The usage of modified asphalt binder in road pavements has been increasing in the past few decades. Therefore, quality control and understanding of modified asphalt binders have become an important issue. This study was conducted as a part of a larger study on the efficient management of these modified asphalt binders by evaluating the characteristics of asphalt binders mixed with styrene–butadiene–styrene (SBS), ethylene-vinyl acetate (EVA), and wax-based warm-mix additives using Fourier transform infrared (FTIR) analysis. For original asphalt binders modified with SBS, response wavenumbers were 700 and 966  Asphalt pavements consist of aggregate, asphalt binder, and air voids; they are a viscoelasticity material affected by loading time and temperature. Generally, asphalt binder is a complex material composed of carbon, hydrogen, nitrogen, and sulfur . The agiPolymer is a material that can improve the properties of asphalt binder and extend the service life of asphalt pavement, and styrene–butadiene–styrene (SBS) is the most widely used type of polymer in asphalt pavements. SBS increases viscosity and improves both elasticity recovery and strain resistance due to its elastic properties . In addiEthylene-vinyl acetate (EVA) is a polymer-based thermoplastic material similar to low-density polyethylene (LDPE) and plasticized polyvinyl chloride (PVC) . EVA is Until recently, most pavements have been constructed using hot mix asphalt, but the use of warm-mix asphalt is increasingly common nowadays because of budget restrictions and environmental conservation. Warm-mix asphalt reduces energy emissions by lowering the temperature during asphalt mixture production . In addiThe study focused on analyzing different types of modified binders and their characteristics using FTIR. Various types of additives were used. For FTIR test, four different types of modified asphalt binder were used. Two types of modified asphalt binder were PG 82-22 (PMA) and 76-22 (PMA) modified by SBS, a commercial polymer. The other modified binders used to analyze the rate of addition of warm-mix additives and to evaluate EVA-modified binders were produced by the process shown in FTIR analysis is a measurement method based on the principle that the molecules that make up a given material molecules structure absorb energy or radiation of a defined wavenumber. FTIR is widely used in many industries related to chemical bonding, such as medicine and food, because it is better suited for the analysis of molecular structure more than any other analytical technique. Typical analysis techniques include the diffuse reflectance inspection (DRIFTS) technique, which analyzes powdered type substances, and the roughing angle inspection specification (RARS) technique, which requires thin film and detailed sample preparation . In the 2 when aggregates and binders are heated at temperatures above 140 °C [Various types of additives have been developed to improve the performance of asphalt. These additives have the advantage of improving resistance to rutting, low-temperature cracking of asphalt pavement, which can extend the service life of a pavement . Styreneystyrene . In addi=O bonds . Hot mixe 140 °C . In the e 140 °C . A numbeThis study was conducted to verify the detectability of the additives discussed earlier in an asphalt binder using FTIR analysis. Furthermore, the additive content of PMA can be roughly inferred by deriving the ratio of additives’ particular peak heights to the particular peak height of the original asphalt binder . In thisSBS-modified asphalt binders with improved performance showed peaks at 700 FTIR analysis of EVA-modified binders revealed that SBS and EVA showed almost identical peaks but at wavenumbers of 1242 According to FTIR analysis of binders modified with warm-mix additives, the peaks appear to be in the 730 This study was conducted to evaluate differences between modified and original binders using FTIR analysis. It was concluded that FTIR analysis constitutes a viable identification method for asphalt binders modified by additives for properties improvement. Further research is needed on other types of additives, as well as to assess the field applicability of FTIR analysis by investigating chemical bond changes under aging conditions.This study was conducted to evaluate the properties of asphalt binders modified with several types of additives using FTIR analysis. Its overall conclusions are as follows:


Because of its simplicity, reliability, and replicability, the Masquelet induced membrane technique (IMT) has become one of the preferred methods for critical bone defect reconstruction in extremities. Although it is now used worldwide, few studies have been published about IMT in military practice. Bone reconstruction is particularly challenging in this context of care due to extensive soft-tissue injury, early wound infection, and even delayed management in austere conditions. Based on our clinical expertise, recent research, and a literature analysis, this narrative review provides an overview of the IMT application to combat-related bone defects. It presents technical specificities and future developments aiming to optimize IMT outcomes, including for the management of massive multi-tissue defects or bone reconstruction performed in the field with limited resources. The reconstruction of segmental bone defects remains a challenge for military orthopedic surgeons, especially in cases of missile or blast injuries characterized by a high proportion of multi-tissue and infected defects –5. TreatIMT is a two-stage procedure, using a cement spacer in the first stage and an ABG in the second stage. The spacer has a mechanical action, obviating fibrous tissue invasion of the recipient site, and a biological action that induces the surrounding membrane due to a foreign body reaction. The membrane then acts as a biological chamber that revascularizes the bone graft and prevents resorption , 9. We bTo date, only a few authors have reported on the Masquelet technique for reconstruction of combat-related bone defects , 12–14. The IM is an encapsulation membrane resulting from a foreign body reaction caused by the implantation of a polymethylmethacrylate (PMMA) cement spacer into the bone defect. This foreign body reaction, composed of macrophages and foreign body giant cells, is the end-stage response of the inflammatory and wound healing responses following implantation of any medical device or biomaterial . The IM From a biological perspective, the IM is a tissue regeneration chamber promoting bone graft integration and bone formation in three ways. First, the membrane acts as a physical barrier between the graft and the surrounding soft tissues, preventing macrophage invasion and subsequent graft resorption. Next, the membrane improves both angiogenesis and osteogenesis by secreting key growth factors including vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and bone morphogenic protein-2 (BMP-2) . BrieflyMasquelet  and GianInfection is the single most frequent cause of IMT failure . Since tCement spacer implantation requires not only the absence of infection but also stable soft-tissue coverage. Any secondary skin necrosis or wound dehiscence inevitably leads to cement spacer contamination, with subsequent infection and IMT failure. Thus, management of combat-related bone defects using the Masquelet technique often involves soft-tissue reconstruction using flap transfer during stage 1. The choice between micro-vascularized free flaps and pedicled  flaps depends on the context of care, surgical resources, and associated injuries. Free flaps are more suited to stable patients managed in the ideal conditions of medical treatment facilities located outside the combat zone . ConversA lack of mechanical stability in the definitive bone fixation at stage 2 is the second cause of IMT failure , 22. InsBasic research of animal found the highest osteo-inductive bioactivity after 2–4 weeks suggesting bone grafting after this short time interval , 35. HowThe IMT global complication rate ranges from 26 to 49% in the literature, with two frequent complications: nonunion and infection , 38, 39.In the literature, IMT is applied for tibia reconstruction in two-thirds of cases. It is also the site at which the complication rate is the highest, with a clear predominance of infection , 39. DueRegarding the risk of infection, most diaphyseal tibia defects are stabilized by external fixation in military practice , 3, 21. Although it runs a higher risk of septic complications, locked intramedullary nailing is the best mechanical option, especially in very large tibia defects Fig. . For MauProximal and distal tibia reconstructions are best achieved using plating. The primary management of such severe multi-tissue injuries usually requires DCO application with temporary external fixation. In the absence of infection, secondary plating can be performed readily during stage 1 Fig. . ConversThe complications rate is lower at the femur, humerus, and forearm levels, where soft-tissue issues are more uncommon . For mecFemur mechanical conditions are less favorable for successful graft integration than those of the tibia. The divergence between mechanical and anatomical axes results in high varus loads, jeopardizing graft corticalization in cases of insufficient fixation stability , 19. A sHumerus, radius, and ulna defects are best stabilized using plates , 33, 44.The complication rate is higher at the hand, similar to the tibia level. The