The propensity to manufacture functional and geometrically sophisticated parts from a wide range of metals provides the metal additive manufacturing (AM) processes superior advantages over traditional methods. The field of metal AM is currently dominated by beam-based technologies such as selective laser sintering (SLM) or electron beam melting (EBM) which have some limitations such as high production cost, residual stress and anisotropic mechanical properties induced by melting of metal powders followed by rapid solidification. So, there exist a significant gap between industrial production requirements and the qualities offered by well-established beam-based AM technologies. Therefore, beamless metal AM techniques  have gained increasing attention in recent years as they have been found to be able to fill the gap and bring new possibilities. There exist a number of beamless processes with distinctively various characteristics that are either under development or already available on the market. Since this is a very promising field and there is currently no high-quality review on this topic yet, this paper aims to review the key beamless processes and their latest developments. There has been a trend in metal additive manufacturing (AM) in recent years and much of the reports on AM are about the growth in metal-AM and its significant impact on design and rapid manufacture of geometrically complicated high-end parts. A recent Wohlers Report  demonstrThe current highlights of metal AM are divided into two main categories: beam-based and beamless techniques . As clasBeamless metal AM techniques  are being employed effectively to overcome the above limitations of the beam-based systems. There exist a number of beamless processes with various distinct characteristics that are either under development or already available in the market. These approaches provide different opportunities in terms of process economics, mechanical properties, achievable geometries and surface quality.Beamless metal AM has been able to draw the attention of both the research community and industry. The growth in beamless research over the past five years is reflected in the number of publications . In addiAs seen in EFAB and FluidFM 3D printing technologies use electrochemical deposition methods that outperform the beam-based systems in terms of resolution and accuracy. Conversely, ultrasonic AM (UAM), additive friction stir deposition (AFSD), friction stir AM (FSAM), and cold spray AM (CSAM) processes use solid state thermo-mechanical bonding without the need for a protective print environment for printing mega scale parts which are not viable through beam-based systems . UAM enaA comprehensive review of the key beamless metal AM systems and the current state of their research and technology are presented in this paper. The review outlines the principle of each beamless metal-AM processes followed by disseminating the efforts and recent progresses. Techniques that either have not been widely adopted  or their application field is mainly confined to writing (such as aerosol jet that needs laser beam for true 3D fabrication) are not addressed in this paper.This review is based on Scopus and Google Scholar searches. Articles published over the period 2012–2019 have been selected and the full papers and other reviews have been analysed. Furthermore, Wohlers Report 2018 [Material jetting which includes processes like liquid metal printing, nanoparticles inkjet printing or aerosol jet process see  is an AMThe material jetting process was initially used for printing parts out of wax  and polyA number of research works have been conducted on droplet-based printing of low/medium stiffness metals such as Sn63-Pb37 alloys ,18,19,20Development of new jetting systems with higher print quality has been the main focus of recent research. In inkjet printing, the drop diameter normally ranges from one to two times that of the nozzle . DropletThe limited crucible volume is the main obstacle for further commercialization of liquid metal 3D printing. To overcome this problem, a wire-based feeding system was proposed by  for geneMolten metal jetting is limited to low/medium melting temperature metals as for other metals a high-temperature resistant printing system is required to keep the metal molten during the jetting process . XJet (RBinder jetting (BJ), known as three-dimensional printing (3DP), was first developed at the Massachusetts Institute of Technology . Binder Binder jetting has been successfully used for printing of different metals such as stainless steel , titaniuMicrostructural analysis and investigation of mechanical properties of the binder jetted metals has been a focal research topic ,65. BasiThe main limitation of metal binder jetting is the relative density of metal parts (95–97%), which can be compared with a typical density of > 99% with laser-based powder bed systems. A number of research works have been conducted to investigate the effect of process parameters  and part orientation ,71, powdThe selection of a binder material system determines green part strength and affects the sintered density and shrinkage . SolventMaterial extrusion refers to the AM techniques that use an extrusion method for dispensing build material selectively. The process known under the trademarked name Fused Deposition Modelling (FDM) technology, also known as Fused Layer Modeling (FLM) or Fused Filament Fabrication (FFF) . Figure The main advantage of FDM is its low technology and operation cost as it does not require expensive components such as lasers or electron sources and vacuum pumps in beam-based machines. Another advantage is the ability to build components combining both thermoplastics and metals within the same build which is not possible with other direct metal systems . The FDMBasically, there are three different approaches for extrusion-based AM. Material extrusion with plungers, screw-based, or filament-based. Metal extrusion AM is mainly used by screw-based or plunger-based approaches since metal filaments present multiple problems mostly due to their mechanical properties such as brittleness.Various material extrusion approaches have been developed with the aim of direct metal 3D printing. Greulich et al.  developePowder-binder mixtures are currently the most promising solutions for extrusion-based AM . Metal o3 which is very close to the typical value for the density of 7.67 g/cm3 specified conventionally processed MIM parts. A CEM printed test part made from 316 as well as an optical microscopy of the microstructure are depicted in A screw-based approach using conventional metal injection molding (MIM)-feedstock was introduced as composite extrusion modelling (CEM) by Lieberwirth et al. . The finAnother plunger-based material extrusion approach is termed 3D gel-printing (3DGP), which selectively deposits a slurry layer-by-layer . The 3DGCold spray (CS) process was originally developed as a solid-state coating method for improving surface properties . In CS pAs illustrated in CSAM of Al and Cu alloys have been widely investigated due to their excellent deformability, and thus cold-sprayability. Dense bulks from Cu and Cu alloys  with good interface bonding and mechanical properties have been printed using optimized CS with helium gas . Post anThe relatively high strength of stainless steel makes it difficult to print dense parts. Coddet et al.  studied It is quite challenging to cold spray dense Ti alloys parts. The CSAMed Ti alloys exhibit a relatively high level of porosity due to their active characteristics and reactions with surrounding air . Wong etIt is very difficult to deposit dense parts from superalloys even using CS at high gas temperatures , so therThis process is a solid-state metal AM process that uses heat and friction to bond materials layer-by-layer , developThe AFSD involves a hollow rotating shoulder, through which the feed material is delivered in the form of either a solid rod or powder . The rapThe peak temperature of AFSD process normally ranges from 60 to 90% of the melting temperature . HoweverMultiple materials such as Nb/Cu and Ta/Cu have been successfully printed using AFSD process. No interfacial delamination was observed when applying a high shear stress at the interface through bending . Also, cUltrasonic additive manufacturing (UAM), known as ultrasonic consolidation (UC), is a hybrid additive/subtractive process that involves the layer-by-layer ultrasonic scrubbing of similar or dissimilar metal foils in the solid state with periodic cutting operation (often end-milling) to fabricate desired part geometry . The proThe common defect in UAM-printed parts is micro-voids that are produced along the interfaces between bonded layers due to foil surface roughness, insufficient or excessive welding energy, and metal foil positioning inaccuracies . AlthougThe use of very high power UAM has been shown to improve elastic properties of the printed AL alloys . SridharThe low operation temperature and the plastic flow of metals provide the opportunity to embed and/or encapsulate various electronic devices in to the metal matrix through the UAM process. Smart material structures could be printed by embedding fibres, conductor, dielectric, and nano-fibres of NiTi shape memory alloy into a metal matrix . Carbon Moreover, solid-state nature of UAM allows joining of dissimilar metals without the formation of brittle intermetallics as seen in powder-bed fusion processes. High performance multi-material parts such as Al/Cu, Ni/Stainless steel, and Al/Ti are routinely produced using high power 9KW UAM . Hehr etThis process is the other type of friction-based technique that is a sheet lamination process. In FSAM, friction stir welding is used to bound stacked multiple metal sheets. A special friction pin is penetrated into the stacked sheets and then a welding line is created throughout the sheets by rotation and traverse movement of the pin. Large parts are produced by repeating the sheet stacking and friction welding, and further machining process to form the accurate external shapes. So, both AFSD and FSAM are intensive techniques as parts are printed through a severe plastic deformation at high temperatures, yet solid state. The main difference is that the FSAM is a hybrid additive/subtractive process that produces microstructures with cyclic variations ,142, whiWire and arc additive manufacturing (WAAM) is becoming more popular due to its high deposition rate, low production cost and the capability for fabricating large-scale components . Contrar3/h or 5–6 kg/h [WAAM is a promising technology for producing large metal components  with moderate complexity. Specifications of this technology can be roughly defined in a high build rate of up to 2500 cm5–6 kg/h . Many di5–6 kg/h ,146,147.5–6 kg/h .Furthermore, WAAM can be distinguished between the use of different heat sources. The WAAM process can use gas metal arc welding (GMAW or MIG), gas tungsten arc welding (GTAW or TIG) or plasma arc welding (PAW). There are different