A fundamentally novel function proposed for extracellular vesicles (EVs) is to transfer bioactive molecules in intercellular signaling. In this minireview, we discuss recent progress on EV-mediated cargo transfer in the central nervous system (CNS) and major gaps in previous studies. We also suggest a set of experiments necessary for bridging the gaps and establishing the physiological roles of EV-mediated cargo transfer. The current state and limitations of research on extracellular vesicles in central nervous system (CNS) signaling are discussed, with particular focus on EV-mediated cargo transfer. EVs can cross the blood-brain barrier under certain conditions such that CNS-originating EVs can enter the circulation and reach other tissues7. Likewise, CNS-resident EVs may come from tissues outside the CNS. EVs enable neurons and glial cells to eliminate excess or harmful membranes and macromolecules11. EVs also mediate intercellular signaling when EV-anchored ligands interact with their receptors displayed on the surfaces of recipient cells15. However, the most fascinating function proposed for EVs is the mediation of intercellular signaling via the transfer of bioactive molecules including proteins, RNAs, lipids, and even entire organelles  are not covered here because they are well established and have been extensively discussed elsewhereaBox 1 Facts and hypotheses of EV-mediated cargo transfer in the CNS22. A typical study of EV-mediated cargo transfer begins with a signaling event elicited by incubation with purified EVs, including changes in cell growth, morphology, metabolism, or gene expression . The cellular response is diminished when EV release from donor cells is reduced or when a putative cargo is deleted56. A notable example is oligodendrocyte-derived EVs that deliver bioactive proteins into neurons to induce metabolic changes58. These studies present exciting observations with the potential to radically change our view of CNS functions. However, despite the progress, much is still unknown about EV-mediated cargo delivery in the CNS.In the past two decades, EV-mediated cargo transfer has been reported in many aspects of CNS physiology60. Hence, a large number of EV fusion events are required to deliver sufficient miRNA molecules to trigger cellular responses in recipient cells. Previous research, however, usually measured crude EV internalization without distinguishing cargoes delivered to the cytosol from those still trapped in the endosome/lysosome. Limited experiments carried out using cultured neuronal and non-neuronal cells showed little or no cytosolic delivery of EV cargoes65. Likewise, cytosolic cargo delivery was detected at very low levels in the CNS using animal models24. The low cytosolic delivery efficiency could be due to a mismatch of EVs and recipient cells  and efficient cargo delivery could occur when EVs are matched with their cognate cell types. Such physiological cognate pairs remain to be definitively established for the CNS.First and foremost, studies of CNS EVs rarely examined cargo delivery to the cytosol of recipient cells. For most signaling functions proposed for EVs, substantial amounts of cargo need to be delivered into the cytosol of recipient cells. In particular, miRNA levels are extremely low in EVs and mammalian cells lack a miRNA-amplifying mechanism66. This issue was often addressed by reducing EV release from donor cells using pharmacological or genetic approaches68. When a signaling event was blunted, the data were often taken as evidence for a role of EVs in the pathway. However, reduction of EV release invariably compromises many other cellular pathways including secretion of non-EV molecules, thus precluding accurate assessment of EV’s role in an intercellular signaling event.Previous studies often sought to determine the EV cargo(es) responsible for eliciting a signaling event, but the evidence was often insufficient. Deletion of a putative EV cargo such as a miRNA from donor cells does not provide a definitive answer because other EV cargoes could be altered as well. Thus, it is difficult to rule out the possibility that a signaling event is mediated by EV ligand-receptor interactions instead of cytosolic cargo delivery. Moreover, a cellular response could be triggered by a soluble molecule co-purified but not associated with EVs. Membrane-free particles often co-purify with EVs and it is challenging to fully separate them70. For instance, Cre proteins or mRNAs can be readily loaded into EVs when a Cre-encoding gene is expressed in donor cells. When delivered to the cytosol of recipient cells, Cre induces recombination of a floxed reporter gene and activates the expression of the reporter70. A major advantage of the Cre/LoxP system is its permanent recording of cytosolic delivery events. Other delivery assays take advantage of engineered luciferase or fluorescent proteins genetically loaded into EVs in donor cells71, permitting quantitative measurements of cytosolic delivery events. If EVs mediate an intercellular signaling event through transferring cargoes, it can be inferred that cytosolic cargo delivery occurs efficiently. Retroviruses are often used to stably express EV cargoes in reporter assays62. To preclude the possibility that EV-mediated cargo delivery is driven by residual viral fusion proteins, neutralizing antibodies against viral fusion proteins should be used as a control. Similarly, if donor cells are transfected with DNA plasmids encoding EV cargoes, caution needs to be taken to remove residual DNA and transfection reagents from purified EVs.To fill the gaps in the studies of EV-mediated cargo transfer, it is crucial to directly examine cargo delivery into the cytosol of recipient cells. Highly sensitive assays have already been developed to detect cytosolic cargo delivery using genetically encoded reportersIf efficient cytosolic cargo delivery is observed, a study needs to determine the cargo(es) responsible for a cellular response. This is a daunting task because as stated above, deletion of a cargo such as a miRNA in donor cells may alter other EV cargoes. This concern could be partially addressed by measuring the protein and RNA profiles of the mutant EVs using proteomics and RNA sequencing. Based on the outcomes of protein and RNA profiling, additional EV cargoes may need to be tested. Moreover, it is critical to adopt stringent EV isolation procedures including density gradient separation and size-exclusion chromatography. To further examine the roles of EVs in a signaling event, EVs or EV subpopulations could be immunodepleted from a sample using antibodies against EV surface markers. In parallel, EV membranes could be disrupted using sonication or detergents .71. Thus, it is tempting to postulate that the fusion of EVs with recipient cells is driven by a fusogen functionally resembling viral fusion proteins. An EV fusogen might be anchored only on EVs, similar to viral fusion proteins, while recipient cells only provide a receptor without directly contributing to the force-generating fusion machinery. Alternatively, an EV fusogen could be a trans-complex formed by proteins rooted in both the EV and recipient cell, analogous to the functions of HAP2 in cell-cell fusion and SNAREs  in intracellular vesicle fusion74. There could be multiple isoforms of an EV fusogen with distinct tissue distributions, enabling EVs to specifically deliver cargoes into their cognate recipient cells. The machinery mediating EV fusion with the plasma membrane may be the same or different from the machinery underlying EV-endosome fusion.The above experiments are essential to interrogate the physiological roles of EV-mediated cargo transfer, but the data obtained are correlative in nature. Ultimately, to definitively address the question, the fusogen mediating EV-cell fusion needs to be identified and perturbed at the molecular level. Like the entry of many enveloped viruses, EV-mediated cargo delivery at the endosome is pH dependent and sensitive to negative regulators of viral fusionOnce the EV fusogen is known, its activity can be selectively disrupted using gene knockout and dominant-negative mutants to determine the role of EV-mediated cargo delivery in an intercellular signaling event. If EV fusogens remain elusive after extensive efforts, an alternative possibility needs to be considered: EVs fuse with recipient cells through spontaneous lipid rearrangements without involving proteinaceous fusogens. Testing this model would require significant conceptual and technical innovations because all known biological membrane fusion processes are driven by proteinaceous fusogens.Despite the controversies and uncertainties associated with EV biology, the potential impacts of the field on fundamental biology and therapeutic delivery are immense. If validated, the significance of EV-mediated cargo transfer would be as high as that of intracellular membrane trafficking, which has been recognized by multiple Nobel Prizes including the 2013 Nobel Prize in Physiology or Medicine. The experiments outlined in this minireview are challenging but critical for establishing the physiological roles of EV-mediated cargo transfer in intercellular signaling. While this minireview focuses on the CNS, all the concepts are applicable to other cell types as well.Further information on research design is available in the Reporting Summary