superficial location of the reconstruction space leads to frequent septic failure secondary to spacer exposure or other soft-tissue coverage issues . ApproprCombat-related extremity injuries are often characterized by extensive soft-tissue and bone defects caused by high-velocity projectiles or explosive devices. These massive tissue destructions are frequently observed in polytraumatized patients whose associated injuries or early complications do not allow for long and complex reconstructive procedures such as micro-vascularized tissue transfer . MoreoveSince micro-vascularized flaps can be hardly achieved on the battlefield or may be contraindicated by associated injuries or vascular complications , soft-tissue coverage of extensive defects is performed using simultaneous pedicled flaps , 32. ThiConsidering the large size of the bone defect and the need for dual or triple flap transfers, external fixation is not suitable. Bone stabilization at stage 1 is best achieved using a reinforced spacer with an intramedullary frame Figs.  and 5. AReconstruction of large bone defects requires highly stable internal fixation during stage 2. Intramedullary nailing is preferred from a mechanical perspective. This also brings the advantage of limiting the graft volume. However, in cases of extensive bone defect, the mechanical stability obtained with a standard locked nail can be insufficient for bone graft integration within the IMT. In such situations, nailing can be combined with a locking plate or a free fibular strut placed within the membrane . This fiThe “Capasquelet” is an alternative strategy described by Combal et al.  for recoContrary to micro-vascularized bone transfers or bone transport procedures, the Masquelet technique can be applied with limited resources in the austere environment of forward surgical units or improvised field hospitals , 17, 22.This is the main difficulty on the battlefield due to delayed surgical management with limited resources. Stage 0 must include serial debridement sessions with appropriate antibiotic medication and negative wound pressure therapy to avoid iterative environmental contamination , 43. NexPMMA cement can be lacking in forward surgical units that are not dedicated to performing bone reconstruction. Similarly, the absence of PMMA often precludes the use of the Masquelet technique in developing countries . To avoiAllograft, bone substitute, or sophisticated bone harvesting devices, such as the reamer/irrigator/aspirator (RIA), are usually not available in battlefield medical treatment facilities , 51. As The use of intramedullary nailing is critically important when the patient’s bone stock is limited or the defect is extensive. The nail acts like a central core limiting the bone graft volume to be applied inside the membrane. When intramedullary nailing is not suitable, a central fibular strut is an interesting option limiting the cancellous bone graft quantity and adding mechanical stability to the bone fixation method, be it for external fixation or plating Figs.  and 5.A non-vascularized fibular graft combined with IMT seems to be a valid alternative to the conventional micro-vascularized fibular bone transfer , 52–55. Under certain conditions, limb shortening may be considered to limit the extent of the bone defect. At the humerus, a 3–4 cm shortening can be tolerated without significant functional impairment Table  6]. In . In 6]. Various approaches are currently being developed worldwide to refine IMT and promote its efficiency. These approaches mainly focus on four different aspects: 1) substitution of the standard PMMA spacer with another biomaterial able to generate an IM exhibiting at least similar osteogenic properties to those conferred by PMMA, 2) enhancement of the quality and the viability of ABG implanted into the IM cavity, 3) substitution or combination of ABG with synthetic scaffolds presenting increased osteogenic properties, and 4) IMT conversion into a single-step procedure by performing an “off the shell” membrane strategy.Various biomaterials have been evaluated for their capacity to generate osteogenic IM in animal models as alternatives to PMMA: smooth or roughened titanium , silicon3 vs. 37 cm3, respectively), with few postoperative complications (including iatrogenic femur fracture)  in combination with grafting materials is commonly applied to treat peri-implant bone deficiencies or alveolar bone reconstruction. GBR membranes act as passive membranes hindering the soft-tissue invasiveness of the defect. Emerging data also suggest that GBR membranes may bioactively contribute to bone regeneration . The appAll these future directions are promising approaches to improving IMT outcomes. However, in a military practice context, surgeons often deal with limited resources. Thus, valuable replacement solutions to the IMT standard of care could include ready-to-use implantable medical devices presenting large-scale manufacturing facilities, easy sterilization processes, and convenient storage modes. We believe disposable polypropylene syringe bodies are an example of a promising alternative spacer to standard PMMA cements in IMT in a military practice context or low-resource environment.The Masquelet IMT offers a successful possibility to solve long-bone defects encountered in military practice. Reconstruction of combat-related bone defects often requires prior infection control and soft-tissue reconstruction during stage 1. Providing those requirements are met, IMT even allows bone reconstruction in the austere environment of field medical treatment facilities. However, specific surgical tactics, such as combined inter-tibiofibular grafting or sequential internal fixation, may be required. We believe that this simple, reliable, and replicable procedure should be considered as a technique of choice for the reconstruction of multi-tissue defects resulting from missile or blast injuries.


Pseudomonas species. Alpha diversity increased but the genes involved in carbohydrate metabolism and glycan biosynthesis decreased. After the symptoms subsided, the gut microbiomes rebounded with their taxonomic and functional communities resembling those of the pre-infection microbiomes. In this study, during disruptive norovirus-associated AGE, the gut microbiome was temporarily altered, returning to a pre-infection composition a median of 58 days later. Our study provides new insights for developing probiotic treatments and furthering our understanding of the role that episodes of AGE have in shaping the infant gut microbiome, their long-term outcomes, and implications for oral vaccine effectiveness.Noroviruses are associated with one fifth of diarrheal illnesses globally and are not yet preventable with vaccines. Little is known about the effects of norovirus infection on infant gut microbiome health, which has a demonstrated role in protecting hosts from pathogens and a possible role in oral vaccine performance. In this study, we characterized infant gut microbiome changes occurring with norovirus-associated acute gastroenteritis (AGE) and the extent of recovery. Metagenomic sequencing was performed on the stools of five infants participating in a longitudinal birth cohort study conducted in León, Nicaragua. Taxonomic and functional diversities of gut microbiomes were profiled at time points before, during, and after norovirus infection. Initially, the gut microbiomes resembled those of breastfeeding infants, rich in probiotic species. When disturbed by AGE, Gammaproteobacteria dominated, particularly  Acute gastroenteritis (AGE), marked by episodes of diarrhea and/or vomiting, is a common ailment of children, especially during infancy and early childhood. Rotaviruses and noroviruses are the most common cause of AGE among children globally . These vVibrio cholera and enterotoxigenic Esherichia coli (ETEC)), the gut microbiomes of children became highly disrupted, but recovery occurred in a predictable pattern . Fo. Fo29]. Nearly all the classified reads (95–99% per sample) were classified as bacteria; Archaea, Eukarya (mostly yeasts), and viruses together comprised less than 1% of the classified reads per sample . The metTo quantify changes in diversity between samples longitudinally, we compared Mash distances in a pairwise fashion between sampling times per child . Followip-adj < 0.05 level  ranked among the top three species in each child’s microbiome prior to infection, together comprising 78 to 89% of all bacterial species  prior to norovirus-associated AGE than during infection were genes involved in the cleavage and transfer of sugar groups   .p-adj < 0.01) when comparing the microbiomes of children before infection with the disrupted microbiomes of children during AGE , with a maximum log2-fold change of 3.6 (p-adj < 0.05).When comparing the functional gene complement of microbiomes before and after disruptive norovirus-associated AGE, only 20 differentially abundant genes were found  and functional genes that were highly abundant prior to AGE (such as those responsible for the metabolism of carbohydrates and lipids in breastmilk), indicating that the healthy infant gut microbiome was restored. One participant (Participant A) did not experience gut microbiome disturbance during norovirus-associated AGE despite having a moderate illness (severity score of 10) and a coinfection with EAEC/EPEC. Instead, microbiome diversity decreased during infection and nearly all the species detected were B. longum (96% relative abundance). It has been reported previously that not all children and adults experience gut microbiome disturbance during norovirus-associated AGE [Following norovirus-associated AGE, there was a return of the probiotic species (i.e., ated AGE ,19,20,23Campylobacter [Norovirus-associated AGE did not alter the trajectory of gut microbiome development for infants enrolled in a birth cohort study in Nicaragua. The consequences of AGE on gut microbiome structure and function were not long-term or severe in these children. Larger and more diverse study populations are needed to substantiate our findings and explore the alternative outcomes that likely exist within and between populations. We acknowledge the small sample size used in this study to be the primary limitation for interpreting our results in a broader context; thus, we consider this work to be a pilot for future studies. Nevertheless, an important aspect of our study was to observe gut microbiome changes within participants that were immunologically naïve and whose microbiomes had not yet been shaped by disruption due to AGE. Additionally, microbiome composition might be associated with an ability to mount a robust immune response following rotavirus vaccination, although supportive data are conflicting . This milobacter . Includi