arc welding technologies that are being used. Motion can be provided either by robotic systems or by computer numerical controlled gantries. TopTIG, for example, is a robotic GTAW welding technology with integrated wire feeder that distinguishes itself with the design of the welding torch. Cold metal transfer (CMT) is a modification of the short-circuit GMAW process which is based on controlled dip transference . PAW canThere currently exist two electrochemical metal-AM processes, namely, electrochemical fabrication (EFAB) and FluidFM 3DP. The EFAB  method uses electrochemical deposition and subtractive planarization in a layer-by-layer process to build 3D micro-objects . It invoTM, EDURA-180TM, copper, and biocompatible noble palladium. More information about the material properties can be found in the literatures [The EFAB has been developed for true 3D manufacturing of metal microparts with features as fine as 20 μm and accuracy of 2 μm. So far, limited metals have been developed by Microfabrica to be processed through EFAB, including: VALLOY-120eratures .FluidFM 3DP process is another electrochemical technique developed recently by Cytosurge AG . It combines electrodeposition, scanning probe microscopy (SPM), and precise liquid ink dispensing . The sysThis process has been developed by researchers at Fraunhofer Institute for Manufacturing Technology and Advanced Materials (IFAM), Branch Lab Dresden (Germany) . It uses2)/metal (17-4-PH) sandwich structure, and copper/ceramic 3D patterns with line width of 150 µm could be printed and sintered without cracks [The metal paste formulation and its rheological behavior is the key parameter to achieve high accuracy detailed parts. The process has limitation in terms of solid particle loading (up to 50 Vol.%) as too high particle concentration can result in undesired shear thickening behavior. A wide range of metals such as stainless steel, copper, titanium, and hard metals have been successfully fabricated with surface roughness of Ra = 5–10 µm . Also, tt cracks .It is believed that the beamless metal AM can improve the development trajectory of metal AM. It can lower technology and operation costs, overcome the intrinsic safety concerns associated with beam-based systems , and posAn in-depth understanding of the strengths/weak points and comprehensive knowledge on capabilities and limitations of each beamless metal AM technology is an important step in selecting the appropriate process for a particular application. A comparison of the key beamless metal AM systems has been presented in The application fields of beamless metal AM systems can be classified into three main areas including micro, medium, and mega-scale parts that are discussed in this section.The EFAB and FluidFM 3DP are the two current beamless methods that have demonstrated great potential for true 3D micro-manufacturing. The current micro-fabrication techniques normally include multiple production steps which are costly and need strictly safety standards. The FluidFM 3DP is a single step process with potential to overcome these barriers and offer higher efficiency to different micro-scale industries. The size of features printed with FluidFM pinpoint 3DP range from 1 µm to 1000 µm  using a The EFAB process has also shown great applicability for true 3D printing of robust sub-millimeter metal parts and mechanical mechanisms for medical, aerospace and semiconductor industries. It provides designers with the new possibilities to commercially produce sophisticated fully assembled medical devices and complex mechanisms with multiple discrete biocompatible components. In particular, as the mechanisms are 3D printed in a finished fully assembled state there is no concern about micro-scale assembly challenges.®  BJ technology at two different sizes to be compared with SLM and EFAB technology. The results of the assessment are presented in In addition, proprietary BJ technology is capable of printing very fine parts with micro-features. For better assessment, the authors conducted a benchmarking study. A tissue staple was used as a benchmark sample and printed using Digital MetalAs can be seen in Metal BJ is well-suited for truly cost-effective serial production of small to medium size parts for different applications such as medical equipment, portable electronics, luxury watches, etc. that is not currently possible using other beam-based metal-AM systems. Metal BJ possess superior benefits over beam-based powder bed AM systems such as reduced costs with higher manufacturing throughput (as there is no need for support), much less residual stress, increased geometrical complexity and print speed.Also, BJ process surpasses metal injection molding (MIM) in terms of design flexibility and part complexity, allowing it to penetrate new application fields that are currently not approachable for MIM. Meanwhile, the BJ can complement MIM rather than compete with it. The current MIM producers already have the infrastructure and facilities to process green parts. So, the BJ can be effectively integrated to MIM process for fabricating prototypes and/or small batches before investing in expensive MIM tools for mass production.®’s BJ technology enables detailed fine metal parts to be printed at high volume with remarkably higher precision and surface quality than SLM or EBM processes. The other commercial systems like ExOne  and GE Additive  have targeted production of medium to large scale parts for automotive and aerospace applications where the BJ technology removes the need for casting and eliminates further expenses such as tooling and molds.Digital MetalIn addition, extrusion-based AM systems can be used for printing of medium size metal parts, in particular, in medical applications such as titanium implants and scaffolds. They can also be served for parts like jigs and fixtures, auto/aero parts, electroforming mandrels, encapsulation molds, dies, electronic joining applications, as well as printing 3D electronic circuitry . Low temTo date, a large number of both research and commercial works on liquid metal jetting have been demonstrated; however, much of the reports confine to 2D and/or 2.5D printing of low melting point metals for electronics applications . The latA great focus has been given to the solid-state beamless metal AM technologies for different mega-scale applications in the past few years as reflected by the number of publications . As therThe spot size of the CS process is currently above 4 mm which is significantly larger than other laser-spots in powder-bed AM systems. Therefore, CS-based AM is considered as an ideal choice for cost-effective printing of near-net-shape features onto the available parts. Meanwhile, the CS head can be integrated with an industrial robot arm for super-fast direct printing of very large metal parts with intricate shapes that is not possible using other metal-AM systems a. TitomiThe other solid-state beamless AM systems, namely, UAM and fiction-based AM systems have been highly regarded by both scientific community and industry. The first commercialized technology of AFSD was developed and patented by Aeroprobe Corporation as “MELD Technology” for printing of a wide range of materials from Al, Mg, titanium alloys, nickel-based superalloys to non-weldable metals at high deposition rates. MMCs can also be deposited through pre-mixing different powders and consolidating them as the feed material. The main application areas of AFSD are repair, rapid manufacture of net-shape large parts b and proUAM has also shown encouraging success in near room temperature fabrication of parts that enables a multitude of thermal-sensitive electronics and sensors to be embedded into the solid metal part, exactly where needed. Also, UAM enables designers to reliably fabricate interwoven channels within a multiple material part printed in a single step. High performance multi-material parts for a wide array of engineering applications, including: coating, heat transfer, strengthening materials, and light weighting can be printed using UAM. To strengthen materials, MMCs can be printed selectively within the part by using continuous fibers at specific high-stress regions c.In contrast to UAM, WAAM is preferred for the production of even larger parts due to its high deposition rate, high material utilization rate, low production and equipment cost, and high equipment flexibility and scalability. In comparison with subtractive processes, WAAM systems have been shown to reduce fabrication time by 40–60% and post-machining time by 15–20% depending on the component size . FurtherAs a consequence of what was discussed, beamless metal AM is well on its way to becoming a viable and established standard manufacturing tool for cost-effective metal part prototyping and rapid manufacturing. The economic and efficiency advantages ensure that beamless metal AM systems will be subjected to extensive investigations in the future. However, the growth of beamless metal AM has been hampered significantly by technical challenges inherent to each process that are currently limiting the wider adaptation of these promising technologies.Despite its advantages, metal BJ has been adopted less than beam-based powder bed systems that might be attributed to the inherent porosity present in the parts, even although post-infiltration and/or liquid phase sintering followed by HIP are normally applied to increase density of the parts. In addition, there are some technical challenges preventing wider use of metal BJ. As-printed green parts are relatively fragile which makes them prone to failure, post thermal treatment increases the lead time remarkably, and for larger parts it results in distortion. Future research may be focused on development of novel techniques for enhancing density without sacrificing homogeneity of material, part accuracy, and/or process scalability. Meanwhile it is worth broadening the range of printable materials through the development of new predictive models and optimization of process parameters, and expanding the application field of BJ into composite materials by overcoming powder segregation and co-sintering issues. Moreover, more efforts on high-speed AM (HSAM) using inkjet-based 3D printing systems is expected to be made in the future although the BJ systems possess a higher speed than beam-based powder bed AM systems. This claim has already been fueled by companies such as HP , and Desktop Metal  with prioritizing the print speed in their new metal BJ commercial 3D printers. HP’s Jet Fusion and MultiJet Fusion (MJF) technologies offer high build speed and quality, and at lower cost than competitive 3D printers. These technologies are quite similar to High Speed Sintering (HSS) process . HP’s MJ3/sec to 375µm3/sec for pure metal parts [The FluidFM 3DP is a newly developed 3D printing method that needs further research and design upgrade to be considered as a mature and reliable technology. The deposition rate needs to be improved for achieving larger build size. Since introduction