We propose a novel approach to disperse and extract small-diameter single-walled carbon nanotubes (SWCNTs) using an aqueous solution of riboflavin and Sephacryl gel. The extraction of small-diameter semiconducting SWCNTs was observed, regardless of the initial diameter distribution of the SWCNTs. Dispersion of SWCNTs occurs due to the adsorption of π-conjugated isoalloxazine moieties on the surface of small-diameter nanotubes and interactions between hydroxy groups of ribityl chains with water. During the SWCNT extraction, specific adsorption of riboflavin to SWCNTs leads to the minimization of interactions between the SWCNTs and gel media. Our experimental findings are supported by ab initio calculations demonstrating the impact of the riboflavin wrapping pattern around the SWCNTs on their interaction with the allyl dextran gel. The unique physical and chemical properties of single-walled carbon nanotubes (SWCNTs) promise multiple high-end applications varying from biomedicine to photonics and electronics –3. Rapidn,m) types, solubilizing and isolating specific carbon nanotube geometries remain one of the paramount technological challenges for their potential applications [Despite significant progress toward the synthesis of monochiral and chirality-enriched carbon nanotubes, further improvements are of unmet need even at the laboratory scale ,8. As SWications ,5,8–10.The noncovalent functionalization of carbon nanotubes promotes their individualization due to hydrophobic interactions between nanotubes and surfactant molecules that also facilitate chirality separation. Conventional surfactants such as sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC), as well as polyethylene glycol-based compositions, are used to obtain high-quality dispersions of individual SWCNTs –510. Alt52) is a precursor of such coenzymes as flavin mononucleotide phosphate and flavin adenine dinucleotide [n,m) SWCNTs from dispersions in organic solvents [Flavin compounds are another class of biomolecules that can be potentially utilized as a surfactant for dispersing SWCNTs. Having relatively low solubility in water, flavins are generally innocuous for living cells. Riboflavin - and -SWCNTs, as well as other resolved optical transitions of nanotubes -chirality -nanotubes resulting in the formation of coaxial helices . Our obsRiboflavin wrapping around SWCNTs of different chiralities was simulated according to Sharifi et al. , where tSephacryl gel is an allyl dextran copolymer, and its dextran links act as adsorption sites for SWCNTs in gel chromatography separation . As SWCNIt was found that in the case of dextran in the top position , weak vaThe strength of bonding between neighboring riboflavin molecules is proportional to the type of riboflavin helix around a particular SWCNT. The yield of extraction, as evidenced by the decrease in photoluminescence intensity, strongly correlates with the diameter of the nanotubes and the density of riboflavin molecules on the surface of the nanotubes. -SWCNTs exhibit the highest density of riboflavin packing, which diminishes the binding between riboflavin and allyl dextran deposited between adjacent molecules of riboflavin due to formation of two additional H-bonds with the isoalloxazine groups of riboflavin molecules . As a reSmall-diameter SWCNT–riboflavin conjugates represent a promising class of nanomaterials for cancer treatment and targeted riboflavin delivery –28. It hWe propose a novel preparative approach to enrich SWCNTs with a small-diameter fraction in aqueous dispersions by a single-step extraction procedure. The adsorption of riboflavin molecules governs the separation effect onto SWCNTs, which determines their interaction with adsorption sites of Sephacryl, and consequently, chirality separation. -SWCNTs wrapped by riboflavin exhibit the densest riboflavin packing, which minimizes the binding between riboflavin and Sephacryl in various spatial geometries. Remarkably, this approach is proven to be effective for small-diameter SWCNTs despite the significant difference in diameter distribution of CoMoCat and Tuball SWCNTs.According to computational results, riboflavin-wrapped -SWCNTs have a minimal interaction energy with Sephacryl’s dextran sites of −10 meV/atom; thus, they are easily extracted in one step without the addition of any typical surfactants. Such optimization of the extraction procedure promotes biomedical applications of -SWCNTs since final dispersions do not contain surfactants or organic solvents incompatible with living systems.CoMoCat  and Tuball (≥80% SWCNTs) SWCNTs were received from Sigma-Aldrich and OCSiAl, respectively, and used without further purification. Riboflavin (98%) was purchased from Alfa Aesar and used as received. Sephacryl S-200 High Resolution was supplied by Sigma-Aldrich.40 mg of SWCNT powder was added to a 2 mM riboflavin solution in deionized water and stored for 14 days at room temperature in a dry chamber without exposure to direct sunlight. The final concentration of SWCNTs in dispersion was 1 mg/mL. Then, the dispersions were processed with ultrasonic treatment using Branson 450 digital sonifier at 90 W for 2 h in an ice bath to obtain dark green dispersions. Notably, dispersions were obtained without additional centrifugation step.A riboflavin concentration of 2 mM corresponds to a 1:1 mass ratio between dry SWCNTs and riboflavin. At higher riboflavin concentrations, it precipitates due to its low solubility in water, which leads to poor solubilization of SWCNT bundles. Lower riboflavin concentrations did not affect the dispersion process of nanotubes in any way. To analyze the dispersing ability of riboflavin, the same amount of SWCNTs was dispersed in 1% w/w SDS aqueous solution by the same ultrasonication protocol.A single-step gel extraction procedure was developed based on the size-exclusive gel chromatography method published elsewhere . Medical22 transitions of SWCNTs. Photoluminescence measurements were performed with a Horiba Jobin Yvon NanoLog-4 spectrofluorometer with a nitrogen-cooled InGaAs detector. A Spectra-physics 3900s Ti:sapphire laser was used for excitation at 725 nm. The measurements were performed with the slit bandpass set to 5 nm and an acquisition time of 30 s.UV–vis–NIR spectroscopy measurements were performed using a Perkin-Elmer Lambda 1050 spectrophotometer in the wavelength range of 400–1300 nm to avoid the immense absorption of water in the NIR and minimize the interference of riboflavin absorption with the E17H20N4O6). The DFT within the local density approximation for the exchange–correlation functional employing norm-conserving Troullier–Martins pseudopotentials and double-zeta plus polarization basis set were used for structural relaxation as implemented in the SIESTA package [Each riboflavin molecule was represented as an isoalloxazine group together with a ribityl side chain -, -, and -SWCNTs were considered since they represent distinct values of riboflavin helical densities discussed by Sharifi and co-workers [n,m)-SWCNT, three riboflavin molecules were deposited on its surface .SWCNT extraction occurs in a ternary system of SWCNT, riboflavin, and the dextran sites of Sephacryl gel. Dextran fragments in Sephacryl are described as a C-workers . For eac surface  and rela surface . DextranSupporting Information features additional data on the reabsorption of photons in CoMoCat/riboflavin dispersions and Tuball/riboflavin photoluminescence spectra.File 1Additional experimental data.


Authors reported peak point prevalence of mental health symptoms in HW of 22.2% forpost-traumatic stress, 29.9% for depression and 26.9% for anxiety, consistent with similardata collected around the World. These findings are mirrored in a recent cross-sectional survey conducted in Abu Dhabiin the United Arab Emirates (UAE) at the zenith of the first wave of the pandemic,suggesting that an overall 39% of HW were affected by symptoms of depression, anxiety andstress, 13% of them severely. An email with a link to an online questionnaire was sent out to all UAE’s largestpublic health sector employees, a total of 18,371. A total of 2184 responses were collected.The questionnaire consisted of the Depression and Anxiety Screening Scale (DASS-21) andbasic demographic questions. The study protocol also included a dedicated psychologicalsupport helpline for staff members. However, this study on a self-selected sample of HW wasa telephone-based helpline for HW, and it is not fully comparable.Dufour and colleagues' work investigated the frequency of psychological distress amonghealthcare workers (HW) in the province of Quebec during and after the first wave ofCOVID-19 pandemic.UAE has a similar gross national income and population size than Quebec. Abu Dhabi is thecapital and the largest emirate. The healthcare system is decentralised like Canada albeitprimarily insurance based rather than publicly funded, offering variable degrees ofcoverage. At times of HW crisis (such as a pandemic) it is imperative to ensure that HW haverapid and effective access to health care including available psychological support, anissue actively discussed in Quebec. In both studies respondents were more frequently women.3Abu Dhabi healthcare services offer free psychological support to HW by virtue of atelephone-based helpline. Data from this service during the first wave suggest that 23% ofcontacts were related to COVID-19. Main reasons for contact primarily included COVID-19 workstress, hazardous work practices, exposure to death-related traumatic experiences,loneliness and insecurity, family separation, self-isolation  and job insecurity.Dufour and colleagues utilised a mobile application which incorporated three main mentalhealth wellbeing indicators to understand the evolution of psychological distress over time.The longitudinal analysis of this data indicated that a small number of individuals (∼8%)experienced a delayed onset. This suggests that the impact of contributing factorsfluctuates in relation to virulence and counteractive measures. The authors also identifiedsub-chronic symptoms (∼7%) supporting the notion of the existence of possible fluctuatingtriggers and maintaining factors which could be addressed for those at risk. The authorsfinally concluded that mental health resilience is the predominant response for most HWfacing the challenge of COVID-19 pandemic (∼67%) and that a large number of affectedindividuals fully recover (∼19%). These interventions could include individualized emotional support . Findings from diverse research conducted in different parts ofthe World offers opportunities to identify mental health needs of HW at critical times.It is important to identify those at risk over time as vulnerability fluctuates. Helplinesand mobile applications could potentially identify risks and maintaining factors, detectpsychopathology and offer cost-effective practical interventions aiming at harm preventionand minimisation.