Particular proteins involved in (i) immune processes, such as neutrophil-related proteins, (ii) protein metabolism, or (iii) proteins related to mitochondrial biology, such as those involved in energy production processes, were upregulated in participants reporting exposure to adversity in childhood. This functional triad was further corroborated by protein interaction- and co-expression analyses, was independent of stress exposure, i.e. observed at both pre- and post-stress time points, and became evident especially in females. In line with the mitochondrial allostatic load model, our findings provide evidence for the long-term effects of childhood adversity on mitochondrial biology.The experience of adversity in childhood has been associated with poor health outcomes in adulthood. In search of the biological mechanisms underlying these effects, research so far focused on alterations of DNA methylation or shifts in transcriptomic profiles. The level of protein, however, has been largely neglected. We utilized mass spectrometry to investigate the proteome of CD14 Exposure to traumatic events in childhood, such as the experience of abuse, neglect, or sexual violence is prevalent , 2 and aFirst, there is compelling evidence linking the experience of adversity to dysregulations of the endocrine stress system , 6 and tSecond, following experimental work in animal models  a growinThird, more recently research started to focus on the transcriptome as an intermediate molecular level in the study of trauma and stress consequences. For example, in post-traumatic stress disorder (PTSD) patients, specific mRNA expression profiles were observed in individuals with a history of childhood abuse . FurtherEnhanced expression of pro-inflammatory immune response genes in leukocytes has been reported for people experiencing loneliness and chronic isolation  and amonThus, findings on the molecular level underscore the relevance of immune and stress response system dynamics, and make a strong case for studying the molecular consequences of adverse childhood experience within the context of acute stress processing. Previously, we observed differential gene expression patterns in monocytes following exposure to social stress, with increased activity of pro-inflammatory upstream signaling in a sample of healthy adults reporting childhood adversity .Yet, although research on DNA methylation or transcriptomic profiles yielded valuable insights, it remains elusive to what extend childhood adversity is reflected within a cell’s proteomic landscape. Currently, there is a lack of knowledge concerning the role of proteins within the biological processes potentially mediating the consequences of stressful or traumatic experiences. Given that it is proteins that carry out molecular functions and given the generally variable correspondence of expression states across the cellular cascade from mRNA to protein levels –34 it is+ monocytes isolated before and after exposure to psychosocial stress in a sample of healthy adults with a history of childhood adversity and a matched control group using a label-free mass spectrometry approach. Based on the aforementioned evidence, we expected to observe differences in protein expression related to immune responses. However, given the imperfect correspondence between transcriptome and proteome level, we also aimed to explore proteins differentially expressed between the adversity and control groups.Here we provide insight into the proteome of CD14n = 30) assessed with the Childhood Trauma Questionnaire (CTQ), and further validated with the Early Trauma Inventory (ETI). The control group (n = 30) reported no experience of childhood adversity. None of the participants met criteria for mental disorders at the time of assessment or in the previous 12 months. Resilience levels were assessed using the Resilience Scale (RS-25) and groups were matched for gender, age, current socioeconomic status (SES), and SES in childhood  between 45 and 60 years of age participated in the study. Half of the participants reported the experience of moderate to severe childhood adversity , a well-validated and standardized laboratory stress protocol . Blood wProteome analysis was performed on 118 monocyte samples taken at a baseline measurement 45 min before (t0) and 180 min following (t1) stress exposure utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. LC-MS/MS analysis detected ~3500 protein groups. Proteins were included in the analysis when identified by at least two unique peptides with valid values in at least eighty percent of participants in at least one of the compared groups or time points. In graphical representations, the evidence level is color coded with yellow indicating the eighty percent criterion and red indicating proteins identified by a hundred percent of valid values. Proteins not meeting the eighty percent criterion as a minimum requirement are colored in grey. Applying these criteria, we identified 1129 proteins at t0 and 1206 proteins at t1. The intersection of both proteomes (the consensus proteome) contained 1119 proteins.a) before and after stress exposure as well as (ii) stress-related changes in protein intensities across time (DEPs) in both groups using the limma package [a sets against the monocytes whole proteome universe was tested using the topGO package [a and hub proteins of interest (see below) were further referenced against mitochondrial-related proteins as provided in MitoCarta 3.0 [All analyses were carried out using the R environment . First,  package . Further package . The sig package  and adju package . To esti package . All DEParta 3.0 .p values were corrected for multiple testing through the Bonferroni method. For each module, we defined hub proteins based on their intramodular kME (the correlation between each protein and each modules eigenprotein) and we fitted multiple regression models using the R package variancePartition [Following WGCNA, we compared module expression differences between sex and condition using linear models based on calculated eigenvalues (eigenproteins) of each module (MEs) that were further correlated with clinical variables. Module preservation analyses across stress induction were carried out using a permutation-based preservation statistic with 500 random permutations of the data . Modulesartition  to charaartition  to inves+ monocytes in healthy adults reporting childhood adversity and a control group before and after psychosocial stress exposure  had fold changes|≥1.3|. We found 34 DEPa to be differentially expressed at t0 and t1, leading to a total of 92 unique DEPa overall. 92% of all DEPa were upregulated in the early adversity group and a common set of 32 proteins was upregulated in the early adversity group at t0 and t1.  or mitochondrial metabolism (nprot= 11). Among the proteins that associate with a role in protein-related processes, two are part of the 40 s ribosomal subunit  and function in protein synthesis, while DDOST, SSR1, and RPN2 play part in protein translocation. These results were stable when using different imputation methods applied to the data to replace missing values , the terminal enzyme complex of the respiratory chain (complex IV) as well as subunits of the mitochondrial ATP synthase (ATP5H), the enzyme catalyzing the synthesis of ATP as universal intracellular energy carrier through phosphorylation. Of all 92 DEPa, 26 (28%) are referenced in MitoCarta 3.0 as mitochondrial Table  and funcs Figure .a identified at t0 (n = 57) and t1 (n = 59) are depicted in Fig. GO-terms associated with upregulated DEPa, we built ten cluster solutions of biological processes , likely account to some extend for sex-specific effects we found. Yet, since sex-specific effects of childhood adversity at the molecular level have already been reported, we did not want to withhold our observations, and thus display them with due caution. As such, comparing adversity-related expression differences separately for sex, we observed 58 DEPa in female compared to 20 DEPa in male participants. Following stress exposure, number of DEPa was higher in female (DEPa = 104), but lower in male participants (DEPa = 18). Larger number of DEPa in females was mainly driven by higher abundance of