of the technology the deposition rate of FluidFM 3DP could be increased from 3µmal parts . The incEFAB process has currently some design constrains such as maximum number of build layers (maximum part height of 1.25 mm) and, more important, parts from very limited metals can be currently manufactured. Smart materials are suggested to be developed in the future for manufacturing of electromagnetism or shape memory-based sensing and actuation systems.The growth of liquid metal jetting has been hampered by some challenges. The scalloped surface topography of solidified droplets and the staircase effect reduce surface quality. The previous studies on understanding the mechanism of metal droplets impacting and solidification, and optimization of process parameters have been very useful. Further research work should focus on the development of new printing mechanisms, and in-depth understanding of the morphology of surface topography through experiment and modeling would be helpful for adaptation of liquid metal jetting. The low-temperature 3D printing of metallic parts is quite promising. However, it suffers from relatively low print speed (and thus limited build size) due to the use of nanoparticles in thin build layers. In addition, preparation of nanoparticle metal inks and reliable printing of the inks without nozzle clogging and/or particle sedimentation seems to be quite challenging.CSAM is a vital breakthrough and revolutionary technology and redefines manufacturing components of complex geometry/3D shapes in a single step. Despite the merits highlighted for CSAM, there exist deficiencies that should be addressed through future research activities. The influence of spraying parameters on mechanical properties of hard-to-form materials such as anisotropic behavior is not well understood and entirely agreed upon by researchers. Further experimental studies are required with the aim of developing a standard fabrication strategy for determining optimal part properties. Moreover, most of the research works have focused on the use of CSAM for part restoration and modification, while direct manufacturing of complex parts is an attractive and challenging research topic. In particular, fabrication of parts at higher spatial resolution through development of new spraying systems with smaller spot size for minimizing post-machining processes is a vital step to be further explored.There are huge application field for friction-based AM in industries where large-scale parts are required such as aviation, defense, automotive, energy, etc. To get the best out of these countless possibilities, development of new practical applications for friction-based AM should be considered as a research focus in the future. The low resolution of material deposition as well as the demand for secondary subtractive machining and/or grinding steps for achieving the final shape are the key barriers to the widespread adaptation of friction-based AM technology for industrial productions. The use of new tool deigns in AFSD with a smaller shoulder diameter might be an effective measure to improve resolution as the amount of deposited plasticized material in each step is decreased. Development of an in-situ quality control system can be another approach to better monitor the FSAM process and enhance resolution and reliability. A closed loop control containing high-speed infrared camera and various sensors is proposed to be used for real-time monitoring of weld formation and in-situ measuring of the peak temperature and cooling rate.Realization of mentioned potentials for wider applications of UAM is stifled by high material wastage, anisotropic properties and low mechanical strength in build direction, as well as design constrains such as height to width ration. The production cost of UAM parts is also relatively high, and thus the process has been adapted to limited industries such as aerospace or automotive where functionality and/or properties of the engineered multiple material parts can compensate the high costs. The high production cost might be decreased in the feature as metal foils are widely available on the market for prices approaching that of billet. However, the process might not be considered as a route for single material parts fabrication since the high wastage of expensive materials in UAM process remains as the key barrier for cost reduction in the future. The future works should focus on more in-depth investigation of the process parameters affecting ultrasonic welding-induced pores in the interface with the aim of improving mechanical properties. Techniques such as laser-assisted pre-heating of the metal foils might be useful for increasing plastic deformation and thus enhanced bonding.Many challenges remain with WAAM technology as well. A part from the low resolution of this technology, microstructural anisotropy and porosity remain some of the key challenges that need to be addressed. At the moment, only components with moderate complexity such as flanges or stiffened panel are feasible. Technical challenges like distortion from excessive heat input, relatively poor part accuracy caused by the “stair stepping” effect and poor surface finish keep the technology from being used in more complex and filigree components. The control of residual stresses and distortions especially for the large-scale WAAM process is of major concern. It not only has an effect on the part tolerances but it can also result in premature failure.The low cost of FDM systems has led to one of the highest adoption rates among AM technologies. However, metal-based FDM systems are not yet developed enough to see widespread use. Further development in print speed through high-aspect-ratio (HAR) nozzles can lead to HSAM of metals . Most apThis paper presented a review on the key beamless metal AM processes, progress and future developments. The sintering-based techniques such as BJ and extrusion-based techniques have demonstrated great potential for cost-effective serial production of small parts and low volume production of medium size parts, respectively. On the other hand, the electrochemical processes such as EFAB and FluidFM 3DP techniques have been able to meet the resolution requirements in micro-manufacturing industry while they provide new possibilities in terms of part complexity. Also, the solid-state beamless techniques provide the opportunity to print mega scale parts with improved mechanical strength. In particular, CSAM process benefits from super-fast metal printing speed, making it well-suited for true industrial production of large parts in the near future.


Complexity of cell-type composition has created much skepticism surrounding the interpretation of bulk tissue transcriptomic studies. Recent studies have shown that deconvolution algorithms can be applied to computationally estimate cell-type proportions from gene expression data of bulk blood samples, but their performance when applied to brain tissue is unclear. Here, we have generated an immunohistochemistry (IHC) dataset for five major cell-types from brain tissue of 70 individuals, who also have bulk cortical gene expression data. With the IHC data as the benchmark, this resource enables quantitative assessment of deconvolution algorithms for brain tissue. We apply existing deconvolution algorithms to brain tissue by using marker sets derived from human brain single cell and cell-sorted RNA-seq data. We show that these algorithms can indeed produce informative estimates of constituent cell-type proportions. In fact, neuronal subpopulations can also be estimated from bulk brain tissue samples. Further, we show that including the cell-type proportion estimates as confounding factors is important for reducing false associations between Alzheimer’s disease phenotypes and gene expression. Lastly, we demonstrate that using more accurate marker sets can substantially improve statistical power in detecting cell-type specific expression quantitative trait loci (eQTLs). deconvolution algorithms for estimating cellular composition from tissue gene expression data have been tested extensively in blood, their performance when applied to brain tissue is unclear. To address this gap, we generated an immunohistochemistry (IHC) dataset for five major cell-types from brain, in order to apply and then assess deconvolution algorithms for application to brain gene expression datasets. We show that these algorithms can indeed produce informative estimates of constituent cell-type proportions. Further, we show that adjusting for estimated cell-type proportions across individuals when conducting differential gene expression analysis is important in reducing false associations.Gene expression data generated from a tissue sample reflects an average gene expression profile across heterogeneous populations of cells. Because composition of constituent cell-types can vary across individuals , differential gene expression analysis requires estimating and adjusting for such cellular heterogeneity. While many  Understanding the molecular aetiology of neurodegeneration and neuropsychiatric diseases holds the promise of developing safe and effective treatments which despite decades of work are still lacking. Large collaborative efforts such as the CommonMind Consortium and the Observed gene expression levels in tissues with high cellular heterogeneity are influenced by the proliferation or death of specific cell-types and also by molecular processes within cell-types. In the context of disease studies, this ambiguity in the origin of gene expression variability can generate spurious disease associations or reduce statistical power to detect true associations. SeparatCell-type deconvolution methods are a spectrum of analytical approaches for separating out changes in gene expression stemming from shifts in cell-type compositions from alterations in gene activity. Typically variations of factor analysis and regression, deconvoA key contribution to the reliability of deconvolution approaches are the cell-type markers, or genes that are expressed predominately in a given cell-type, that are used in the modelling. Recent single-cell RNA-seq–9 and cehttps://github.com/ellispatrick/CortexCellDeconv.In this work, we constructed a benchmark brain dataset for quantitative evaluation of deconvolution algorithms. Specifically, we generated an immunohistochemistry (IHC) imaging dataset and quantified the proportions of five distinct cell-types from the cortex of 70 individuals, for which bulk-tissue RNA-seq data have also been acquired. With the IHC dataset as the benchmark, we evaluated four state-of-the-art deconvolution algorithms whose effectiveness has not been assessed in brain tissue. We applied these algorithms to brain tissues by using three sets of marker genes derived from human brain single-cell RNA-seq data, and celS1 Table), which likely enhances the heterogeneity of cell-type proportions .To establish a benchmark for cell-type proportions in heterogamous brain tissue, we used immunohistochemistry to