The discovery and quantification of mRNA transcripts using short-read next-generation sequencing (NGS) data is a complicated task. There are far more alternative mRNA transcripts expressed by human genes than can be identified from NGS transcriptome data and various bioinformatic pipelines, while the numbers of annotated human protein-coding genes has gradually declined in recent years. It is essential to learn more about the thorough tissue expression profiles of alternative transcripts in order to obtain their molecular modulations and actual functional significance. In this report, we present a bioinformatic database for interrogating the representative tissue of human protein-coding transcripts. The database allows researchers to visually explore the top-ranked transcript expression profiles in particular tissue types. Most transcripts of protein-coding genes were found to have certain tissue expression patterns. This observation demonstrated that many alternative transcripts were particularly modulated in different cell types. This user-friendly tool visually represents transcript expression profiles in a tissue-specific manner. Identification of tissue specific protein-coding genes and transcripts is a substantial advance towards interpreting their biological functions and further functional genomics studies. NGS platform is particularly essential in the advancement of modern genomic studies that mainly focus on determining genome sequences and deciphering the significant genome features of millions of nucleotide sequences. Genome sequence determination is now the simplest part of genome researches. However, obtaining the comprehensive annotations on all protein-coding gene loci and their gene structures are still challenging3. With the introduction of deep learning-based artificial intelligence (A.I.) machine learning approaches, exon structure prediction accuracy has improved5. Nevertheless, this is not enough to fully comprehend the molecular mechanisms in terms of protein-coding gene modulations. In humans and other higher organisms, manual interpretation and experimental evidence are yet needed to completely decipher transcribed mRNA transcripts of protein-coding genes6; this often involves the generation of alternative transcripts by using complex mRNA transcription maturation mechanisms7. Currently, precisely authenticating all possible alternative transcripts of protein-coding genes is still difficult because of the limitations of NGS platforms. Furthermore, diverse tissue expression modulations of these alternative transcripts complicate the authentication process. Protein-coding genes are regulated by developmental temporal programs and restricted tissue spatial patterns in addition to their common cellular physiological functions9. Because this is a complicated process with variations occurred in different genes, visual informatic tools are desirable to carefully investigate alternative transcript expression profiles in different tissues.With the advancement of next-generation sequencing (NGS) platforms, unprecedented progress has been achieved in the fields of biology and medicine10, the exact human protein-coding gene structures and their mRNA transcript expression modulations would need to be thoroughly investigated in different tissues11. Therefore, we aim to develop a user-friendly web tool for exploring the tissue expression profiles of the alternative transcripts in human protein-coding genes. We used the NGS dataset from the Genotype-Tissue Expression (GTEx) project12; it is a well-known international consortium that provides essential research resources on genetic variations and global RNA expression data in multiple normal tissues. The GTEx project aims to create datasets for the systematic evaluation of genetic variations and examine their relationship with gene expression in multiple tissues14. In the current GTEx data release (V8 version), 54 human tissue subtypes are available. We believe this is a helpful gene expression dataset containing information on various tissue types and a better unified data resource than earlier datasets for the evaluation of tissue expression variations in alternatively transcripts. The use of a single gene expression dataset can help prevent complicated mRNA isoform quantification problems in NGS transcriptome analysis pipelines that occur if heterogenous sources are used. Besides, few databases provide transcript-level alterations with limited spliced transcript information and visualization for users15.Although the sequencing portion of human genome project has been accomplished16, thus, it is desirable to have graphical visualization tools to interactively examine the top-ranked transcript expression in diverse tissue types. We hypothesized that such alternative transcript modulations would have biological significance in protein product expressions, and biomedical researchers would be benefited from visual bioinformatic tools on these data.Therefore, GTEx provides an exceptional resource through the study of transcriptome among various normal tissues. Tissue-specific transcript expression profiles could vary among different tissues for certain genes during development and oncogenesis. Previous tissue expression profile studies often used expression information at the gene level17. One can easily inspect modulations about expressed transcripts of one human protein-coding gene by their ranks as well as ratios among different tissue types. It is useful to observe switch events of top-ranked transcripts in certain protein-coding genes, which would implicate particular modulations on selected transcripts. While this web tool also provides visual tissue expression profiles on human protein-coding genes, it is lacking tissue expression level comparison information to specifically recognize tissue-specific genes or transcripts. There are needs to interrogate the tissue expression profiles on particular mRNA transcripts, since tissue-specific gene or transcript modulation would implicate distinctive biological functions in selected tissue types. Thus, we would like to provide an improved tool for interrogating the representative tissue expression profiles of human protein-coding genes. This new database would enable researchers to explore the top-ranked transcript expression profiles in different tissues as well as identification of significantly expressed genes or transcripts in selected tissues.Previously, we generated a web tool (TREGT) for visually illustrating the expression information on top-ranked transcripts of protein-coding genes using the GTEx dataset14. The GTEx dataset was mainly used to avoid dysregulated expression information regarding cancer cells, and GTEx can provide outstanding tissue expression information from many different human tissue subtypes13. Version 8 of the GTEx dataset was mapped to the 199,324 transcripts and 58,219 genes in the GENCODE 26 human reference set. Within this dataset, there are 150,749 mRNA transcripts belonging to 19,591 protein-coding genes and we mainly interrogate protein-coding genes in this study. Among the 150,749 transcripts of protein-coding genes, there are various transcript types, including protein-coding, processed_transcripts, nonsense_mediated_decay, retained_intron etc. Only 80,354 transcripts are defined as actual protein-coding transcripts by the GENCODE transcript type feature. Herein, we started with 145,571 transcripts of protein-coding genes after removing further 5178 transcript records without expression values as described in the Methods section. A previous study indicated that approximately seven alternative spliced transcripts exist for human protein-coding genes17. Among all the alternative transcripts within a given protein-coding gene, only few transcripts are dominantly expressed. In most cases, the top five transcripts could occupy more than 90% of protein-coding gene expression levels. Subsequently, examination of the expression profiles of alternative transcripts in each protein-coding gene would reveal restricted expression patterns in dominantly expressed transcripts in selected tissue subtypes. Because transcripts could have different coding potential or regulatory significance (such as microRNA target sites)19, the tissue expression profiles of each highly expressed transcripts must be carefully examined. Therefore, we would like to develop a new bioinformatic tool to specifically examine the distinct expression profiles of dominant transcripts in protein-coding genes. We believe these dominant tissue specific transcripts, if translated into protein isoforms, could represent significant biological functions in particular tissues. We designated these tissue specific transcripts as representative tissue transcripts in this database.Studies have used the GTEx dataset for the analysis of alternative transcripts to systematically determine their expression profiles in human noncancer tissuesTo evaluate specifically expressed transcripts as representative tissue transcripts in various human tissues, we applied the standard score (Z-score) criteria to examine the expression data of 145,571 transcripts. The standard score is frequently used for outlier identification and calculated as the original raw data value minus the mean value divided by the standard deviation. Thus, the standard score illustrates the difference in the TPM value between a particular tissue expression amount and the mean average of all tissue types for any given transcript isoform. We applied the Z-score value of ≥ 3 as the cutoff to obtain specifically expressed transcripts in diverse normal tissues. The Z-score value of ≥ 3 indicated that the difference in expression is more than 3 standard deviations in that observed tissue type.We then calculated the respective Z-score value for each transcript of protein-coding genes among different tissue types. Interestingly, we found that over 80% of the 145,571 transcripts had a Z-score value of ≥ 3  in at least one of the 54 tissue types. Only 28,457 transcripts were commonly expressed transcripts in all tissue types, with their Z-score being < 3. This finding indicated that most of the human genes had distinctive tissue expression profiles. Among them, dominantly expressed transcripts found in a single tissue type were the major category, accounting for approximately 50% of tissue-dominant transcripts. In total, 77,606 transcripts had a Z-score of ≥ 3 in a single tissue class . There are reported literatures on the enriched expression protein genes in both brain and testis tissues by large scale proteomic and transcriptomic studies22. Alternative splicing and polyadenylation events were also highly elevated in the brain and testis tissues24. Compared with some other brain regions, the amygdala, hippocampus, and putamen had less than 300 dominantly representative tissue transcripts. Thus, highly differential gene expression profiles were observed even in various tissue subregions (such as the brain), which implied the spatial gene expression patterns.Among the analyzed tissue expression results, the testis, cerebellum, and cerebellar hemisphere had the greatest numbers of dominant tissue expression transcripts Fig. A. The teFor individual transcript expression patterns (TPM levels), the overall profiles differed between tissue types. Hemoglobin subunit beta is the highest expressed tissue-dominant transcript (Rank1 transcript TPM = 5012.79). Although the testis had the maximum number of dominant tissue transcripts, their average expression level was only 1.2. The ovary had the highest TPM expression level of tissue transcripts, with an average TPM value of 28.6  terms were enriched in the acute-phase response, phospholipid efflux, and reverse cholesterol transport  database specifically concentrated on the visualization of the tissue expression profiles of protein-coding genes. The GTEx dataset provides a unique gene expression resource that would be suitable for the functional investigation of alternative transcripts expression in noncancer tissues.We mainly identified dominantly expressed transcripts by their Z-score values according to various tissue types by using an easy-to-recognize graphical illustration. We observed that the majority of significantly expressed transcripts were among the top-ranked transcripts. In the main web entry window, information regarding the top five ranked transcripts was provided to reduce the crowding of