downregulated proteins (+35 DEPa) after stress exposure. The largest overlap of DEPa between pre- and post-stress time points was observed for female participants a show clear differences in structure, density, and functional annotation between female and male participants. As such, baseline DEPa networks of female participants are comprised of interrelated but distinct clusters of proteins functionally involved in protein synthesis, mitochondrial biology, and immune system processes. Following stress, this tri-parted network is further enriched and complemented by a cluster of proteins that more specifically play a role within the establishment of protein localization. In contrast, networks generated from DEPa of male participants are rather loosely connected and of minor density. The proteins interrelated within this assembly play a role in mitochondrial biology, which suggests that altered mitochondrial protein expression is associated with childhood adversity in both females and males.The imbalanced sex distribution in our sample and the smaller number of investigated males (29) Fig. . String nprot = 1119). The Zsummary statistic as a permutation-based preservation indicator was used to compare the preservation of identified t0 baseline modules in the t1 stress network and vice versa. Within the t0 network  in the t1 network and we found the highest preservation scores (Zsummary > 15) for the salmon, the light cyan, and the purple module  identified as differentially expressed between the groups using limma. Consequently, the module eigenprotein  of both the salmon  and the purple module  were positively correlated with experience of early adversity , the salmon  and the purple module  correlated positively with participants' score on the CTQ physical neglect subdimension.The purple module (M7: 82 proteins) is comprised of proteins implicated in mitochondrial biology and cellular respiration and further contains proteins involved immune responses and viral gene expression or translation Fig. . The sality Fig. . While ta is strongly conserved (Zsummay > 10) in the stress network, and mainly involved in gene expression and the regulation of RNA metabolic processes but as well contains a few proteins involved in cellular respiration. The module’s eigenprotein value was negatively associated with the experience of adversity and correlated negatively with the CTQ physical neglect subdimension .Both, the light cyan module (199 proteins) and the midnight blue module (45 proteins) were more strongly expressed in control individuals and particular male control participants showed significantly stronger expression of the midnight blue module. The light cyan module, among others, harbors a set of proteasome proteins implicated in the mediation of immune system processes such as antigen presentation and proteins involved in regulation of mRNA stability. The string network generated from this module showed a dense structure including proteins involved in vesicle-mediated transport and a cluster of proteins functioning in the context of RNA splicing Fig. . The midZsummay = 8.4) in the baseline network than the other way around (Mean Zsummay = 11.2)  Fig. . In four.2) Fig. .a identified using limma analysis and paid particular attention to hub proteins. Hub proteins are part of highly correlated protein assemblies in modules and are likely important from a co-expression perspective. We focused our analysis on the four most conserved modules indicating differential activities between groups and report both, top ten and top ten percent of proteins with the highest kME values for each module in Fig. Next, we analyzed to what extent proteins of conserved baseline and post-stress co-expression networks overlapped with the DEPa identified before and after stress exposure were part of the midnight blue or the light cyan module and none of these proteins represented a hub protein. The salmon module on the other hand comprised altogether 30 DEPa. Among these, S100A11, a calcium-binding protein, is mechanistically involved in toll-like receptor-mediated inflammatory responses of macrophages [a of which eight were hub proteins . Note that half of these proteins locate in the mitochondrial membrane and all are highly connected in protein-protein interaction networks  and the salmon (2.91%) modules expression. Vice versa, participants’ RS25 score explained 10.09% of variance of the light cyan modules expression, while the variance accounted for by the CTQ score was negligible. Both, CTQ (3.17%) and RS25 scores (7.85%) explained variance in the midnight blue module´s eigenprotein, as did biological sex (7.44%) and the experience of childhood adversity (18.63%) after controlling for all other variables. These relations mirror the results of our previous analyses and are as well reflected within the expression of the top 10 module hub proteins Fig. . Among tTo summarize, utilizing WGCNA to calculate baseline (t0) and post-stress (t1) consensus protein-co-expression networks, we identified several modules representing specific aspects of monocytes cellular biology. These modules harbor, e.g., proteins relevant for immune response mechanisms or implicated in mitochondrial biology. Comparing protein co-expression with differential protein expression analyses, consistent results emerged. Proteins previously identified as differentially expressed between the early adversity and control condition were mostly co-expressed, predominantly assigned to distinct modules, often represent hub proteins, and are thus likely to play an important function within the co-expression network.biologically embedded. Our proteome analysis of CD14+ monocytes revealed three major findings: First, we observed differentially expressed proteins in participants with a history of childhood adversity, which mapped to specific functional domains: mitochondrial biology, immune system processes, and protein synthesis. Second, we observed indications for sex-specific expression differences between groups suggesting stronger effects of adversity on protein expression within female participants while differentially expressed proteins in males were restricted to mitochondrial biology. Third, stress exposure was not associated with major changes in protein expression. The fact that we observed clear effects of stress on the RNA level in our previous work [Exposure to adversity in childhood not only causes immediate harm but is also associated with a lifelong increased risk for a range of health problems, raising the question of how early psychosocial experiences are ous work  and givea revealed adversity-associated proteins to function within three particular domains of the monocytes biology: (i) immune relevant processes , (ii) protein metabolic processes  and (iii) mitochondrial biology and the cell’s energy metabolism .Specifically, GO analysis of upregulated DEPa identified at t0 and t1 were strongly enriched for GO terms indicating neutrophil degranulation and activation. This study analyzed isolated monocytes, so it is unlikely that these GO annotations reflect alterations in neutrophil biology per se. However, monocytes and neutrophils share a common developmental pathway as myeloid lineage immune cells, and both include an extensive system of granule proteins involved in cellular activation, chemotaxis, adhesion, reactive oxygen responses, and inflammatory biology. Hence, we believe that the identification of proteins previously associated with neutrophil activity in our isolated monocyte samples is indicative of the broader interplay of myeloid lineage innate immune cells involved in the orchestration of acute immune responses and inflammatory activity.Of note, DEPa showed distinct but interrelated clusters of interconnected proteins related to cell respiratory energy metabolism or to protein biosynthesis. These protein-interaction network structures were conserved across stress exposure and built around a set of 32 upregulated consensus DEPa. Importantly, proteins in this set are derived from two repeated measurements and can thus be considered technical replicates.Further, protein interaction networks generated from t0 and t1 DEPa in female participants exceeded the numbers found in male participants. Furthermore, proteins found to be differentially expressed between females with a history of childhood adversity and control group participants form clearly structured interaction networks mirroring the functional triad described above. In contrast, proteins differentially regulated between groups of male participants form only loosely connected network structures including proteins involved in mitochondrial biology. The smaller number of investigated males , and thus lower statistical power, might explain these disparities. On the other hand, sex-specific regulation of immune-relevant processes was documented before on basis of a mega-analysis across several transcriptomic studies [The number of DEP studies , supportBaseline protein co-expression analysis validated findings from the differential protein expression approach and revealed modules representing distinct aspect of monocytes function related to childhood adversity and resilience.Childhood adversity-associated expression differences became evident within four modules. A salmon module (M1:166 proteins) was implicated in the regulation of immune system responses and harbors proteins involved within IL-12 signaling. This pathway promotes inflammation and facilitates innate as well as adaptive immune