experimentally measure the proportion of neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells from dorsolateral prefrontal cortex (DLPFC) tissue of 70 older individuals. These individuals are a subset of the larger ROSMAP cohort with bulk RNA-seq (n = 508) from the same region; donors GFAP), microglia (IBA1), oligodendrocytes (OLIG2), and endothelial cells (PECAM). Automated image analysis (EBImage) was applied to identify DAPI stained cells and the cells that were positive for each antibody . The proportion of each cell-type was derived by averaging 30 images taken from 6 μm slides per individual. Confirming the quality of the IHC-based cell-type proportions, we observed that the proportion of the five major cell populations per subject approximately sums to one, despite separate staining performed for each cell-type . That is, because each cell-type is stained and counted independently, both natural and technical variability (counting error or staining efficacy) can result in the sum of the proportions being greater or less than one. The sum of the cell-types approximately reaching one also implies that the five measured cell-types make up the bulk of the DLPFC, with no other major cell population unmeasured.To generate IHC-based cell-type proportions, antibodies were chosen to identify neurons (NeuN), astrocytes . Moreover, when looking at global gene expression levels, the IHC-based cell-type proportions explain ~11% of the variance in gene expression levels  and are found to correlate with the expression levels of a large number of genes . These results confirm that cell-type heterogeneity is indeed a major contributor to the variation in bulk-tissue gene expression data from the brain, also verifying that our expression data is a relevant test bed for evaluating deconvolution algorithms.We find that variability in the ROSMAP gene expression can be explained by changes in cell-type proportions. We previously derived cell-type specific modules of covarying genes from this cohort which weRecent studies have shown that cell-type proportions in blood samples can be reliably estimated through computational means, but the accuracy of these estimates for brain cell-type deconvolution is currently unclear. To establish the validity of computational estimation of cell-type proportions in brain tissue, we used the IHC data as the benchmark to assess the accuracy of four deconvolution methods. The methods fall into two classes: 1) “supervised” reference-based methods, which included non-negative least squares (NNLS), CIBERSOTo adapt these deconvolution algorithms to brain tissue, we examined 3 sets of cell-type marker genes derived from: (1) human brain single-cell RNA-seq data (“Zhang”), (2) human brain cell-sorted RNA-seq data “Darman.relative proportions across individuals is assessed, but not the absolute values of the proportions. Having high correlation is often adequate for the estimated proportions to be useful for downstream analysis; for example when estimates are used as confounding factors in association analysis, only the accuracy of relative proportions is important. On the other hand, if the abundance of different cell-types in varied brain regions is of interest, the absolute difference between the estimated and ground truth proportions as measured with MSE is more appropriate.We assessed the concordance between the IHC-based cell-type proportions and estimates generated by the deconvolution algorithms with two metrics: (1) correlation and (2) mean squared error (MSE) between the inferred and measured proportions for each cell-type across individuals. With correlation, we assess whether individuals with higher proportion estimates also display higher proportion in the IHC data, i.e. the Figs 2A and S3). We also observed that the various algorithmic approaches yield highly correlated estimates as assessed more robustly across a larger set of 508 ROSMAP samples . However, CIBERSORT and NNLS are “outliers” in this respect for estimation of microglia cells, which might stem from their difficulty in estimating such low abundant cell-type . Overall, correlation results are similar for the different sources of marker genes , and so we report the results with “Zhang” markers in the main text and “Darmanis” markers in the supplement. We note that we found removing technical confounds from gene expression data generally improves the accuracy of the tested algorithms  which provides evidence that there may be other technical factors such as sample quality or effectiveness of image analysis algorithms that are contributing to the moderate effect sizes.We observed that the correlations between IHC and deconvolution estimates are mostly significant, with moderate effect sizes, but variable results for endothelial cell proportions . The observed correlations between IHC and deconvolution estimates are thus reasonably within the expected range if noise in image analysis is considered.Although statistically significant, the magnitude of the correlations are only moderate and could have been reduced by technical variability. To approximate upper bounds for the correlation values, we constructed “artificial mixtures” from a brain single cell RNA-seq dataset. WithoutFig 2B), and are consistent with estimates based on single cell data from cortex[. To test the generalizability of the results, we further estimated cell-type proportions across nine brain regions from GTEx data[Fig 2B and 2C). For other brain regions, we observed strong variations in the estimated cell-type proportions, with adjacent regions tending to yield similar proportions , which demonstrates the stability of the computational estimations. Although not much is conclusively known about the variation in cell-type proportions across human brain regions, encouragingly, these estimates matched what was expected based on cell counts using a single-cell RNA-seq dataset[S9 Fig).In addition to evaluating the proportion estimates for each cell-type independently, we also assessed the estimates based on relative magnitude across cell-types. When averaged across samples, the relative magnitude across cell-types show high concordance between the IHC and deconvolution estimates S10 Fig). DSA was the only algorithm that provided significantly accurate absolute proportions for 3 out of 5 cell-types, as assessed by permutation tests , and interestingly single cell markers (“Darmanis”) generally yielded more accurate absolute proportions .In terms of MSE, which is a much stricter criterion for evaluation, we observed greater differences in performance between methods . While neurons are the most abundant cell-type in each assay, there are very few endothelial cells labelled in the snRNA-seq data. There are also very few astrocytes relative to oligodendrocytes. While this comparison does not make it clear which assay is more accurately identifying proportions, unlike the IHC data, none of the deconvolution algorithms are significantly correlated with the snRNA-seq proportions . Additionally, MuSiC[S11 Fig) neither method estimates absolute proportions well  suggesting that either the snRNA-seq data needs more cleaning, the cell markers used need to be optimized or the snRNA-seq is simply not appropriate for deconvolution in this case. These results indicate that this particular snRNA-seq dataset is not as useful for calculating proportions or informing specialized deconvolution algorithms for estimating proportions in the ROSMAP cohort.Cell-type proportions estimated using single-nuclei RNA Sequencing (snRNA-seq) data perform poorly in this study. Single-nuclei RNA Sequencing has previously been performed on 48 individuals from the ROSMAP cohort which prly, MuSiC and BSEQly, MuSiC are two Fig 4A), so long as genes that span multiple marker sets are filtered out . Second, we compared the neuronal sub-type proportions with the overall proportion of neurons, and found that the relative proportion of excitatory to inhibitory neurons is consistent with prior reports[Fig 4B). Overall, these results suggest that estimation of excitatory and inhibitory neurons is feasible from bulk-tissue RNA-seq. However, the availability of robust markers with cell-type specific expression is critical for accurate inference.Single cell and single nuclei transcriptomic studies of the brain have revealed notable heterogeneity in neurons, with a dozen different neuronal sub-types and states identified based on clustering of gene expression data. Althougr reports, and the, suggesting that the genes found without adjusting for cellular heterogeneity are likely false positives since their variance can be significantly explained away by variability in cell-type proportions. Supporting this, we observed significant correlations between amyloid levels and proportions of oligodendrocytes and neurons . Similar trend, but to a lesser degree, was observed for association with clinical AD.To assess the relevance of the estimated cell-type proportions in disease studies, we re-analyzed the ROSMAP dataset to identify genes whose expression levels are associated with Alzheimer’s disease (AD) and its related neuropathology, namely amyloid beta and tau proteins. By including the estimated proportions as confounding factors, we observed substantial reduction in the number of genes associated with amyloid beta .In addition to using the estimated cell-type proportions to correct for cellular heterogeneity in disease studies, another important application of these estimates is the discovery of cell-type specific genetic regulation of gene expression. Recent blood-based studies have shown the feasibility of inferring cell-type specific eQTLs from bulk-tissue gene expression data, so long as accurate estimates of cell-type proportions are available. To demonstrate the feasibility of this approach for brain, we performed cell-type-specific eQTL analysis with theHere we addressed the apprehension surrounding interpretation of gene expression measurements from post-mortem brain tissue by demonstrating that existing deconvolution algorithms can be used for estimating cell-type proportions in the brain. The application of these algorithms enable better utilization of the large number of existing well-annotated bulk post-mortem RNA-seq datasets to study brain-related disease and gene regulation.The benchmark dataset that we produced provides a resource that can be used to further optimize cell-type deconvolution algorithms for use in brain tissue. Cell-type deconvolution algorithms have predominately been developed and evaluated in whole blood, PBMC and tumors, where ssample and population level. For the ROSMAP cohort specifically, this provides support of the inferences that have been made associating proportion of cell-types in the prefrontal cortex of subjects with pathological burden or cognition[Our results indicate that cell-type deconvolution algorithms can be used to make inferences about cell-type composition at the cognition. We noteUnfortunately, single-nuclei sequencing data did not appear informative