display information for genes with many transcripts Fig. . We beliAt the beginning of the web page, users can first choose a particular tissue icon of their study interest Fig. . A new wIn this graphic protein-coding gene expression information page, besides essential gene information, summarized gene functions are listed including protein class; molecular function and disease involvement. Users can further compare the features of all alternative transcripts based on their expression rank, transcript length, ORF length, and dominantly expressed tissues (Z-score ≥ 3) in the transcript list section. In the expression graphic display, the GTEx tissue expression levels of the top 5 ranked transcripts are displayed ; PGA5, PGA4, and GIF genes are expressed in the stomach tissue; and DEFA6, DEFA5, and FABP6 genes are expressed in the small intestine tissue. In addition, genes expressed in particular physiological systems, such as GUCA2B gene, which is expressed more in the small intestine, colon, stomach, and liver, can be observed with our web interface. Therefore, this RTTPG web tool could be beneficial for biomedical researchers in functional analysis.26. Human protein-coding genes have more alternative transcripts than previously estimated3. However, due to the read length limitations of NGS platforms, studying all alternative transcripts, particularly expression profiles among various cell types, may be challenging. With accumulating transcriptome data, concerns regarding the expression and biological significance of these transcripts are arising. It is likely that transcriptional variations would occur inside cells, and not all transcripts would conduct proper biological functions. Intriguingly, recent proteomic studies on the expression and distribution of human protein isoforms have implied that only limited numbers of protein isoforms for protein-coding genes have been detected27. Using several proteomic datasets, Ezkurdia et al. suggested that the majority of protein-coding genes express only a main protein isoform28. The APPRIS database is an excellent web resource for identifying main principal protein isoforms of protein-coding genes in many species29. In a recent comprehensive study using proteomic and transcriptomic datasets, Rodriguez et al. demonstrated that tissue specific alternative splicing events (ASE) in the alternative splicing transcripts and subsequent protein isoform productions in human protein-coding genes30. This phenomena is highly conserved evolutionally in some genes and would have important functional implications. We also observed that overlapping significant tissue types of reported ASE genes with our RTTPG data. Furthermore, our web tool could be valuable in assisting visual interrogation of GTEx tissue expression profiles among different top-ranked transcripts.With the advancement of the NGS technique, scientists have generated a large amount of transcriptome data. Increased transcriptome data have helped scientists to learn more about the expression patterns of human protein-coding genes31 Although more alternatively expressed mRNA transcripts could be discovered using NGS sequence data, limited protein molecules were observed by using the proteomic platform data. This observation might be attributable to the sensitivity and throughput of the current shotgun-based mass spectrometry platform in the detection of all minor protein isoforms in tissues as well as the complicated issues in identification of posttranslational modified peptides. Some translated protein variants might have low abundancy or short half-life. Furthermore, another reason may be the distinctive expression profiles of different mRNA transcripts observed in only particular cell types15. It is likely that transcripts are preferentially expressed in certain cell or tissue types, this is particularly evident in developmental stages9, or pathological malignant conditions32. This point suggests the importance of investigating detail transcript expression in different tissue or cell types. Understanding the difference in expression between each transcript can provide more insights into putative functional effect on different transcripts in respective tissue types. The RTTPG web tool presented here would be beneficial for biologists to perform a thorough analysis of transcripts in many tissue types.This generates discrepancy and debate on the numbers of protein-coding transcripts and their translated products, especially concerns about the actual biological significance of alternative transcripts. 33. Thus, protein coding sequences might not be altered in most transcripts with alternative UTR regions. Furthermore, this opinion supports the findings of proteomic studies regarding a dominant protein isoform28. Our previous findings demonstrated that the dominant transcripts of protein-coding genes were the top-ranked transcripts and often represented the major expression transcripts for most protein-coding genes34. It is suggested that the transcriptome complexity of protein-coding genes may not be as high as estimated earlier based on the number of alternative transcript s identified by NGS reads. Moreover, not all top-ranked transcripts are protein-coding transcripts34. Many alternative transcripts are actually noncoding transcripts, and we observed that only 80,354 of 145,571 these transcripts annotated as protein-coding transcripts by the GENCODE biotype. Therefore, expressed transcript isoforms must be examined in more detail in addition to their tissue expression profiles. This further strength the applications of the RTTPG database.Besides expression levels, our web interface provides additional easy to read transcript information, especially the length and coding protein information. This feature is absent in most web databases. In a recent report, distorted transcription start and termination sites were the main classes in novel transcripts instead of conventional alternatively spliced exon–intron selection usagesWe have utilized the GTEx dataset to establish a web database for visualization of alternative transcripts expression in various normal human tissue subtypes. This web tool would be useful in analyzing distinctively expressed transcripts in a tissue specific fashion. Knowledge learned about the tissue specific expression profiles would be valuable in cellular function analysis for further single cell sequencing studies.12. The data used for the analyses described in this manuscript were obtained from the GTEx Portal on 05/20/20. The transcript expression data file  was retrieved for our study. This public release dataset here do not contain participant information and also follow the NIH Genomic Data Sharing guideline13. Only the gene expression data and average values were used. We have then preprocessed the GTEx public dataset as described previously17. In brief, GTEx datasets were downloaded from their data portal website, which contained 54 different human tissue types from 948 donors. Some tissues were divided into subregions; notably, the brain tissue had the most subtypes14. GENCODE 26 (GRCh38 genome build) was the human gene and transcript annotation standard applied for the GTEx expression analysis pipeline—Version 8. The data file was first pre-processed using Python scripts to generate tissue based expression information according to each transcript and gene. Originally, total numbers of transcript records in this file was 199,324 records. We then removed 5178 transcripts without any expression information among all tissue samples. We used the GENCODE biotype labels to further classify the transcript and genes35. Non-coding genes and their downstream transcript records were filtered and removed. At this stage, we selected only 145,571 expressed transcripts from protein-coding genes for this study, and there are 19,591 protein-coding genes in this processed dataset.The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS17. Python and R packages were used for subsequent statistical analyses36. For selecting distinctively expressed tissue transcripts, we used the standard-score (Z-score) values, which was computed as the raw data value in each tissue minus the average TPM value, divided by the standard deviation. We used the Z-score cutoff of ≥ 3 in order to select distinctively representative tissue transcripts. The Z-score value of ≥ 3 indicated that the difference in expression was larger than 3 standard deviations with a p value of 0.00135.Due to the large numbers of data, the transcript expression information was initially processed and divided according to the different tissue subtypes using Python scripts. We then summarized the average expression values of each transcript within all protein-coding genes by respective tissue subtypes due to the unequal numbers of donor samples. The overall average expression information of every transcript was then tabulated from all GTEx tissues subtypes. Ranking of transcripts in protein-coding genes was mainly determined by their expression values. Accordingly, assignment of Rank1 transcript is annotated to the most abundantly expressed transcript. In some cases, the ORF length or transcript length information of particular transcripts were utilized if the average transcript expression TPM values were identical among transcripts. The transcript with longer ORF length or transcript length was preferably selected and annotated with higher order in Ranks. Expression characteristics of all ranked transcripts in each protein-coding genes were further collected and investigated as reported previouslyThe Z-score value allow us to learn about the significant difference of each transcript in a given tissue type. For further identifying uniquely tissue representative genes or transcripts across all tissues, the coefficient of variation (C.V.) value sorting function was applied to easily reveal tissue-specific transcripts or genes in the gene list user interface. Higher coefficient of variation value would indicate a more unique tissue expression profile among all tissues.The variance between the transcripts among different tissues would indicate the tissue-specific expression patterns.37. The DAVID Bioinformatic Resources 6.8 (https://david.ncifcrf.gov) was used to obtain significantly enriched GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Statistical significance of such pathway enrichment analysis was set with p value of < 0.05. Selected genes were uploaded to the DAVID analysis pipeline, and default parameters were used for identifying enrichment clusters.The DAVID  functional analysis was performed to identify functional enrichment classes38. The software program was obtained from the FunRich web site (http://funrich.org). The top 100 genes of Rank1 transcripts from respective tissues were chosen for enrichment analysis. We used the GO-term biological process function for comparison according to published instructions.We used another published bioinformatic tool, FunRich (3.1.4), which is useful for functional enrichment and gene network analyses39. All transcript expression data on the webserver are stored in a flat file format and loaded into MySQL database for RTTPG web interfaces. In order to further investigate tissue representative transcripts, we provided the list of putative genes based on the tissue subtypes. Several important features of transcripts were displayed for users to examine their significance, including transcript TPM value and ORF region length. The graphic expression page then displayed the 54 GTEx tissue expression information on top five Ranked transcripts. Users can analyze expression information by using TPM or Z-score values. Furthermore, additional gene functional annotations on protein-coding genes were retrieved from the Human Protein Atlas database and processed22. We matched the GTEx and Human Protein Atlas datasets with the Ensembl Gene ID and provided the Gene Name; Gene Synonym; Gene Description; Protein Class; Molecular Function and Disease Involvement features in the individual gene expression page. The web-hosting Docker engine was utilized in an Ubuntu Linux server. All information regarding representative transcript expression is accessed with no restriction and is available at https://rttpg.ibms.sinica.edu.tw.The RTTPG database was implemented using PHP language on an Apache webserver framework in conjunction with the MySQL database as described previouslySupplementary Information.