responses, modulating differentiation of T helper cells and NK cell toxicity. The salmon module was significantly stronger expressed in participants with a history of childhood adversity and correlated positively with the CTQ total score. S100A11, a member S100 protein family, is one of the proteins of this module. S100A11 has been reported to stimulate the production of the pro-inflammatory cytokine IL-6 by peripheral blood mononuclear cells (PBMC) and overexpression of S100A11 was associated with inflammation-related health problems such as osteoarthritis or rheumatoid arthritis .Both a light cyan (ME 5: 199 proteins) and a midnight blue (ME 4: 45 proteins) module were more strongly expressed in male control participants. While the first module seems implicated in immune-relevant processes and harbors proteins involved in, e.g., vesicle-mediated transport, both modules contain proteins that play a role in gene regulation, particularly influencing RNA metabolic processes.Notably, resilience scores explained substantial proportions in variation in the light cyan and the midnight blue modules top 10 hub proteins, while CTQ scores accounted for additional variance in the midnight blue modules top 10 hub proteins. Hence, both modules might represent promising targets for future studies regarding risk and resilience mediating processes and sex-specific responses to adversity on the molecular level.a (35% percent of the sizes of the modules), is significantly enriched with mitochondrial proteins, significantly stronger expressed in the early adversity group, and positively correlated with the CTQ total score. This is in line with a number of studies implicating alterations in mitochondrial biology associated with the experience of early adversity [+ monocytes specifically.Lastly, the purple module (M7: 82 proteins) harbors altogether 29 DEPdversity . For exadversity  found hidversity  reporteddversity , 14, 15 + monocytes play a role in mediating the systemic effects of early adversity. On the subcellular level, mitochondrial recalibrations might indicate higher cellular energy demands due to such functional shifts.Monocytes are antigens representing immune cells and progenitors of macrophages implicated in the first line of defense as part of the innate immune response and play a critical role in immune-to-brain communication processes , 51. Fur+ monocytes could as such reflect rather stable adaptions towards the increase in physiological energy costs resulting from higher pro-inflammatory activities. As we observed an increase in proteins involved in mitochondrial biology in participants with a history of childhood adversity independently from acute psychosocial stress exposure, our results point in this direction. This is consistent with findings from Gumpp et al. [Since childhood adversity is strongly linked to persistent low-grade inflammation, a greater amount of mitochondrial respiratory chain proteins in CD14p et al. , showingp et al. , howeverThe following limitations need to be considered. The sample size was small. Nevertheless, 118 mass spectrometry analyses could be realized, which is relatively large and therefore quantitatively robust by the standards of global proteomics studies. Further limitations arise from the chosen methodology and the preparation strategies used. Here, we applied a rather generalist protocol for protein extraction, designed to collect a representative part of the monocytes protein assembly instead of targeting specific proteins bound to membranes or presented in distinct cell organelles. Consequently, the proteins identified represent a fraction of the monocytes proteome and give valuable insights but inevitably do not display the complete picture. Lastly, it is important to keep in mind that the significant findings presented did not survive correction for multiple testing. The fact that results from two analytical strategies showed convergent findings, and the fact that each sample was measured twice, lend confidence to our findings. However, future confirmatory studies will be required to follow up on these results, and the present findings should be considered hypothesis-generating rather than definitive. Further, reported results come from a correlational analysis and are subject to the short-fallings of this design. Thus, it is not clear whether the observed proteomic differences represent a causal effect of early life adversity, or stem from some other factor that is correlated with early adversity but was not controlled for in this study.To conclude, we provide evidence for altered protein expression associated with psychosocial adversity in childhood, especially implicating proteins related to mitochondrial biology, cellular energy metabolism, and inflammatory biology. The present results suggest that these effects may have some degree of sex-specificity. Lastly, network-based approaches identified adversity-related differences in protein co-expression, which largely converged with differential expression analysis on the level of single proteins. Our results identify hub proteins that can be used in directed hypothesis testing in future studies, and they support the idea of mitochondria as sub-cellular targets for the effects of adverse psychosocial influences. These data also contribute an unbiased proteomics perspective towards a rich body of work trying to elucidate how long-lasting consequences of early adverse rearing conditions are biologically sustained.Supplementary Information


Due to a large technical improvement in the past decade, transcatheter aortic valve replacement (TAVR) has expanded to lower-surgical-risk patients with symptomatic and severe aortic stenosis. While mortality rates related to TAVR are decreasing, the prognosis of patients is still impacted by ischemic and bleeding complications, and defining the optimal antithrombotic regimen remains a priority. Recent randomized control trials reported lower bleeding rates with an equivalent risk in ischemic outcomes with single antiplatelet therapy (SAPT) when compared to dual antiplatelet therapy (DAPT) in patients without an underlying indication for anticoagulation. In patients requiring lifelong oral anticoagulation (OAC), the association of OAC plus antiplatelet therapy leads to a higher risk of bleeding events with no advantages on mortality or ischemic outcomes. Considering these data, guidelines have recently been updated and now recommend SAPT and OAC alone for TAVR patients without and with a long-term indication for anticoagulation. Whether a direct oral anticoagulant or vitamin K antagonist provides better outcomes in patients in need of anticoagulation remains uncertain, as recent trials showed a similar impact on ischemic and bleeding outcomes with apixaban but higher gastrointestinal bleeding with edoxaban. This review aims to summarize the most recently published data in the field, as well as describe unresolved issues. Since the first implantation in an inoperable patient, by Alain Cribier in 2002 , transcaWith the improvement of procedural technique and extension to lower-surgical-risk patients, mortality rates associated with TAVR kept decreasing in the last decade. Nonetheless, available data on long-term outcomes after TAVR from the first trials report a high rate of long-term mortality (28 to 72% at 5 years) . MortaliStroke is one of the most dreaded complications after TAVR and is associated with an increased rate of mortality ,9,10. ThAtrial fibrillation (AF) is common in patients undergoing TAVR and is associated with worse outcomes. Both pre-existing AF and new-onset AF are predictors of adverse ischemic cerebrovascular events, mortality and bleeding post-TAVR ,37,38,39p = 0.017) . C. C150]. n = 170) will evaluate the safety and efficacy of anticoagulant therapy alone (VKA or DOAC) versus anticoagulant plus aspirin (NCT02735902) in patients requiring OAC who underwent successful TAVR, after a 12-month follow-up. This trial is expected to end in April 2023. The POPular PAUSE TAVI randomized trial previously cited will provide information on the optimal anticoagulant strategy to adopt during the TAVR procedure, with a comparison of the effect of peri-operative discontinuation versus continuation of OAC on VARC-2 ischemic and bleeding outcomes in patients undergoing TAVR with prior OAC therapy (NCT04437303). Finally, in low-risk patients undergoing TAVR with no indication for OAC, the ongoing LRT  trial is currently exploring the effect of VKA in addition to aspirin, compared to aspirin only, on clinical outcomes and valvular heart deterioration .The main ongoing trials evaluating the antithrombotic regimen in TAVR patients are summarized in TAVR is expanding towards a low-risk patient category as a result of technical advances and operators’ improved skills. However, the post-TAVR antithrombotic regimen remains challenging. Single antiplatelet therapy appears to be the best compromise when there is no compelling indication for chronic oral anticoagulation. Whether it should be aspirin or clopidogrel is not established. There is no supportive evidence to use oral anticoagulation when there is no established indication for oral anticoagulation other than the TAVR procedure. The gap in evidence as to whether DOACs should be preferred over VKA remains when there is an indication for OAC use. It seems that DOACs are not the same and randomized trials are awaited. Likewise, whether oral anticoagulant therapy should be continued or interrupted during the procedure remains unclear.