for inferring cell-type proportions. It is well accepted that single-cell sequencing technologies have varying levels of cell-type recovery for different cell-types with substantial optimization often required to detect cell-types of interest. ImagingFinally, we demonstrated the practical benefits of estimating cell-type proportions in the brain. Alzheimer’s disease and other neurodegenerative diseases have a substantial impact on brain structure and so observations of changes in gene expression in bulk cortical tissue are likely to be masked by changes in cell-type composition. We showed that including cell-type proportion estimates as confounding factors is imperative for reducing false association between gene expression and disease phenotypes. Further, we demonstrated that accounting for this confounding using estimates from cell-type deconvolution algorithms produced significant improvements in cell-type specific eQTL detection by using more accurate marker sets based on multiple genes. These results emphasize that estimates of cell-type proportions generated by deconvolution algorithms have the potential to increase the power, stability and interpretability of gene expression studies using brain tissue.During the dissection, one hemisphere is cut into coronal slabs and frozen in a −80°C freezer. The other hemisphere is placed in 4% paraformaldehyde. The frozen middle frontal gyrus (MF) has been used for RNA-seq while the paraffin-embedded fixed tissue from the exact same region, but other hemisphere has been dedicated for immunohistochemistry (IHC). The white matter has been removed from the grey matter of the frozen sample. For both RNA-seq and IHC, only the grey matter has been analyzed.Six μm sections of formalin-fixed paraffin embedded tissue have been stained for NeuN (Millipore), GFAP (Dako), Iba1 (Wako), Olig2 (Sigma) and PECAM-1  using antigen retrieval Buffer (Citrate Buffer pH 6.0) for each marker. Sections have been blocked with blocking medium containing 3% BSA and incubated with primary antibodies for overnight at 4oC. Sections have been washed three times with PBS before incubation with Fluorophore-conjugated secondary antibody (Thermofisher) for one hour and coverslipped with anti-fading reagent containing Dapi . Using fluorescence upright microscope (Zeiss Axio), 30 images have been captured in grey matter for each section at magnification x20 with a set exposure time in a systematic zigzag pattern to ensure that all layers of the cortex have been included in quantification.https://github.com/ellispatrick/CortexCellDeconv as well as the corresponding IHC images.EBImage was usedThree datasets were used to define marker genes and cell-type reference profiles. Cell-specific reference profiles were collected from single-cell RNA sequencing data (Darmanis) and RNA-The deconvolution algorithms in this study were applied to the 508 RNA-seq samples from ROSMAP cohort, processed as previously described. BrieflyCD31 wasn’t expressed in the gene expression data, CD34 was used as the gene marker for endothelial cells instead. See above for the details of the marker set selection approach and https://github.com/ellispatrick/ CortexCellDeconv for R scripts.In total, six cell-type deconvolution algorithms were applied to the data; CIBERSORT, dtangleTo assess the robustness and magnitude of correlations observed in our study, we compared these to an artificial mixture analysis. A single nucleus sequencing dataset from the Allen Brain Atlas was downS10 Fig). Given the small number of markers that were truly cell-type specific, we decided to combine markers to two sets: excitatory and inhibitory. Using the DSA algorithm, we estimated the proportion of 4 major cell-types as well as excitatory and inhibitory neurons simultaneously.We obtained markers for 7 neuronal sub-types, 2 excitatory and 5 inhibitory, from Darmanis dataset[We assessed the correlation between gene expression levels and each phenotype in two ways: a univariate model that associates gene expression and a single phenotype, and a multivariate model that includes additional covariates to adjust for cellular heterogeneity. The covariates are the predicted proportions of five major cell-types from the DSA algorithm with Zhang markers. The gene expression data was already adjusted for technical and biological confounding factors as previously described. The scay is a vector of gene expression levels, g is the genotype for the test SNP, c is the proportion of test cell-type, and g x c is the interaction term between genotype and the proportion of cell-type. The statistical significance of the interaction term, modeled by γ, implies the existing of a cell-type-by-genotype effect. As suggested by Westra and colleagues[cis xQTL (main effect)[We used the approach described by Westra and colleagues to identolleagues, to redun effect) using a n effect).All participants signed an informed consent approved by the Institutional Review Board of RUSH University.S1 Table(XLSX)Click here for additional data file.S1 FigCell-type proportions calculated from IHC data are compared between individuals with and without Alzheimer’s Disease (AD).(TIF)Click here for additional data file.S2 FigEach dot depicts an individual. Our previous study defined a set of modules with gene members that were enriched for each of the five cell-types examined : the average expression of each of these modules (across genes) represents a relative score for each individuals that can serve as a proxy for proportion of the corresponding cell-type. The module average expression is shown on the x-axis and the IHC-based proportions are shown on the y-axis.(TIF)Click here for additional data file.S3 FigScatter plots show the inferred and measured proportions for five cell-types across four different methods.(TIF)Click here for additional data file.S4 FigPlots show the pairwise correlation between pairs of deconvolution methods using the Zhang markers, assessed based on 508 samples.(TIF)Click here for additional data file.S5 FigFigure shows the performance of (A) DSA and (B) dtangle methods, based on different sources for marker gene set selection: scRNA-seq based markers (Darmanis), human cell sorted (Zhang) and mouse microarray and ISH (NeuroExpresso). Y-axis shows the correlation between the prediction and IHC across 70 ROSMAP samples. (C) Figure shows the correlation between all 4 methods and single gene markers, as inferred using “Darmanis” markers, with measured IHC data.(TIF)Click here for additional data file.S6 FigFor data from post-mortem brain, in addition to RNA integrity number (RIN), other technical factors such as PH and post-mortem interval (PMI) are known to have a major impact on the estimated gene expression levels. To assess whether correction for these variables impacts the accuracy of cell-type proportions, we applied deconvolution algorithms on “raw” and “adjusted” data see . Figure (TIF)Click here for additional data file.S7 FigFigure shows the achievable range of correlation coefficient on the simulated experiment. Artificial bulk gene expression data was created for deconvolution by sampling with replacement from a population of 1576 annotated cells from the human lateral geniculate nucleus and averaging. After a dispersion parameter was estimated from the ROSMAP cohort, these average profiles were resampled from a negative binomial distribution to emulate technical and biological noise. These pseudo-bulk profiles were then deconvolved using the original single nucleus sequencing data and the estimated proportions were compared to the truth.(TIF)Click here for additional data file.S8 Fig(A) Population-level range of prediction of absolute proportions with variable size of marker gene sets based on Darmanis markers. (B) Correlation between prediction of cell-type proportions with variable sizes of marker gene sets. Differential expression analysis using single cell data was used to define marker gene sets.(TIF)Click here for additional data file.S9 FigFigure summarizes the proportion of nuclei assigned to various cell-types . A: astrocytes; E: endothelial cells; M: microglia; N1,N2,N3,N4: different neuronal populations; O: oligodendrocyte; OP: oligodendrocyte progenitor cells.(TIF)Click here for additional data file.S10 FigMean squared error (MSE) quantified across 70 individuals, using (A) Zhang and (B) Darmanis markers as input to deconvolution algorithms. STD refers to the standard deviation of the IHC measurements for each cell-types. (C-D) Figures show the significance  for the estimated MSE, as assessed by permutation tests using 10000 permutations, where Zhang (C) and Darmanis (D) markers are used.(TIF)Click here for additional data file.S11 FigThe Spearman correlation coefficient between IHC derived cell-type proportions and six deconvolution algorithms. Included here are two methods, MuSic and BSEQ-sc. Both of these methods are designed to use single-cell sequencing data as a reference set to deconvolute bulk expression. Here they are using the ROSMAP snRNA-seq data as a reference to deconvolute the ROSMAP bulk gene expression data.(TIF)Click here for additional data file.S12 FigIn order to assess the performance of deconvolution of cell-type subsets we obtained markers of excitatory and inhibitory, neurons from reported by Darmanis et al. These sub-type markers were not specific to neurons (A) and so they were filtered to those that were specifically highly expressed in neurons in the Darmanis data (B).(TIF)Click here for additional data file.


Pain is prevalent and associated with adverse outcomes in older adults. The moderating effect of pain on cortical control of locomotion has not been assessed. This study examined the effects of subjective pain on changes in fNIRS-derived oxygenated hemoglobin (HbO2), gait velocity, and cognitive accuracy from single to dual-task walking conditions among older adults. Participants  were community-residing older adults. Participants completed two single tasks [Single-Task-Walk (STW) and Cognitive Interference (Alpha)] and the Dual-Task-Walk (DTW), during which participants performed the two single tasks simultaneously. The MOS- PSS and PES were used to assess pain severity and interference. Gait velocity was assessed with an electronic gait mat, cognitive accuracy was measured using the rate of correct letter generation. Linear mixed effects models revealed that HbO2 increased in DTW compared to STW (p < .001) and Alpha (p < .05). The presence of perceived pain was associated with an attenuated increase in HbO2 from Alpha to DTW (p < .05). Amongst those with pain, worse pain severity was associated with an attenuated increase in HbO2 from STW to DTW (p < .001). Pain interference did not moderate the increase in HbO2 from single to dual tasks. Pain did not have a moderating effect on behavioral outcomes. These results indicate that task-related changes in the hemodynamic response in the prefrontal cortex during walking may be a sensitive marker to the effects of subjective pain on brain function in older adults.