Posterosuperior rotator cuff tears range among the most common causes of shoulder complaints. While non-operative treatment is typically reserved for the elderly patient with low functional demands, surgical treatment is considered the gold standard for active patients. More precisely, an anatomic rotator cuff repair (RCR) is considered the most desirable treatment option and should be generally attempted during surgery. If an anatomic RCR is impossible, the adequate choice of treatment for irreparable rotator cuff tears remains a matter of debate among shoulder surgeons. Following a critical review of the contemporary literature, the authors suggest the following evidence- and experience-based treatment recommendation. In the non-functional, osteoarthritic shoulder, treatment strategies in the management of irreparable posterosuperior RCT include debridement-based procedures and reverse total shoulder arthroplasty as the treatment of choice. Joint-preserving procedures aimed at restoring glenohumeral biomechanics and function should be reserved for the non-osteoarthritic shoulder. Prior to these procedures, however, patients should be counseled about deteriorating results over time. Recent innovations such as the superior capsule reconstruction and the implantation of a subacromial spacer show promising short-term results, yet future studies with long-term follow-up are required to derive stronger recommendations. In the management of tears of the posterosuperior rotator cuff, characterized by tears of the tendons of the supraspinatus and infraspinatus muscle, there exists consensus that an anatomic rotator cuff repair (RCR) is the most desirable treatment option in active patients in the absence of severe degenerative changes . HoweverIf an RCR is deemed impossible prior to surgery due to an irreparable situation, treatment options such as non-operative management, the debridement of the rotator cuff with or without the tenotomy or tenodesis of the long head of the biceps tendon (LHBT), partial repair, tendon transfers (TTs), the implantation of a subacromial biodegradable spacer (SBS), superior capsular reconstruction (SCR) and the implantation of a reverse total shoulder arthroplasty (RTSA) are considered viable alternatives ,12,13,14The purpose of this article was to review the current literature and to provide a current concepts overview of available contemporary treatment options for irreparable posterosuperior RCTs as well as to present an evidence- and experience-based clinically applicable treatment algorithm.p < 0.05). The range of motion in forward elevation improved from a mean of 40° (range 30–60°) prior to treatment to a mean of 160° (range 150–180°) [Given a failure rate of up to 70% after RCR of chronic and massive tears  and ackn50–180°) . PersistIrreparable RCT may also be treated by an arthroscopic debridement of the remaining rotator cuff in combination with a (reversed) subacromial decompression (SAD) and a tenotomy or tenodesis of the LHBT  21,22,2,222,23. Even if a complete anatomical repair is impossible, a partial repair of an irreparable RCT is an option to restore the rotator cuff’s force couple, which is created by the subscapularis muscle anteriorly and the infraspinatus and teres minor muscles posteriorly. Even in cases of a torn supraspinatus tendon, a sufficient concavity compression of the humeral head into the glenoid can be obtained by a balanced force couple and thus prevent the superior migration of the humeral head . SeveralTendon transfers such as the latissimus dorsi tendon transfer (LDTT) or the lower trapezius tendon transfer (LTTT) are considered viable treatment options in young patients without glenohumeral osteoarthritis (OA) but with functional deficits, especially in terms of external rotation, caused by an irreparable posterosuperior RCT . As the p < 0.001). Additionally, the frequency-weighted mean active forward elevation improved from 101.9° preoperatively to 137.4° postoperatively (p < 0.001), while the external rotation improved from 16.8° to 26.7° (p < 0.001). The overall reported complication rate was 9.5%, including surgical site infection, neurapraxia, hematomas, wound dehiscence and tears of the transferred tendon. Predictors of favorable outcomes included a lower degree of teres minor fatty infiltration, an LDTT as a primary procedure and the presence of an intact subscapularis tendon [In its physiologic function, the latissimus dorsi muscle-tendon unit acts to adduct, internally rotate and extend the humerus. Its large muscle excursion makes the latissimus dorsi feasible for a muscle transfer procedure . Transfes tendon .In 1934, L’Episcopo introduced the combined latissimus dorsi and teres major tendon transfer to regain external rotation in pediatric patients with plexus paralysis. Comparable to isolated LDTT, the transfer of both tendons allows the closing of the rotator cuff defect and allows them act as an external rotator and a humeral head depressor. Especially in patients where the teres minor muscle is already degenerated, the combined teres major and latissimus dorsi tendon transfer might be beneficial to balance the rotator cuff’s compromised force couple . Lichtenp < 0.01) and Disabilities of Arm, Shoulder and Hand (DASH) Score . One patient failed and required shoulder fusion subsequently. All successfully treated patients improved their ability for glenohumeral external rotation. Interestingly, patients with more than 60° of preoperative flexion had more improvement in range of motion than patients with less than 60° of glenohumeral flexion [p = 0.045), patient satisfaction, progression of arthritis (SCR: 22.7% vs. with LTTT: 2.8%) and graft integrity (retear rate: SCR 63.6% vs. LTTT: 8.3%) at minimum 2 years follow-up [The theoretical advantage of the lower trapezius tendon transfer (LTTT) compared to the LDTT is the synergistic function of the lower trapezius muscle and the infraspinatus muscle, sharing a similar force vector trajectory. Accordingly, both muscles contribute to scapular retraction and glenohumeral external rotation . Compare flexion . A more  flexion . At a me flexion . Valenti flexion  investig flexion . Compareollow-up .The SCR has recently been introduced as an alternative treatment for irreparable posterosuperior RCT that is less invasive compared to tendon transfer surgery . BiomechTwo recent systematic reviews of early postoperative clinical outcomes reported convincing clinical results in an early stage ,58. ClinThe SBS, also known as the balloon system, is a preshaped spacer made of a copolymer of poly-L-lactide, which biodegrades over approximately 12 months . SBSs arAs surgeons have become more experienced with reverse total shoulder arthroplasty (RTSA), surgical indications have expanded over the past decade ,75,76. WThe optimal management of irreparable posterosuperior RCT remains a challenge for shoulder surgeons. If possible, the anatomic reconstruction of the torn rotator cuff should be attempted. As discussed above, if anatomic reconstruction is not feasible, several treatment modalities including non-operative and operative strategies are available and should be tailored to the patient’s individual preconditions and functional demand. Despite a large body of evidence on the management of irreparable posterosuperior RCT, there is still controversy about the ideal decision-making algorithm. In this current concepts review article, current evidence on surgical and non-surgical treatment strategies for irreparable posterosuperior RCT was summarized. As a result, a treatment algorithm with clinical steps and decision criteria is suggested to guide the shoulder surgeon in the decision-making for the management of irreparable posterosuperior RCTs .In general, in the setting of an irreparable posterosuperior RCT, an initial non-operative treatment based on a standardized exercise program with the possibility for delayed, consecutive surgical treatment is recommended. However, purely non-operative treatment is often unsatisfactory and is mainly limited to patients not suitable for surgery or elderly patients with low functional demands. For active patients, a more selective approach is necessary as a posterosuperior RCT results in altered glenohumeral contact mechanics with the superior migration of the humeral head and accompanying force couple insufficiency. Consequently, abnormal glenohumeral contact pressure and microinstability cause rapid and early onset glenohumeral OA ,81.Thus, the evaluation of glenohumeral OA in patients with irreparable posterosuperior RCTs based on the classification proposed by Hamada et al.  should bIf a patient with low-grade glenohumeral OA presents with pseudoparesis or a significant loss of function as the chief complaint, the next diagnostic step is an injection of local anesthetics in patients, to determine whether the main reason for the functional deficit is primarily due to insufficient rotator cuff function or secondary to pain. In the case of a resolution of functional deficits upon injection, a partial repair combined with LBHT surgery as well as debridement are indicated. In the situation of persisting deficits following injection, it is important to determine the main limitation in the range of motion or main direction of pseudoparesis . In the case of deficits primarily in flexion or abduction, joint-preserving treatments such as a SCR and SBS implantation or (if possible) partial repair with margin convergence are recommended as possible treatment options, while an RTSA may also be considered in patients age > 65 years. However, although SCR and SBS implantation have become increasingly popular in recent years, as they preserve the native anatomy and do not compromise future treatment options, additional studies with long-term follow-up are required to sufficiently assess clinical outcomes, as existing early