Dying tumor cells shed DNA fragments into the circulation that are known as circulating tumor DNA (ctDNA). Liquid biopsy tests aim to detect cancer using known markers, including genetic alterations and epigenetic profiles of ctDNA. Despite various advantages, the major limitation remains the low fraction of tumor-originating DNA fragments in a high background of normal blood-cell originating fragments in the cell-free DNA (cfDNA) pool in plasma. Deep targeted sequencing of cfDNA allows for enrichment of fragments in known cancer marker-associated regions of the genome, thus increasing the chances of detecting the low fraction variant harboring fragments. Most targeted sequencing panels are designed to include known recurrent mutations or methylation markers of cancer. Here, we propose the integration of cancer-specific chromatin accessibility states into panel designs for liquid biopsy. Using machine learning approaches, we first identify accessible and inaccessible chromatin regions specific to each major human cancer type. We then introduce a score that quantifies local chromatin accessibility in tumor relative to blood cells and show that this metric can be useful for prioritizing marker regions with higher chances of being detected in cfDNA for inclusion in future panel designs. Dying cells in the body, including those originating from tumors, shed DNA fragments into the blood and make up a pool of short circulating fragments known as cell-free DNA (cfDNA). Fragments of a diseased tissue of origin harbor genetic and epigenetic markers of the disease, e.g., somatic mutations or DNA methylation markers. Detecting these markers simply from the plasma of cancer patients offers a number of advantages over classic tissue biopsy, including the elimination of biopsy bias, convenient and minimally invasive identification of personalized markers for therapeutic decisions, monitoring of relapse, tissue of origin determination, early detection and screening among others1. However, the clinical power of cfDNA-based technologies is limited by the fact that fragments originating from a diseased tissue of interest typically contribute only a small fraction of the cfDNA pool, especially in early stages, given the over 80% presence of cfDNA fragments from blood cells during normal turnover3. The high background of non-marker-harboring fragments in cfDNA necessitates the use of very sensitive experimental methods. Next-generation sequencing can reach this sensitivity provided sufficient sequencing depth is reached at marker regions4. To avoid cost-prohibitive deep whole genome sequencing, targeted sequencing panels are designed to enrich and sequence only small portions of the genome that are likely to harbor disease markers9.Liquid biopsy refers to the minimally invasive detection of cancer biomarkers in bodily fluids11. However, this limits the utility of liquid biopsy to diseases associated with somatic genetic aberrations, and even among those, only to subtypes with multiple known recurrent mutations or known driver genes. To improve the sensitivity of mutation detection, several diverse methods have been proposed, including barcoding fragments with unique molecular indices (UMI) followed by error suppression12, using the phasing of multiple somatic mutations in individual DNA fragments13, and machine-learning based scoring to prioritize driver mutations for inclusion in panel designs14.Targeted sequencing panels for human cancers are typically designed through mining of tumor genomic data for identification of recurrent modifications. Most of the currently existing designs are based on pan-cancer or cancer-type-specific genetic lesions17. Given the large number and wide distribution of tissue-specific epigenetic markers across the genome that are not normally present in cfDNA in abundance, this significantly increases the sensitivity for detection. Furthermore, this strategy is generally applicable across conditions associated with tissue-specific cell death, regardless of the existence of mutations in diseased tissues. Previous studies have shown the utility of DNA methylation markers in various diseases, including cancer24. However, specific targeting of differentially methylated regions of interest typically involves bisulfite conversion, which causes degradation of DNA in already low levels of cfDNA, and also is not readily integrable with somatic mutation panels.Cancer mutations are rare across the genome, can be hard to distinguish from non-cancer mutations in blood cells that arise from clonal hematopoiesis, and most do not inform tissue of origin. To further broaden the utility of liquid biopsies and improve their performance, focus has shifted to epigenetic markers either alone or in combination with mutations27. Distinct nucleosome positioning and chromatin compactness at tissue-specific regulatory regions that lead to fragmentation profiles in cfDNA have been proposed as alternative epigenetic markers32.It has been established that open chromatin regions undergo higher degradation and fragmentation in cfDNA due to the function of nucleases. The differences in accessibility of chromatin across genomic regions are manifested as differences in fragmentation patterns in cell free DNA and can inform tissue of origin. Features of this non-random fragmentation pattern include a decrease in sequencing depth and fragment lengths in open regions compared with regions protected by DNA-binding proteins35. For instance, given an overall increase in fragmentation of tumor-originating DNA36, size selection of cfDNA fragments to enrich those between 90 and 150 bp in length helped improve somatic mutation detection33. Another study on pediatric cancers with low mutational burden also showed improved detection by integrating fragment length (ratio of short to long) as well as coverage across the genome35. Improved detection when combining mutations with fragmentation features was also reported for and additional 7 cancer types17. Another recent study combined fragment length features at personalized mutation sites in Glioma to improve detection36.To further improve sensitivity for early detection, integration of genetic markers with fragmentation information has been proposed37. This allows for the use of machine-learning based approaches to identify cancer-type-specific accessibility profiles. These fragmentation features can be incorporated into future targeted sequencing panel designs for liquid biopsy purposes. Such panels would offer several advantages over the existing alternatives, including not being limited to known genetic lesions, being conveniently integrable with mutation regions for complementing existing panel designs, and offering precise regulatory region enrichment without requiring bisulfite conversion. Here, we introduce a novel pipeline that incorporates accessibility information toward identification of marker regions for cancer detection in cell-free DNA. We develop a score based on relative chromatin accessibilities of blood and tissue and show its utility in optimization of panel designs.In the recent years, an unprecedented amount of data on chromatin accessibility profiles (ATAC-seq) of primary human tumors has become available through the cancer genome atlas (TCGA)27. Since cfDNA in healthy individuals is known to mainly consist of blood-cell originating chromatin fragments2, we reasoned that blood cell accessibility across the genome should be associated with cfDNA fragmentation patterns. We, therefore, obtained ATAC-seq data of healthy PBMC from EGAS0000100260538, neutrophils from GEO15352039, and endothelial cells from GSE14577440  across the genome  in prostate, Apolipoprotein A1 (APOA1) in liver, NK6 homeobox 3 (NKX6.3) in stomach, paired box gene-8 (PAX-8) in kidney, and Surfactant Protein B (SFTPB) in lung cancers were among the selected markers from our pipeline  WGS data on cfDNA samples from cancer types in our study in which such data was publicly availablepattern “”. In eacinaccessible regions. We reasoned that this information can be incorporated in various contexts of panel designs for prioritizing among genomic regions. We considered the following scenarios for each of the 7 cancer types in the study: panels based on fragmentation patterns of inaccessible regions, and panels based on known mutation or differentially methylated region (DMR) markers.Beside cancer-specific ATAC-seq peaks corresponding to tissue-specifically accessible regions, we also set out to analyze this dataset for identification of tissue-specifically 27. Cancer-specifically closed chromatin regions may serve as a class of epigenetic biomarkers if their fragmentation patterns are sufficiently distinct from that of background cfDNA from blood and other cells. To identify such regions, we used the cancer-specific local relative accessibility scores as previously described . Typically, targeted sequencing panels for liquid biopsy are designed to cover recurrent somatic mutations30. Based on this, we hypothesized that mutation- or DMR-harboring regions that fall into inaccessible chromatin contexts in the cancer of interest , but fall in accessible chromatin regions in blood cells (low noise) have a higher chance of being detected given the increased fraction of tumor-derived fragments in cfDNA.One of the factors that explains part of this variation in detectability and prevalence of different tumor markers in the plasma is chromatin compaction state. It has previously been established that compared to open chromatin regions from the same cells, closed chromatin and nucleosome occupied regions are over-represented in cfDNA due to lower amount of degradation and fragmentationCOSMIC and tissue-specific DMRs from methHC42 that