During aging, alterations of the intestinal microbial ecosystem can occur contributing to immunosenescence, inflamm-aging and impairment of intestinal barrier function . In the context of a diet-microbiota-IP axis in older subjects, food bioactives such as polyphenols may play a beneficial modulatory role.MaPLE is a project centered on a randomized, controlled cross-over dietary intervention trial [polyphenol-rich diet (PR-diet) versus control diet (C-diet)] targeted to older people (≥ 60 y) living in a well-controlled setting (i.e. nursing home). The 8-week interventions are separated by an 8-week wash-out period. Three small portions per day of selected polyphenol-rich foods are consumed during intervention in substitution of other comparable products within the C-diet. Biological samples are collected before and after each treatment period to evaluate markers related to IP, inflammation, vascular function, oxidative stress, gut and blood microbiomics, metabolomics. A sample size of 50 subjects was defined based on IP as primary outcome.Evidence that increasing the consumption of polyphenol-rich food products can positively affect intestinal microbial ecosystem resulting in reduced IP and decreased translocation of inflammogenic bacterial factors into the bloodstream will be provided. The integration of data from gut and blood microbiomics, metabolomics and other IP-related markers will improve the understanding of the beneficial effect of the intervention in the context of polyphenols−microbiota−IP interactions. Finally, findings obtained will provide a proof of concept of the reliability of the dietary intervention, also contributing to future implementations of dietary guidelines directed to IP management in the older and other at risk subjects.ISRCTN10214981); April 28, 2017.The trial is registered at ( Age-associated changes significantly compromise health status and increase the risk of chronic diseases. Within these modifications, recent research has been focusing on those that specifically occur at the gut epithelium level with impact on intestinal immune homeostasis and related systemic responses . The maiThe intestine acts both as a barrier (to keep harmful substances out of the body) and as a selectively permeable surface that allows the controlled passage of substances from the gut lumen through the gut wall and into the body. This controlled flux across the intestinal wall is known as intestinal permeability (IP) . InapproIncreasing evidence suggests that dietary patterns can represent a relevant factor in shaping the intestinal microbiota and modifying the relative abundance of specific bacterial taxa –13. ConsOlder subjects are often characterized by alterations of the intestinal microbial ecosystem , 19, whiIn the context of a diet-microbiota-IP axis, food bioactives may have a key role in regulating the numerous interconnected processes involved. Particularly, polyphenols exert antioxidant, anti-inflammatory/immunomodulatory properties at intestinal and systemic levels, and there is increasing mechanistic evidence suggesting their potential to modulate IP , 22. In The aim of the MaPLE project (Microbiome mAnipulation through Polyphenols for managing Leakiness in the Elderly) is to evaluate the hypothesis that an increased intake of polyphenol-rich foods can reduce IP and lower inflammogenic bacterial factors in the bloodstream promoting an overall protective/beneficial metabolic phenotype in older subjects. Three approaches have been taken; the main study, a dietary intervention randomized controlled trial described here, combined with pre-clinical studies in an animal model of aging to test the impact of the polyphenol-rich diet on IP associated markers, and also in cultured human intestinal cells (caco-2) to investigate the capacity of single polyphenols to modulate IP.The MaPLE RCT is a single-blind, randomised, controlled, cross-over trial [polyphenol-rich diet (PR-diet) versus control diet (C-diet)] in older people (≥ 60 y) living in a nursing home. Each intervention period consists of 8 weeks and is separated by an 8-week wash-out period in which participants consume their habitual diet to avoid carry-over effects.The PR-diet and C-diet were developed to provide adequate and comparable levels of energy and nutrients. The PR-diet was achieved by replacing three portions per day of low polyphenol foods/beverages with specific polyphenol-rich foods/beverages (as detailed below). During the study, subjects are asked to fast overnight before each scheduled time-point of blood, urine and feces collection. In addition, daily menus and weighted food records (WFRs) are collected throughout the trial. An overview of the study design is represented in Fig. ISRCTN10214981).The trial has been prospectively registered ; analyses and data elaboration are still ongoing.The intervention has been performed at Civitas Vitae  which hosts a large number of older people living in residential care buildings or in independent residences located in the same area, depending on individual willingness and level of disabilities. The OIC Foundation provides several facilities and dedicated area for meal preparation. This allows to collect accurate information with regard the composition of the diets from the recipes used for each of the foods in the meals delivered daily to the participants. We were able to accurately assess food intake using weighed food records in the intervention study.Before recruitment, a meeting with the medical staff and nurses’ coordinators at OIC Foundation took place in order to present and widely discuss the aim, methodologies and technical aspects related to the development and the management of the MaPLE RCT. After this meeting, several formal presentations of the project aim and some general information on the intervention planned were organized at OIC Foundation for the hosts and their families. Finally, an accurate evaluation of the host characteristics was performed in collaboration with the physicians/geriatricians and nurses’ coordinators to pre-select based on the verification of the main inclusion and exclusion criteria (see below) and to identify plausible candidates for the study. Subjects who were interested in participating in the study signed an informed consent reporting all the information on the dietary intervention, the analysis and protocols that they were asked to undertake/follow.More specifically, volunteers were selected according to the inclusion and exclusion criteria reported below:Age ≥ 60 yearsAdequate nutritional status evaluated with Mini Nutritional Assessment (MNA), score ≥ 24Good cognitive status tested with Mini Mental State Examination (MMSE), score ≥ 24Self-sufficiency assessed with validated tests Increased intestinal permeability evaluated by serum zonulin levelCeliac diseaseSevere liver disease with cirrhosisSevere renal insufficiency Presence of severe Chronic Obstructive Pulmonary Disease  or severe cardiovascular disease (heart failure class III or IV NYHA - New York Heart Association)Antibiotic treatment in the last monthMalignant tumor that required treatment in the previous 2 yearsEach subject enrolled has been assigned to an ID number. The encoding of samples is hidden to both the investigators and the participants. All clinical and personal data, including the biological samples, of the subjects involved in the study are collected and stored anonymously.In order to define the polyphenol-rich dietary protocol, an initial estimation of nutrient and total polyphenol intake was performed through the analysis of the daily menu provided at the OIC Foundation.Subsequently, an identification of the specific polyphenol-rich food products to be included in the diet was carried out in order to consider not only the amount and contribution of the different polyphenols but also the food preparation in order to ensure their bioavailability. In addition, an evaluation of conditions to enable optimal texture (e.g. considering the use of purées instead of the whole product) and an assessment of the product acceptability by the target population was also undertaken.The polyphenol-rich dietary protocol (PR-diet) was finally developed by including in the C-diet 3 portions per day of the following selected polyphenol-rich foods: berries and related products, blood orange, pomegranate, green tea, Renetta apple, and dark chocolate.A schematic plan of the type and serving sizes of polyphenol-rich products consumed daily in the PR-diet is shown in Table Even though no reports of adverse effects due to a polyphenol-rich diet had been registered or reported in the literature, subjects were advised to annotate and communicate any adverse symptom perceived during the intervention period. Since green tea was selected within the polyphenol-rich food sources to be used in the intervention study, there was a comprehensive discussion to define the dose to use. Green tea extract is a rich source of epigallocatechin-3-gallate (EGCG) known for many different protective effects; however, the intake of very high doses of EGCG/green tea extracts as supplements has been reported to cause liver toxicity. Recently, it has been proposed an EGCG upper level (UL) based on human intervention studies of 300 mg EGCG/day in healthy adults . The proFood intake before (enrollment phase) and during the intervention periods was recorded through the evaluation of OIC Foundation daily menus and the use of WFRs. The daily menus, covering different seasons, were analyzed to quantify nutrient and polyphenol content. Moreover, the day before each time-point, a WFR was completed and both nutrient and polyphenol intake were estimated. At least 3-WFRs were completed during each intervention period. Daily menus and WFRs were assessed using MetaDieta®  to estimate energy and nutrient intake. Total polyphenol estimation was performed by using the Phenol Explorer database (phenol-explorer.eu) to provide estimates of polyphenol concentrations in each food, and where there were no useful values, using our proprietary data or values obtained from the literature. Total polyphenol content of the foods was estimated directly using the Folin-Ciocalteau method .Blood, urine and fecal samples were collected at each time-point as defined in Fig. In addition, a brush was used to collect an oral mucosal sample from each participant for further evaluation. The brush with the collected tissue was stored in a cryovial containing a buffered saline solution, which was immediately frozen.The primary selected outcome of the study was zonulin as an IP marker, whereas other IP related markers , inflammatory markers , oxidative stress and vascular function markers , metabolomics and microbiomics (16S rRNA gene quantification and taxonomic profiling) were included as secondary outcomes to support and validate our study hypothesis.Body weight, height and BMI calculation were assessed at the beginning and the end of each intervention period following the international guidelines of Lohman et al. .Each participant was monitored at the beginning and the end of each intervention period measuring both systolic and diastolic pressure obtained in a resting, seated position following the validated JNC 7 guidelines .At enrollment and at each time-point, metabolic and functional parameters  were assessed by a standardized validated protocol, using an automatic biochemical analyzer . Low density lipoprotein cholesterol (LDL-C) concentration was estimated using the Friedewald formula , while nIntestinal permeability was evaluated by quantifying serum zonulin concentrations. Human zonulin is a protein (i.e. prehaptoglobin-2) released by enterocytes able to promote the activation of the signaling transduction pathway that cause tight junction protein disassembly enabling potential bacterial factor translocation . In thisThe concentrations of several markers related to inflammatory processes were quantified using specific ELISA kits . CRP (DCRP00), IL-6 (HS600B), TNF-α (HSTA00E) were quantified in serum at the beginning and the end of each intervention periods.In order to assess vascular function, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were quantified in serum samples at each intervention time point using an ELISA kit .The levels of endogenous and oxidatively-induced DNA damage, as markers of oxidative stress, were assessed in PBMCs by the comet assay. The samples are collected before and after each intervention period. Levels of endogenous DNA damage were assessed using a specific enzyme  that can be used to detect 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and ring-opened formamidopyrimidine nucleobases. Oxidatively-induced DNA damage was measured by treating the cells with hydrogen peroxide and by evaluating the capacity of cells to counteract an oxidative insult. Both Comet assay protocols have been previously described by Del Bo’ et al. .Bacterial DNA quantification and sequencing reactions were performed by Vaiomer SAS  using optimized blood-specific techniques as described earlier –43. SpecAll of the following steps were performed in-house at QIB. Fecal samples were weighed into Lysing Matrix E bead beating tubes  and extraction was completed according to the manufacturer’s protocol for the FastDNA™ SPIN Kit for Soil (MPBio) but extending the bead beating time to 3x60s. DNA was quantified using a Qubit® 2.0 fluorometer , normalized to 5 ng/μl and the V3/V4 region of the 16S rRNA was amplified using the primers detailed below. Sequencing was performed using a 600 cycle MiSeq v3 reagent kit  giving approximately 100,000 reads per sample.Bioinformatic analysis was conducted using VSEARCH ; reads wPrimers:16S 341F – TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG.16S 806R – GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.In addition, taxonomic profiling was carried out through shotgun sequencing. In brief, metagenomic DNA isolated from fecal samples was sequenced using an HiSeq instrument  by CosmosID . Microbial community composition was determined through the analysis of shotgun metagenomic datasets with the CosmosID metagenomic software as previously described .Urine samples collected prior and after each intervention period were subjected to targeted metabolomics analysis by applying the Quantitative Dietary Fingerprinting approach recently developed by González-Domínguez et al.  with thep-value adjustment is performed using the Hochberg-Benjamin correction. Significance is set at P ≤ 0.05; significance in the range 0.05 < P < 0.10 is accepted as trend. Potential gender differences will be also considered in all the analyses.According to data literature , 52 it wThere is growing evidence of a link between IP impairment and increased inflammation . Since aBy considering this premise, the MaPLE RCT here described aimed to investigate whether a PR rich-diet can improve the intestinal microbial ecosystem of older subjects characterised by an increased IP. In addition, it is hypothesized that such modulation could promote an overall beneficial impact on IB function, a decreased IP and translocation of inflammogenic bacterial factors in the blood.The development and management of well-controlled and adequately balanced dietary intervention studies is not an easy task and it becomes even more difficult when the target population is older subjects. Consequently, the first task of the project was dedicated to the optimization of the trial in order to overcome the possible problems related to compliance with the dietary instructions and to other relevant potential confounding factors (e.g. periods of illness or the use of drugs that may be relevant in this target group). For this reason, the MaPLE RCT was planned in a residential area for older people, since it provided a favourable and controlled environment in which it was possible to optimize and standardize most of the important experimental conditions. For example, since outcome data from dietary intervention studies are prone to being affected by individual differences in diets and lifestyle behaviour over time (e.g. during the two eight week periods of dietary intervention), we were able to ensure both strict compliance with the dietary intervention and a consistent dietary pattern among participants by including the polyphenol-rich products in their usual meals provided by the residential home. In addition, the selection of polyphenol-rich foods was based on three important considerations: (i) That the types of foods selected were largely universally liked, (ii) that the texture of the selected products was suitable for older subjects , and (iii) that the portion of food would reliably provide a high dose of polyphenols. In addition, weighed food intake was also assessed to provide us with data to allow accurate estimates of actual nutrient and polyphenol intake in the two periods of treatment (PR- and C- diet). This allowed a high degree of control and substantially reduced between treatments differences.As regard the primary outcome, serum zonulin concentrations were used as the marker of IP because of the low reliability and applicability of the multi-sugar test in the older population (i.e. due to a high rate of incontinence amongst the elderly participants and the need for adherence to a strict dietary protocol before the test) .It is also noteworthy that the MaPLE RCT is testing, for the first time, the hypothesis that a dietary intervention may modulate quantitatively the bacterial DNA in bloodstream and qualitatively the blood microbiota composition. This should provide further evidence of the impact of the dietary intervention on IP being potentially associated with a reduction in translocation of bacterial factors. Other objectives of the MaPLE RCT are to integrate microbiota profiling data with inflammation and metabolomics data to improve understanding on the impact of the dietary intervention. In addition, the inter-individual response to the treatment will be investigated and food metabolite profiling data will be exploited for the identification of a set of potential biomarkers with relevance in the context of preventing or treating impaired IP.Finally, results will be pivotal for the development of new dietary approaches and guidelines for managing IP related conditions in the complex context of healthy aging.