clinical outcome data are currently insufficient to provide final recommendations. In patients with deficits primarily in external rotation, an LDTT or LTTT is recommend as the treatment of choice, while a partial repair with a possible augmentation with an SCR may be a viable alternative. Of note, the clinical benefit of the combined LDTT and teres major tendon transfer (L’Episcopo technique) compared to the isolated LDTT is uncertain . In patiIn patients with high-grade glenohumeral OA , only two surgical treatment options are recommended depending on the presence of pseudoparesis. A combined arthroscopic debridement, LHBT tenotomy/tenodesis, and partial rotator cuff repair is considered the first-line treatment in patients with good glenohumeral motion and no pseudoparesis, in case pain is the main symptom and/or functional deficits secondary to pain resolve upon the intra-articular injection of a local anesthetic. If severe functional deficits in any direction are present and persist after the injection of a local anesthetic in patients with high-grade glenohumeral OA, an RTSA is recommended with optional concomitant LDTT in patients with high physical demands and existing preoperative limitations of external rotation.The management of irreparable posterosuperior RCTs remains a challenge for shoulder surgeons and treatment strategies should be tailored to the individual patient. In the non-functional, osteoarthritic shoulder, treatment strategies in the management of irreparable posterosuperior RCTs include debridement-based procedures and reverse total shoulder arthroplasty as the treatment of choice. Joint-preserving procedures aimed at restoring glenohumeral biomechanics and function should be reserved for the non-osteoarthritic shoulder. Prior to these procedures, however, patients should be counseled about deteriorating results over time. Recent innovations such as the SCR and the implantation of an SBS show promising clinical short-term results, yet current comparative studies indicate limited efficacy comparing the SCR or the in-space balloon to partial repair procedures. Taking the much higher costs for these innovative procedures into account, no fundamental recommendation can be given for either the SCR or the balloon. Future prospective comparative studies at long-term follow-up are required to delineate the sustainability of these procedures and derive stronger recommendations.


Exposure to military blast and repetitive head impacts (RHI) in contact sports is associated with increased risk of long-term neurobehavioral sequelae and cognitive deficits, and the neurodegenerative disease chronic traumatic encephalopathy (CTE). At present, the exact pathogenic mechanisms of RHI and CTE are unknown, and no targeted therapies are available. Astrocytes have recently emerged as key mediators of the multicellular response to head trauma. Here, we investigated interface astrogliosis in blast and impact neurotrauma, specifically in the context of RHI and early stage CTE. We compared postmortem brain tissue from former military veterans with a history of blast exposure with and without a neuropathological diagnosis of CTE, former American football players with a history of RHI with and without a neuropathological diagnosis of CTE, and control donors without a history of blast, RHI exposure or CTE diagnosis. Using quantitative immunofluorescence, we found that astrogliosis was higher at the grey-white matter interface in the dorsolateral frontal cortex, with mixed effects at the subpial surface and underlying cortex, in both blast and RHI donors with and without CTE, compared to controls. These results indicate that certain astrocytic alterations are associated with both impact and blast neurotrauma, and that different astroglial responses take place in distinct brain regions. Mild traumatic brain injuries (mTBI), including concussive and subconcussive injuries, are the most common type of TBI, accounting for an estimated 80% of all TBIs . Blast-aIn addition to cognitive and behavior changes, exposure to RHI and military blast is tied to several neuropathological alterations. Athletes with a history of contact sport-related RHI have increased neuroinflammation compared to non-athlete controls, indicated by higher levels of CD68 + macrophages and microglia, that progressively increase in the presence of mild and severe CTE . Other hA growing body of evidence implicates astrocytes in many of these post-traumatic injury processes. Astrocytes interact with blood vessels to regulate BBB integrity , 4, neurIn response to head trauma, astrocytes rapidly undergo structural and functional changes to adopt a reactive phenotype in a process known as astrogliosis , 53. MorAstrogliosis is a hallmark neuropathological feature found in virtually all neurodegenerative disorders , includiIn the present study, we investigated astrogliosis in brain donors after RHI and RHI-related CTE and compared the results to the severity of the astrocytic alterations after blast and blast-CTE. We hypothesized that astrogliosis would be significantly higher in donors with a history of RHI, regardless of co-existing military blast exposure or CTE diagnosis, compared to age-matched controls without TBI or RHI. We also hypothesized that these effects would be most pronounced in impact-vulnerable regions, such as the grey-white matter interface at the depth of the cortical sulci . We specPostmortem human brain tissue was obtained from a convenience sample of 50 donors from 2 brain biorepositories at the VA Boston Healthcare System . The Blast-RHI CTE group included individuals with a history of military blast exposure and non-blast related RHI exposure from contact sports, and a diagnosis of low CTE . The RHI group included individuals with a history of RHI exposure , all from participation in American football. The RHI CTE group included individuals with a history of RHI exposure from American football and a postmortem diagnosis of low CTE .Due to the small number of brain donors with military blast exposure in the UNITE brain bank, donors with sport histories other than American football were included in the Blast-RHI and Blast-RHI CTE groups. As most donors in the UNITE brain bank have RHI exposure from American football, all donors selected for the RHI and RHI-CTE groups had a history of playing American football.The neuropathological diagnosis and staging of CTE followed the NINDS/NIBIB criteria for the pathological diagnosis of CTE , 35, incTissue processing, neuropathological examination, and tissue storage procedures are harmonized across the UNITE and PTSD brain banks. All brain tissue was comprehensively analyzed neuropathologically to detect the presence of any neurodegenerative disease, according to previously described criteria and protocols . BrieflyTissue sections underwent antigen retrieval with AR6  for 20 min at 95 degrees Celsius, then blocked for 30 min in 3% donkey serum in phosphate buffered saline with 0.4% triton (PBST). The primary antibody anti-glial fibrillary acidic protein  was applied for one hour, followed by a 30-min incubation with horseradish peroxidase (HRP)-conjugated mouse secondary antibody. Slides were incubated with a fluorescent dye catalyzed by HRP  for 10 min, followed by the nuclear counterstain Spectral 4’, 6’‐Diamidino‐2‐phenylindole (DAPI) for 5 min before coverslipping with Prolong Gold Anti-fade Mounting Media (Invitrogen). All staining took place at room temperature.All slides were scanned and imaged using a Vectra Polaris (Akoya Biosciences) multispectral fluorescent slide scanner and analyzed using the HALO image analysis software platform version 3.1 . Astrocyte immunoreactivity, measured in terms of mean GFAP fluorescent intensity and percent positive staining area, was assessed in three representative regions of the cortical ribbon in the dorsolateral frontal cortex: at the top of the cortical surface in the subpial glial plate (SGP)  or layerThe selection of each annotated region of interest is illustrated in Fig. Astrogliosis, indicated by level of GFAP immunoreactivity, in each of these representative cortical compartments was quantified using a modified version of the Area Quantification FL analysis settings algorithm in HALO . The algorithm was thresholded based on detection of positive pixel staining while minimizing background. All slides were analyzed using the same analysis algorithm and the percent positive area and mean fluorescent intensity of GFAP-positive pixels in each annotation region at the crest and sulcus were recorded for each image. All analyses were conducted blinded to donor identity and diagnosis.Statistical analyses were run using Graph Pad Prism (version 9). Data were tested for outliers and normality using a Shapiro–Wilk test. Group means reflecting levels of astrogliosis in the different tissue compartments  were compared using one-way ANOVAs with Dunnett’s post-hoc tests for normally distributed data, or Kruskal–Wallis with Dunn’s post-hoc tests for non-parametric data. Potential differences in astrogliosis present at the gyrus versus the sulcus were calculated within each cohort using a ratio of sulcus:gyrus GFAP. Ratios were log transformed and compared using one-way t-tests.We conducted two sets of comparisons, outlined in Fig. p = 0.0297, RHI CTE vs Control: p = 0.0025). The Blast-RHI CTE and RHI CTE groups had significantly lower mean GFAP intensity in this same area . There were no significant differences detected between groups in GFAP percent positivity or intensity within the grey matter immediately subjacent to the subpial plate . All neurotrauma cohorts had significantly higher levels of astrogliosis in terms of mean fluorescent GFAP intensity at the grey-white matter interface compared to controls , though the percent GFAP-positive staining in this region did not reach statistical significance .There