are frequently used in panel designs  should be better represented in cfDNA than those with the opposite pattern (RelAccS > 0), allele frequencies in cfDNA should better reflect those in matched tumors for the former scenario. We indeed observed a higher correlation between AF of mutations that were detected in both tumor and cfDNA for regions with a negative RelAccS (r = 0.49) compared with regions with a positive RelAccS (r = 0.15)  but exposed in blood cells (background noise) are more likely to be detected in cfDNA as a higher proportion of cfDNA fragments in those regions originate from the diseased tissue. We translate this information from available ATAC-seq data into a score that can be used for prioritizing marker regions in liquid biopsy panels. Genetic alterations found in tumors can serve as liquid biopsy markers and are the basis of most targeted sequencing panel designs. However, some genetic lesions in tumors are hardly detectable in cfDNA and those that are detected tend to have variable fractions in cfDNA which complicates panel designs. Most panel design strategies rely on target selection based on recurrent alterations in the population. However, the concordance between variants detected in cfDNA with that of solid tumors is limited even in matched samples, rendering such panel design strategies inefficient and questionable. Many factors contribute to this variability in variant allele representation in cfDNA data all of which decrease the signal-to-noise ratio in cfDNA either by lowering the contribution of tumor fragments or by increasing the contribution of fragments from other cells throughout the body. Here, we introduce a metric named RelAccS to quantify this relative contribution and show that target selection for liquid biopsy panels can use this score for ranking and prioritization of regions that are more likely to be detected in cfDNA.Liquid biopsy holds great promise as the future of cancer screening and monitoring. However, in most cancer types, the sensitivity of the current liquid-biopsy based tests suffer due to the low signal-to-noise ratio from a high background of fragments with hematopoietic origins in cfDNA. Much focus has been devoted to the improvement of the biochemistry of library preparation methods as well as data analysis steps, such as UMI barcoding, short fragment enrichmentIn summary, we illustrate the application of RelAccS in two different contexts of panel design. In the first context, regions are identified to be directly targeted according to RelAccS for classifying a cancer type from other types, in which case we are suggesting the tumor-open regions would offer more specificity. In the second context, the goal is to sensitively detect cancer from normal based on either fragmentation features or other features  and using RelAccS to guide the choice of candidate targets. In this latter context, we suggest features that fall in the tumor-closed chromatin regions to be prioritized.It is important to note that here as a proof-of-principle, RelAccS was simply calculated using ATAC-seq counts from tumors and blood cells by cpm normalization. This normalization takes differences in library sizes across samples into account, but ignores differences in composition of libraries. Thus, in the future, a more sophisticated version of RelAccS may add to its utility. Furthermore, the application of RelAccS for panel optimization can be expanded to epigenetic markers selection as well, including panels targeting differentially methylated regions, transcription factor binding sites, and transcription start sites. Such panels hold great clinical promise not only in the fight against cancer, but more generally for the future of non-invasive disease screening and monitoring across a diverse spectrum of human conditions.52.The incorporation of chromatin accessibility states in targeted sequencing panel design is a promising new approach to improve cancer detection from cfDNA. As a proof of principle, we first investigated chromatin accessibility states of blood cells with cfDNA fragmentation patterns. We observed lowly accessible regions are associated with higher fragment length and increased local sequencing depth. This association was recently shown to become stronger with deeper sequencing of cfDNAWe present a robust pipeline which utilizes ATAC-seq profiles of tumors and blood cells to identify tissue specifically accessible and tissue specifically inaccessible chromatin regions for seven major human cancer types. In silico analyses suggest that these regions show different fragmentation patterns in corresponding cancer patient cfDNA and healthy individuals. We introduced a metric called RelAccS, which measures relative chromatin accessibility in tumor vs. blood cells and can quantify the relative contribution to cfDNA pool.We illustrate the application of RelAccS in two different contexts of panel design. In the first context, regions are identified to be directly targeted according to RelAccS for classifying a cancer type from other types, in which case we are suggesting the tumor-open regions would offer more specificity. In the second context, the goal is to sensitively detect cancer from normal either solely based on fragmentomic features or based on other features  and using RelAccS to guide the choice of candidate targets. In this latter context, we suggest features that fall in the tumor-closed chromatin regions to be prioritized.In brief, we introduced novel epigenetic marker regions based on relative chromatin accessibility. We suggest that these markers can be used alone or with other genetic and epigenetic markers to help further improve the limit of detection in cancer detection from plasma. To our knowledge, this is the first use of chromatin accessibility states with machine learning approaches to identify cancer-specific markers with potential utility in liquid biopsy testing. Although further research is needed to address early-stage cancer detection challenges, our results provide a useful strategy for future targeted sequencing panel designs.We collected measures of chromatin accessibility in genome of cells that contribute most to the cfDNA pool in healthy individuals. To this end, high quality ATAC-seq profiles from PBMC and neutrophils were obtained. Raw ATAC-seq data were downloaded from PBMC of 120 healthy individuals from dbGap (accession code phs001934.v1.p1) and raw ATAC-seq data from unchallenged neutrophils of 4 healthy individuals were downloaded from Gene Expression Omnibus (accession code GSE153520).54 (http://pepatac.databio.org/en/latest/) similar to TCGA ATAC project37. First, adapters were removed with skewer55, then reads were mapped to GRCh38 build of human genome using bowtie2 with parameters-very-sensitive-X 2000. Samtools was used to sort and isolate uniquely mapped reads and duplicates were removed using picard56. Mark Duplicates with options VALIDATION_STRINGENCY LENIENT -REMOVE_DUPLICATES true. Next the resulting bam files were corrected for Tn5 offset using deeptools57 alignmentSeive with parameter-ATACshift. Then peak calling was performed using MACS258 with parameters-shift-75-extsize 150-nomodel-call-summits-nolambda-keep-dup all-p 0.01. Peak summits were extended 250 bp in both directions and then filtered by ENCODE blacklist (https://www.encodeproject.org/annotations/ENCSR636HFF/).Fastq files were processed to peak call with PEPATAC pipelineWe decided to identify accessible regions in cells that have the highest contribution to normal cfDNA. Accordingly, we took advantage of PBMC and neutrophil ATAC-seq data set obtained and processed in the former step. As vascular endothelial cells also shed small amounts of cfDNA to blood stream, we took human umbilical vascular endothelial cell (HUVEC) ATAC-seq peaks into our analysis in the following steps. MACS2 peak calls of HUVEC from five healthy individuals was obtained from Gene Expression Omnibus (GSE145774). We then extended peak summits 250 bp in both directions to get a fixed length peak set for all samples. Then we used PEPATACr library to generate non-overlapping peak set for each sample of PBMC, neutrophil and HUVEC. The PEPATAC pipeline uses an iterative algorithm to handle overlapping peaks. First the most significant peak (peak with highest score) is kept and any overlapping peak is removed then the algorithm repeats this process for the next significant peak. This process is continued until there are no overlapping peaks left in the peak set.Next, specific peak sets for neutrophils, PBMC and HUVEC were generated. To do so, MACS2 peak scores in each sample were divided by sum of all peaks scores divided by one million, then all the peaks from the same cells were combined and overlapping peaks were trimmed using the same iterative process explained above. Peaks present in at least two samples with scores higher than 5 were retained. This process resulted in a reproducible high quality “cell type specific peak set”.Finally, we wanted to define a consensus blood peak set to identify accessible chromatin regions of genome of cells that contribute the most to cfDNA pool of healthy individuals. First, scores of all “cell type specific peak set” were renormalized. Then the resulting peak sets from PBMC, neutrophils and HUVEC were combined into a integrated peak set and overlapping peaks were removed with the same iterative process. In this study we refer to this final fixed width peak set as the “consensus blood cells peak set”.59 and then normalized to each sample library size. Then average of normalized ATAC-seq counts of PBMC and average of that of neutrophils were calculated in every peak. As the neutrophil population in blood is higher than PBMC (approximately in a 2:1 