Objective: To describe the clinical efficiency of transluminal Nd:YAG laser embolysis (TYE) and hyperbaric oxygen (HBO) as an off-label combined treatment for branch retinal artery occlusion (BRAO) with visible emboli.Methods: A 77-year-old woman had a history of seeing a “shadow” in the lower visual field of the left eye for three days. Platelet-fibrin embolus at the arterial bifurcation was disintegrated by TYE technique and the patient was referred to HBO treatment for 20 sessions.Results: One week after treatment, best-corrected visual acuity improved to 0.8 while a good arterial blood flow in the affected branch was seen. Platelet fibrin plaques had disappeared at fundus, and the pale appearance in the retina had decreased. Conclusions: TYE and HBO combination treatment may be an effective and feasible treatment for restoration of blood flow and vision in BRAO cases caused by visible platelet-fibrin emboli.  Branch retinal artery occlusion (BRAO) may result from obstruction with platelet-fibrin emboli, cholesterol plaques or calcific emboli, typically located at the arterial bifurcations or areas of narrowing. Patients with BRAO present with sudden loss of vision. Fundus examination usually reveals a sectorial superficial whitening and a visible intralumenal embolus . BRAO coDue to the visual impairing nature of this condition, an effective emergency treatment capable of reversing the visual loss is needed. Considering prior studies, TYE and HBO as an off-label combined treatment was planned for a delayed BRAO case. Herein, we aim to report the clinical efficiency of TYE and HBO combined treatment for BRAO with visible emboli.A 77-year-old woman presented to our clinic complaining of vision loss for three days in the inferior field of her left eye. She had well-controlled atrial fibrillation and hypertension in her systemic history. She also had a history of cataract surgery in both eyes with no complications. In the examination, best-corrected visual acuity (BCVA) was 0.9 in the right eye and 0.3 in the left eye on the Snellen scale. Intraocular pressure (IOP) was 14 mm Hg and 12 mm Hg in the right and left eye respectively, and both eyes were pseudophakic. Dilated fundus examination revealed a superior temporal artery branch occlusion with retinal paleness in the upper region of the macula, with a visible transluminal platelet-fibrin embolus at the arterial bifurcation, and numerous other small fibrin emboli scattered along the arterial branch  can be listed as other treatment options . Some stThe results of our case revealed the satisfactory visual improvement following TYE and HBO treatment in patients with BRAO that have poor initial VA. Even if TYE and HBO combination treatment is an off-label modality, it may be considered in BRAO cases caused by visible platelet-fibrin emboli. It may be a fast, effective and feasible treatment for restoration of blood flow and vision.The authors declare that they have no competing interests.Detailed informed consent has been obtained from the patient.


Viral encephalitis, an inflammation of the brain parenchyma caused by virus infection, poses a serious threat to global public health. The major causal agents worldwide are herpes viruses and arboviruses. HSV-1 is the leading cause of adult encephalitis in developed countries. While Japanese encephalitis is the most common encephalitis worldwide, West Nile virus is the most widespread virus. Recently, Zika virus, chikungunya virus, and Dengue virus have co-circulated in many regions of the Americas Silva, .Despite the advance in studying the complex interplay between viruses and infected cells, the pathogenesis of viral encephalitis is largely unknown. For example, the regulation of blood brain barrier (BBB) permeability and the regulation of immune responses to virus infection in the CNS remain to be explored.This Research Topic combines 10 publications from 64 authors, including 3 review articles and 7 research articles, covering many aspects of RNA and DNA virus-associated encephalitis.Chen B-S et al. and Majer et al. provide readers a convenient way to understand the most up-to-date knowledge of non-polio, enterovirus-associated encephalitis. These two articles cover all aspects of recent advances in enterovirus-induced encephalitis including the routes of CNS infection, tropism, virulence, immune response, and molecular pathogenesis. These two reviews provide insightful information for the development of effective antiviral strategies and vaccines.The two in-depth reviews by Chen J. et al. described the epidemiological and phylogenetic analysis of the very first large-scale Echovirus 30  outbreak in northwest China in 2015. The study shows children aged 6–15 years are more susceptible to E-30 infection. The highly similar E-30 genomes in this outbreak suggested an aggregate outbreak of E-30. The dominant lineage has a complex genetic transmission that indicates the infection initiated from coastal provinces of China and then spread to other parts of the country. The study provides valuable information for future surveillance.Giannitti et al. characterized the first case of bovine astrovirus-associated encephalitis in Uruguay (South America). Phylogeographic analysis suggests the virus was introduced to South America from Europe and later spread to North American and Japan. The study advances the understanding of the geographic distribution and genetic diversity of astroviruses.Globalization promotes virus transmission and disease spreading among continents. The ongoing COVID-19 pandemic is a devastating example  restricting virus replication in West Nile virus (WNV)-induced encephalitis. WNV is the leading cause of viral encephalitis in the United States. ZBP1 plays an essential role in triggering robust immune responses. WNV infection dramatically up-regulates ZBP1 expression in mouse brain and primary mouse cells. Deletion of ZBP1 results in higher morbidity and mortality after WNV infection in mice. Infection of ZBP1–/– mice with the virus is lethal, indicating that ZBP1 is required for survival after WNV infection.The vast majority of papers in this Research Topic are related to Flavivirus-associated encephalitis with distant emphasis on different viruses. Tomar et al. reported the development of a polymerase spiral reaction assay for real time detection of WNV from clinical samples. As there is no FDA-approved vaccine or antiviral against WNV, early diagnosis of WNV infection is critical for clinical management and disease control. The reverse transcription polymerase spiral reaction (RT-PSR) assay rapidly and accurately detects the envelope gene of WNV using real-time turbidimeter or visual detection by the addition of SYBR Green I dye. The assay is validated and is able to detect as low as one copy of RNA, which is 100-fold higher than conventional RT-PCR. The assay has a potential to be used for rapid testing of a large number of clinical samples.Chiu et al. used human placenta trophoblasts cells (JEG-3) and human brain-derived endothelial cells (hCMEC/D3) as in vitro models to study the mechanisms ZIKV uses to cross the physiological barriers. The study shows ZIKV infection changes the permeability of JEG-3 cells by disrupting the tight junction and by transcytosis. ZIKV crosses the BBB by transcytosis.Zika virus (ZIKV) is another important member in the Flavivirus family that causes severe neurological complications such as meningoencephalitis in adults and microcephaly in fetus  mice and in RIPK3-knockdown neuro2a cells with a significantly increased level of interferon (IFN)-induced protein 44-like gene (IFI44L). Over-expression of IFI44L decreases JEV replication in neuro2a cells and double knockdown of RIPK3 and IFI44L promotes virus replication. On the other hand, RIPK3 inhibits IFI44L expression to promote JEV propagation in neurons.Japanese encephalitis virus (JEV) is the major cause of viral encephalitis worldwide. JEV infection targets neurons and receptor interacting serine/threonine-protein kinase 3 (RIPK3), which contributes to neuron inflammation and death. Oladunni et al. reviewed the discovery of the virus, the latest developments of treatment and disease control, and summarizes recent advances in the research of EHV-1 pathogenesis. The information presented is useful for development of strategies to limit the spread of EHV-1 in equine populations.DNA virus infection can induce encephalitis as well. Equine herpesvirus-1 (EHV-1) is one of the most important pathogens of horse and causes a constant threat to the equine industry worldwide. There are increasing numbers of devastating equine herpesviral myeloencephalopathy outbreaks. Qi et al. used a fosmid library as a platform and Red/ET recombination technology to generate recombinant PRV fused with EGFP. The rescued recombinant virus is used to monitor retrograde and anterograde moving in the axon, making it a powerful tool for neuronal circuit analysis. The study is helpful for development of vaccines against PRV and other herpesviruses.Pseudorabies virus (PRV) is a member of the Herpevirus family. We hope that this Research Topic provides readers a better understanding of virus-associated encephalitis. A deeper understanding of viral encephalitis will facilitate the development of innovative antiviral approaches.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


High-frequency oscillatory ventilation (HFOV) is used in our unit for any type of PARDS when the patient meets specific criteria as outlined in our manuscript . Our data confirmed that it was possible to do this in all patients, irrespective of age  and tolerance of high FiO2 instead. However, the best strategy to optimise CMV in children with severe PARDS remains uncertain [We agree that in a subgroup of patients in our cohort, especially those with mild-to-moderate PARDS optimisation of conventional mechanical ventilation settings might have been attempted. The median OI of 38 as pointed out by the reviewer is the OI ncertain . To datencertain . We alsoncertain . Hence, http://www.prospect-network.org) on patient outcome [We eagerly await the results of a 2-by-2 factorial randomised controlled trial comparing the effects of ventilation strategy (CMV vs HFOV) with or without prone positioning ( outcome .


