst a moderate correlation was found between dose exposure and maximum setup errors in AP and any dimension. For ipsilateral lung and contralateral breast, no significant correlation was found between dose exposure and setup errors. The relation between OAR exposure and setup errors on days 1–3 is illustrated in Our data indicates that neither mean nor maximum setup errors on treatment days 1–3 can sufficiently predict the magnitude of setup errors during subsequent treatment fractions. In addition, setup errors on treatment days 1–3 correlate poorly with dose coverage of the CTVs and dose exposure to the OARs.Despite being an increasingly relevant topic in modern adjuvant breast radiotherapy, guidelines on imaging frequency remain sparse. The United States National Comprehensive Cancer Network (NCCN) recommends weekly imaging, yet complementing that under “certain circumstances” more frequent imaging might be appropriate . Not leaPrevious studies have tried to identify risk factors for extensive setup errors, yet none of them managed to establish efficient suggestions on customized breast IGRT. Yang et al.  conducteBased on our data, it can be concluded that the magnitude of setup errors during the first three treatment fractions is, by itself, not suitable to determine CBCT frequency for the entire treatment. Even large deviations during the first three fractions are not necessarily associated with larger deviations down the treatment course or significant dose deviations in the CTVs and OARs. The CTV-PTV margin has an important impact on CTV coverage. In the current study, a CTV-PTV margin of 10 mm was implemented for the breast, and given the poor correlation between setup errors , it can Neither setup errors over the course of treatment nor dose deviations in the CTVs and OARs correlate sufficiently with the setup errors  during the first three treatment fractions. CBCT frequency in adjuvant breast cancer radiotherapy should therefore not be determined solely based on the magnitude of setup errors during the first three treatment fractions.