ratio), for each peak in the consensus blood cells peaks set, weighted average of mean of normalized ATAC-seq count of PBMC and neutrophils was calculated  as follows:i is the region of interest.ATAC-seq insertion counts of PBMC and neutrophil samples in consensus blood peak set were calculated from corrected bam files using featureCountsWe considered the lowest quartile of blood consensus peak set as “lowly accessible regions in blood cells” and the highest quartile as the “highly accessible regions in blood cells”.https://gdc.cancer.gov/about-data/publications/ATACseq-AWG) and samples from desired cancer type were selected. Then ATAC-seq insertion counts were normalized to library size of each sample. For each region of interest, average of normalized ATAC-seq counts across TCGA samples of desired cancer was divided by normalized ATAC-seq counts of blood cells as follows:RelAccS score measures relative chromatin accessibility of genomic region of interest in tumor vs. blood cells. First, raw ATAC-seq data matrix of primary tumor TCGA samples was downloaded from  were obtained with accession number of GSE71378. Among all the samples, IH01, IH02, IC35, IC37, IC17, and IC20 have been chosen for further analysis due to their high sequencing depth. The bam files of the mentioned samples were used which were mapped to the human reference genome (GRCh37) by ALN algorithm in BWA.27. Then the depth of the target regions in the healthy cfDNA bam files was measured by using samtools bedcov. Counting the median fragment length was carried out by get_fragment_size function of ctDNAtools package and our implemented in-house python script65. The obtained results were visualized by Prism66.In the first phase, we tried to determine the depth and fragment length distributions of two types of regions sets that correspond to regions of low and high chromatin accessibility in blood cells. For this aim, two healthy cfDNA samples  with distinct library preparation methods were obtainedBoxplots with 10–90 percentiles were used to show the mentioned two parameters distribution in healthy cfDNA samples. As it is illustrated in the Fig. In the next step, we calculated ATAC-seq counts of blood cells in each region as weighted mean of average of neutrophils ATAC-seq counts and average of PBMC ATAC-seq counts Eq. . Then, tFor this analysis, we log2-normalized the counts and then removed outlier regions (less than 1 log2). Then a smoothing approach was applied on the normalized data. The smoothing parameters were optimized to consider 15 neighbors to average and second order of the smoothing polynomial. Finally, the correlation was measured based on Pearson Correlation Coefficients.We propose two types of target selection strategies (accessible and inaccessible) for each cancer type. Total depth and fragment lengths at the cancer specific open and closed regions for BRCA, COAD, LIHC, and LUSC were assessed in their related cancer cfDNA along with the healthy cfDNA samples. For depth calculations, raw counts were normalized to total sequencing depth of their related cancer and healthy cfDNA samples.In this part, 48 targeted sequencing data files from prostate cancer patients including cfDNA, white blood cell (WBC), and matched tumor data were obtained under the accession number of EGAD00001004526 from EGA. The obtained fastq files were quality-checked by FastQC and high-quality reads  were used for downstream analyses.56. Finally, joint calling of cancer data  with normal samples (WBCs) from the same individual was done. Then, low quality variants were filtered from VCF files . Same as in Cario et al.14, we considered a threshold of 20% for germline variants. Accordingly, we removed variants with higher allele frequency in cfDNA from cfDNA VCF and corresponding tissue VCF. We assigned prostate cancer (PRAD) RelAccS score of the overlapping peak to corresponding variant. We then divided variants into two groups: (1) variants with RelAccS > 0, (2) variants with RelAccS < 0.Next, variant calling was carried out based on the best practices of GATK for somatic variant calling. This pipeline comprised several steps as follow: first, fastq files from cfDNA, WBC, and tumor were mapped against hg38 reference genome. Next, the pre-processing of the generated bam files were done using picardWe then selected the detected variants (variants present both in tumor and matching cfDNA samples). Allele frequency correlations between tumor and matched cfDNA was calculated in R.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.


Fusarium species are ubiquitous fungi, both saprotrophic and pathogenic to plants, animals and humans. They are also potent mycotoxin producers which makes them one of the most devastating plant pathogens. Mycotoxin biosynthesis and regulation has recently become one of the mainstream research topics, since knowledge concerning individual metabolic pathways became available and modern ’omics’ techniques allowed us to expand this even further. Independently, high-throughput sequencing methodology helped researchers gain insight into the complex phylogenetic relationships among closely related genotypes comprising Fusarium populations, species and species complexes. Molecular tools have so far been very powerful in species identification and phylogeny, as the great diversity of the Fusarium genus has forced scientists to continuously revise previously described taxons.  Fusarium Species“ ten research articles have been published concerning pathogens from the Fusarium oxysporum species complex (FOSC) [F. oxysporum f.sp. conglutinans isolate from diseased cabbage and analysed the genome-wide SNP polymorphisms to track the diversity of the populations in China [F. oxysporum f.sp. asparagi isolates from southern and northern Europe failed to distinguish the populations from different climate regions. Moreover, isolates from varying geographical origins showed similar pathogenicity towards asparagus plants [2+ signalling machinery components in the regulation of the germ-tube formation, required for initial F. oxysporum colony establishment. These preliminary results may pave the way towards using molecular engineering to control the infection process through signalling pathway component management [egfp-tagged quantitative imaging) coupled with quantitative pathogen detection (qPCR) are often deployed to detect the early stages of infection in plant tissues and track the sequential spread of the pathogen in invaded organs, a strategy employed for F. oxysporum R1 identification in saffron roots and corms [In the present Special Issue entitled “Plant-Pathogenic x (FOSC) ,2,3,4. Lin China . FurtherFusarium verticillioides is an significant pathogen of maize occurring worldwide and limiting grain yield and quality. The Vietnamese F. verticillioides population was screened to evaluate the efficacy of the resistance components of two maize genotypes varying in susceptibility. Using a well-established procedure of gene expression profiling of the essential defence-related genes, Tran et al. showed that a higher expression may contribute towards the performance of more resistant maize genotypes [Fusarium fungi can be dispersed in various ways, mostly by air, soil, water and seeds. One of the less frequent transmission routes include insects acting as vectors for plant pathogens. Recent study have proven that beetles from the species Xylosandrus morigerus can be effective vectors for the Fusarium solani species complex (FSSC), even though the two isolates showed a considerable level of diversity and were reported to be pathogenic to different plant species [enotypes . Fusariu species . Fusaria and other fungal species or plant hosts and has become a fundamental tool in functional studies, as employed in identifying CFEM  protein domains or KP4-like (killer toxin-like) proteins of Fusarium graminearum [F. graminearum effectors [Fgkp4l genes were up-regulated during direct interaction between F. graminearum and Trichoderma gamsii (pathogen’s antagonist), only one ortholog was expressed during the interaction in wheat spikes. Interestingly, these genes were more common in Fusarium species with a broad host range than in the specialized ones [Bioinformatics has given insights into the evolutionary and functional divergence of fungal proteins playing specific roles during the interspecific interactions between minearum ,8. The Cffectors . ExpressFusarium growth and metabolism. All metabolites tested, including coumarin, spermidine, p-coumaric acid, iso-orientin, and quercetin were reported to inhibit the synthesis of fumonisin B1 and beauvericin but did not impair isolate growth [Fusarium mycotoxins was noticed by Badr et al., namely deoxynivalenol (DON) and zearalenone (ZEN), produced by the Fusarium culmorum strain. However, pure standards of the individual compounds exerted a higher impact than the complex extracts made from fruit by-products [Environmental factors have a significant impact on fungal physiology and compounds produced by plants, either primary or secondary metabolites, are among the most researched agents influencing fungal growth and mycotoxin biosynthesis. Therefore, various kinds of plant extracts are currently being examined for their antifungal properties. Among others, plant extracts from susceptible and resistant pea cultivars have been compared in terms of their components and influence on e growth . A similproducts . Fusarium research can be seen and is expected to develop even more in the future in all areas of fungal biology, pathology and toxicology, especially with the aid of modern techniques to uncover the mechanisms of secondary metabolic regulation and interspecific molecular communication.In conclusion, constant progress in