Our OPA is a much simpler setup than either the “gold-standard” laser and optical parametric oscillator (OPO) combination traditionally used for CARS imaging, or the more recently developed OPA systems pumped with femtosecond pulses [1]. Rapid and accurate tuning between resonances was achieved by changing the poled channels and temperature of the periodically-poled lithium niobate (PPLN) OPA crystal together with the OPA seed wavelength. The Pump-Stokes frequency detuning range fully covered the C-H stretching band used for the imaging of lipids. By enabling three multiphoton techniques using a compact, synchronization free laser source, our work paves the way for the translation of label-free multi-photon microscopy imaging from biomedical research to an imaging based diagnostic tool for use in the healthcare arena.We demonstrate a continuous wave (CW) seeded synchronization-free optical parametric amplifier (OPA) pumped by a picosecond, 1 µm laser and show its performance when used as a simple yet powerful source for label-free coherent anti-Stokes Raman scattering (CARS), concurrent second harmonic generation (SHG), and two-photon fluorescence microscopy in an epi-detection geometry. The average power level of above 175 mW, spectral resolution of 8 cm Despite the many attractive features of the spectroscopically specific CARS imaging modality, its uptake is restricted by the cost, size and complexity of the ‘gold-standard’ synchronously pumped OPO systems currently used . To addrCARS is label-free and has significant advantages including chemically specificity and z-sectioning capability compared to the prevalent fluorescence based one-photon imaging methods. It also offers significantly faster signal acquisition times compared to Raman spectroscopy ,7. In thCARS is a nonlinear optical process and requires pulsed lasers at a pump frequency and at an offset Raman-Stokes frequency. The anti-Stokes signal is generated when the pump and Stokes are offset by the Raman resonance frequency of the molecule of interest. In order to efficiently drive the vibrational coherence and thus to achieve rapid image acquisition, CARS requires incident pulses of pump and Stokes be temporally and spatially overlapped when tightly focused into the sample. In general, pulses of a few ps duration are optimal for CARS bio-imaging because they have bandwidths that match well with the Raman resonance peak in typical cellular structures and yet still provide the high peak powers needed to efficiently excite this nonlinear process. Due to the technical complexity, high cost and large footprint of such bulk laser based OPO sources, a dedicated optics laboratory is normally required for CARS microscopy, which prevents it from being more widely used. There is an urgent need for a simplified laser system that will provide high performance as needed for CARS but in a small and robust form while at low cost.A notable recent development was the report of a femtosecond crystal-based laser pump and a CW-seeded PPLN-based OPA that provided high gain and was even sufficiently stable for SRS microscopy without a balanced detection system . The 152In our work, we incorporate several aspects of that crystal-based OPA system design , but simWe found that in addition to providing good CARS imaging contrast, the OPA also enabled concurrent SHG and TPEF imaging. The powers and beam quality were both well optimized, enabling us to obtain strong signals in the backward direction compared to the signal beam (epi-detection) rather than using forward detection, which is easier due to the strong forward signal, but requires thin samples to allow the signal to be collected in that direction. Hence the system is suitable for imaging clinical-diagnostics-relevant and simple-to-prepare thick samples. The system was tested initially on chemically pure beads to verify the chemically specificity enabled by CARS-spectroscopy. Then, by imaging mouse adipose tissue with epi-detection, the multimodal capabilities were shown in bio-images combining concurrent CARS, SHG and TPEF signals detected in 3 separate output channels on our inverted microscope platform.2.The multimodal CARS laser source and microscope system is shown in achieved . The speThe output signal of the OPA was separated from the residual pump and was focused into a second PPLN crystal for SHG, which provides the pump beam for CARS imaging. The SHG PPLN crystal had a length of 10 mm and 5 grating periods from 3.3.1We first tested the OPA performance as a function of pump power and seed power. When the seed laser was set with wavelength of 1594.4 nm and output power of 8 mW, the minimum pump needed for gain to be measured was 2.7 W and the OPA output power increased with pump power beyond this reaching 400 mW at the maximum pump power of 3.9 W, as shown in We used the tunable laser source (TLS) to demonstrate the wavelength flexibility of the OPA. Note that we also did experiments with a much less costly 1592 nm wavelength telecoms laser diode with similar power to the TLS and temperature-tuned the diode wavelength by up to 6 nm and achieved similar output power and pulse characteristics from the OPA as when using the TLS.  This verified that, in principle, the costs of the system could be reduced and the tuning range maintained by using a switchable array of telecoms seed diodes with the wavelengths selected to target different molecular vibrational frequencies in the bio-samples of interest.3.2We next tested the chemical specificity and imaging ability of our system by recording the CARS spectra and microscopy images of 40-µm diameter polystyrene beads. The SHG of the OPA output was tuned from 778 to 803 nm enabling the CARS anti-Stokes signals shown in The spontaneous Raman reference spectrum for polystyrene was recorded with a home-built Raman micro-spectrometer system utilizing a Shamrock303 spectrograph with an Andor iDus 420 camera as the detector and a 785 nm excitation laser. The spectrum was acquired with a single 10 second exposure  imaging in an epi-detection configuration. Biological samples were prepared by excising tissues from culled mice. All procedures were carried out in accordance with the Animals (Scientific Procedures) Act 1986 set out by the UK Home Office. Female C57BL/6 mice aged between 4-6 months were culled via Representative images for adipose tissue from mice are shown in Images are acquired with a Pump-Stokes frequency detuning of 2845 ffolding , (detectde (FMN) ). These de (FMN) . We veride (FMN)  and our de (FMN) . Figure de (FMN)  through Currently tissue diagnostics makes use of colored stains on thin tissue slices  which are suited for the transmission images recorded so that samples require very careful preparation and sectioning. The ability to use label-free imaging on clinically relevant thick tissue samples for diagnostic applications  would be greatly simplified if the epi-detection became viable. To highlight this capability, we show in The axial depth of focus is expected to be different for CARS (three-photon process) and SHG/TPEF (two-photon processes): However, with the 1 µm z-resolution of our microscope stage we did not observe any significant effect. Overall, the ability to concurrently generate high contrast images with multiple modalities using the new OPA based laser source should make it a valuable tool for bio-science users. The epi-detection capability with an OPA-based setup demonstrates the relevance to biological and clinical samples without the need for extensive sample preparation which would be required to create the thin sections normally used with forward detection.4.In summary, we show the demonstration of a compact and reliable, tunable, CW seeded synchronization-free PPLN OPA with a robust, commercial ps pump laser. This laser and OPA combination is shown to be well suited for label-free CARS and concurrent SHG and two-photon fluorescence microscopy in an epi detection geometry. Rapid and accurate tuning between Raman resonances was achieved by changing the channels and temperatures of OPA and SHG crystals concurrently with tuning of the CW OPA seed wavelength. The corresponding pump-Stokes frequency detuning range was from 2700 to 3200 By combining elements of multiphoton techniques in an inexpensive and compact configuration, our work paves the way for the simple implementation of multimodal CARS with concurrent SHG/TPEF imaging capability. Power level, spectral resolution and dwell times are all comparable with previous reports , while u