was a significant decrease in subpial GFAP percent positivity at the gyral crest among the RHI and RHI CTE groups compared to controls . However, differences were detected between groups within the subjacent cortex and at the grey-white matter interface. Specifically, the Blast-RHI, RHI, and RHI-CTE groups had significantly higher mean fluorescent GFAP intensity in the sulcal cortex compared to controls , and similar changes were found in the Blast-RHI, Blast-RHI CTE, RHI, and RHI-CTE groups compared to controls at the grey-white matter junction .No significant differences in GFAP at the subpial surface were found between groups at the depth of the sulcus in either percent positivity or fluorescent intensity  and RHI CTE (p = 0.0022) groups both had significantly higher percent positivity of GFAP at the sulcus  group also had significantly higher mean fluorescent intensity in the same area compared to the gyrus  had more in the sulcus in terms of percent positivity  and mean fluorescent intensity . Meanwhile, at the GWMI , Blast-RHI (p = 0.0458), Blast-RHI CTE (p = 0.0368), and RHI CTE (p = 0.0241) cohorts had significantly higher mean fluorescent GFAP intensity at the depth of the sulcus compared to the gyral crest .When comparing relative ratios of sulcus:gyrus GFAP within each cohort at the subpial surface Fig. A, D, onlcus Fig. A, while rus Fig. D. Withinrus Fig. B, E, theity Fig. B, while WMI Fig. C, F, the p = 0.048, Blast- p = 0.048, Blast-We found increased GFAP fluorescence intensity indicative of reactive astrogliosis at the grey-white matter boundary in subjects with a history of RHI or blast injury compared to controls without TBI. We also found mixed results, including significantly lower GFAP at the subpial surface at the crest of the gyrus in the same groups compared to controls, but no change at the sulcus. These findings suggest that neurotrauma alone, even in the absence of CTE pathology, might cause astrocytes to alter their GFAP expression , and thaAstrocytes become reactive in response to varying external stimuli, such as microglia-released cytokines , 31 and Our findings support and extend previous studies , 50, 54.We expected all donors with a history of neurotrauma to have elevated astrogliosis compared to controls, as previous studies have shown increases in neuroinflammation, reactive astrogliosis and GFAP expression after head trauma. Furthermore, we expected this increase to be highest in tissue compartments most subject to shearing forces from RHI, such as the grey-white matter junction at the depth of the cortical sulcus. However, not all our results were consistent with these predictions. While the RHI-CTE group did have significantly higher GFAP at the grey-white matter interface in both the sulcus and gyrus compared to controls, when compared within group they also had a lower density of astrogliosis at the sulcal GWMI compared to the gyral GWMI. The loss of GFAP immunoreactivity detected at the gyral subpial surface in the neurotrauma cohorts was also unexpected. It is unclear from the current studies why these discrepant results occurred, as they relied on GFAP as the sole marker of reactive astrocytes.A recent study looking at genetic alterations in the sulcus versus crest in RHI, low CTE, and controls reported altered glial responses as an innate feature of the sulcus . This stThere are several limitations to this study, including the use of restricted cortical sampling and the small number of cases assessed in the Blast-RHI, Blast-RHI CTE, and Control cohorts due to tissue availability. Additionally, the unique nature and heterogeneous mechanisms of blast injury make quantification of exposure challenging and non-uniform. For instance, blast exposure may involve multiple mechanisms of trauma, including blast wave (primary), acceleration of debris (secondary), body displacement (tertiary), and burns, toxic gases, and crush injuries (quaternary). While wThe lack of a CTE-specific effect on GFAP expression observed in the Blast-RHI CTE and RHI CTE groups, compared to their non-CTE counterparts, might be due to inclusion of only mild CTE cases  that have significantly less p-tau pathology than later stage disease . In addiFuture studies should incorporate a multipronged approach using a combination of markers and techniques, including quantitative morphological analyses, to fully capture the effect of neurotrauma on regional reactive astrogliosis . Integra1H-MRS) to detect astrocyte metabolism [1H-MRS study of retired professional football players found significant correlations between clinical cognitive symptoms and different neurochemicals associated with inflammation, including the neurometabolite myo-inositol (mIns) [This work has translational significance considering the potential clinical utility of astrocyte-focused biomarkers. Advanced imaging techniques are used to follow individuals exposed to neurotrauma, such as proton magnetic resonance spectroscopy  , 28, andl (mIns) , 56.In summary, although considerable research has been directed towards neuronal and axonal injury and accumulations of neurodegenerative proteins following head trauma, the role of astrocytic alterations in post-traumatic injury processes are just beginning to be recognized. The results presented here quantitatively highlight increased GFAP expression at the cortical grey-white matter interface as a neuropathological hallmark of mild blast or impact neurotrauma.


Leishmania parasites cause a variety of discrete clinical diseases that present in regions where their specific sand fly vectors sustain transmission. Clinical and laboratory research indicate the potential of immunization to prevent leishmaniasis and a wide array of vaccine candidates have been proposed. Unfortunately, multiple factors have precluded advancement of more than a few Leishmania targeting vaccines to clinical trial. The recent maturation of RNA vaccines into licensed products in the context of COVID-19 indicates the likelihood of broader use of the technology. Herein, we discuss the potential benefits provided by RNA technology as an approach to address the bottlenecks encountered for Leishmania vaccines. Further, we outline a variety of strategies that could be used to more efficiently evaluate Leishmania vaccine efficacy, including controlled human infection models and initial use in a therapeutic setting, that could prioritize candidates before evaluation in larger, longer and more complicated field trials.  Leishmania, obligate intracellular macrophage parasites, are an important genus of parasites that can affect humans and canines for which disease manifestation is dependent upon the infecting parasite species. Each Leishmania species demonstrates a geographic range that is naturally determined by the presence of their specific sand fly vector, and thus differing forms of leishmaniasis are dispersed across endemic regions. Given migration and that the parasites can be transferred in blood, however, cases are occasionally observed in non-endemic regions. Most infections remain asymptomatic  with a key element for parasite containment being an effective antigen-specific T cell response that can prevent advancement to, or resolution from, the diseased state [Leishmania vaccines [ed state ,2. Recovvaccines ,2.Leishmania infection models have served as an important tool for general immunological understanding. Seminal work in the 1980s used these models to define the Th1/2 paradigm, identifying that experimental L. major infection becomes established then typically clears as antigen-specific Th1 cells develop in resistant C57BL/6 mice whereas infection continues unabated despite the Th2 cells that predominate in the susceptible BALB/c mice [+ T cells and reveal genetic mechanisms involved in the development of both disease and adaptive immunity [+ T cells, there is evidence that CD8+ T cells also participate in protection in both experimental and physiological situations [Leishmania infection provides a strong basis for the evaluation of T cell-inducing vaccines and determining their durability.In addition to their own clinical significance, experimental B/c mice ,4. TheseLeishmania targeting vaccines to clinical trial. The sheer volume of potential vaccine platforms and antigen targets identified, the substantial economic impact of producing these as GMP-grade, and the lack of availability of safe and effective adjuvants with which to enhance or sustain responses, has led to reticence in advancing to trial candidates that appear to have ‘room for improvement’ [Leishmania vaccine trials that have disease prevention as an endpoint may be mitigated by a predictive controlled human infection model (CHIM).An increase in the clinical availability and use of antileishmanial drugs has been observed in recent years, with a positive impact observed in the reduced severity of disease especially in the most lethal visceral leishmaniasis manifestation. Such trovement’ ,13,14,15Leishmania, where multiple antigens are known to afford at least some protection in animal models, it may be possible to evaluate leads, then quickly revise them, in response to emerging clinical data. Relative ease in the design and manufacture of nucleic acid-based vaccines also suggests the potential for inexpensive and somewhat generic production. One considerable logistical advantage of RNA-based vaccines over the majority of other platforms is that the RNA can be produced in a cell-free environment by in vitro transcription, removing the need for cultured cells in the manufacturing process and avoiding the quality and safety issues associated with their use. In this way, it is possible to perform simple downstream purification to provide both faster and more cost-effective manufacturing, and robust manufacturing processes have now been established for both mRNA and self-amplifying replicon RNA (repRNA) constructs. Thus, RNA vaccines possess an inherent nimbleness that recombinant proteins of defined subunit vaccines do not. Further, the composition of T cell responses elicited by different vaccine platforms are qualitatively distinct: the intracellular localization of RNA vaccines allows for MHC I presentation and generation of associated CD8+ T cell responses that is not typically observed in response to immunization with subunit vaccines  the SARS-CoV-2 Spike protein was rationally selected as the target antigen given previous work on SARS and MERS , and (b)vaccines . The utirotection.Leishmania vaccine candidates, including live-attenuated or whole-killed parasites (first generation), recombinant proteins (second generation) and DNA vaccines (third generation). Theavailability of genetic information has significantly aided the development process. Several live attenuated Leishmania species have been rendered by genetic modification of critical parasite virulence or survival genes, perhaps most notably the Centrin gene-deleted series that includes L. braziliensis, L. donovani, L. major and L. mexicana parasites [Leishmania. Selected targets have been delivered as formulated protein, or as DNA sequences either alone or within vectors such as adenoviruses or even within Leishmania themselves, and measurements have included cellular immune responses and protection [Numerous diverse technological platforms have been explored as arasites . Signifiotection ,29,30,31otection ,33,34. ARNA technology has the potential to provide an effective and practical solution to vaccine development for a multitude of diseases, including many neglected tropical diseases . RNA vac+ T cell responses, as for example in mice immunized with a naked repRNA derived from a vaccine strain of the alphavirus Venezuelan equine encephalitis virus (VEEV), TC-83, which has a long history in pre-clinical and, more recently, clinical development [The first studies of mRNA and repRNA in the context of immunization demonstrated antigen-specific cell-mediated and humoral-adaptive immune responses against the influenza A ,44,45. Uelopment ,47,48. TM family of highly stable cationic emulsions was developed. LIONTM enables electrostatic association with RNA molecules when combined by a simple 1:1 (v/v) mixing step and the formulation is colloidally stable for at least 12 months when stored at 4 and 25 °C  are supporting the safety of LIONTM formulated repRNA vaccines (unpublished data).In contrast to the relative ease in generating responses in mice (where even naked RNA can generate immunity if larger enough doses are provided), several formulation strategies have failed upon evaluation in primates and humans . Thus, ad 25 °C  and provided immunity sufficient to protect against L. donovani challenge [Building upon a long-term antigen selection program that encompassed cutaneous and visceral leishmaniasis patients, New and Old World leishmaniasis, and associated hallenge . These dLeishmania transmission within much larger overall endemic regions, establishing controlled human infection models (CHIM) provides an opportunity to evaluate vaccines in the context of assured infection rates, with a marked impact in terms of reducing study complexity and cost. For example, estimates suggest that use of a CHIM for sand fly transmitted Leishmania major may require as few as 30 subjects per arm to detect vaccine efficacy of 60%. This compares favorably to the need for many hundreds if not thousands of subjects in conventional natural exposure trials. CHIM studies, by virtue of the known time of exposure, also provide an excellent opportunity to identify immunological correlates of protection and to further understand disease pathogenesis. Such findings from CHIM studies can then be evaluated and used to interpret data within larger field trials. CHIM studies and prospective human infection studies exist for a diverse array of pathogens, including for SARS-CoV-2 [Addressing the on-the-ground reality of inconsistent pockets of local RS-CoV-2 ,68,69,70RS-CoV-2 .Leishmania spp. is extremely well established for both needle challenge and sand fly initiated infection [Leishmania species have been characterized genetically and multiple antigens have been proposed as vaccine candidates; despite improving drugs, drug resistance has and continues to emerge; and finally, establishing well characterized, and preferably GMP compliant, parasite banks for clinical use is being addressed [Leishmania CHIM then present themselves. A study involving non-infected sand fly biting was used to establish parameters for challenge and importantly to gauge and incorporate public perceptions of this type of study into a challenge protocol ) vaccines may be tested as stand alone treatments. In moreLeishmania parasite lysates have been advanced to approval for canine leishmaniasis (CanL). Injection of a vaccine comprising total antigens of Leishmania amazonensis plus saponin (LaSap) to infected dogs alleviated clinical symptoms and reduced parasite loads in the skin for at least 6 months [Leishmania A2 protein plus saponin (LeishTec®) has also indicated a 25% reduction in risk of developing CanL in asymptomatic dogs and a 70% reduction in mortality among younger dogs (<6 years old) [Leishmania braziliensis antigens and TLR4 agonist MPL (LBMPL vaccine) demonstrated reductions in both parasite burden and the intensity of disease in the treated dogs, accompanied by a blocking of their transmission of L. infantum to sand flies (observed in 66% (6 of 9) dogs evaluated 3 months later [In contrast to the human situation, veterinary vaccines based on 6 months . Post-inars old) . Similarhs later . Leishmania-affected dog influences the response to treatment and study outcomes could therefore be impacted by infection level at time of treatment. Supplementation of subunit vaccines with MPL has promoted success, or contrarily has had no impact, in halting disease progression [Leishmania infection to progressively worsening CanL [Leishmania-induced cellular exhaustion could be overcome by potent immunization [It is well established that infection and clinical status of each gression ,82,83. Aing CanL . Ex vivonization . The invnization .L. infantum infected dogs for clinical and parasitological outcomes following short-term treatment with allopurinol, either alone or in combination with a defined subunit vaccine. Dogs treated with allopurinol alone alleviated their CanL symptoms but had only a transient reduction in parasites that rebounded after a few months. Concomitant immunization with Leish-F2 + SLA-SE, however, not only improved clinical status but also elicited L. infantum clearance from lymphoid tissues and systemic organs in the long term (A further complication in providing effective immune therapy may arise from the observation that even after ‘cure’ parasites are retained in very low numbers and maintain immune status. The hypothesis that allopurinol-induced parasite reduction in CanL-affected dogs would render animals more able to develop robust immunity to provide additional longer term control of infection, was tested by evaluating As we have outlined, RNA vaccines could address several current limitations in generating a truly field applicable vaccine for leishmaniasis. RNA vaccines can be made more cost-effectively than either defined subunit or vector-based vaccines and, given the growing familiarity and acceptance of RNA platforms with regulatory agencies, progression to trial is likely to become simpler and more rapid. In this way, we envision that trials can be planned and conducted in a manner more responsive to the identification of relatively small and transient “hot-spots”, permitting a much more efficient design for the evaluation of efficacy. As an alternative, proof-of-concept of safety and efficacy could be generated in human infection models or inferred among patients undergoing therapy.


EFT was further significantly higher in patients with severe liver steatosis versus patients with mild–moderate liver steatosis . Through the meta-regression analysis, we found that patients with increasingly higher blood levels of aspartate aminotransferase displayed an increasingly higher depth of association. The current meta-analysis suggests that EFT may represent a useful surrogate for assessing the presence and severity of NAFLD in a non-invasive manner.We performed a systematic review and meta-analysis to assess the association between epicardial fat thickness (EFT) and nonalcoholic fatty liver disease (NAFLD). This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) and was based on a registered protocol (CRD 4201809 5493). We searched Medline and Embase until December 2021 for studies reporting on the association between EFT and NAFLD. Qualitative reviews, meta-analyses and meta-regressions were performed to explore this association. Effect sizes are reported as standardized mean differences. We included 12 studies, comprising 3610 individuals. EFT was evaluated with trans-thoracic echocardiography in nine studies, two studies using cardiac computed tomography and one study using magnetic resonance imaging (MRI). The presence of NAFLD was evaluated using transabdominal liver ultrasound in nine studies. Other studies used histology, magnetic resonance spectroscopy and MRI-derived proton density fat fraction. Liver biopsy was performed to assess the severity of NAFLD in four studies. The random-effects meta-analysis indicated that, as compared to control patients with lean livers, patients with NAFLD displayed significantly higher EFT (standardized mean difference 0.61, 95% confidence interval: 0.47–0.75,  Epicardial adipose tissue is a visceral fat deposit positioned between the heart and the pericardium. Epicardial fat covers around 80% of the surface of the heart, representing 20% of the organ’s total weight . PutativNonalcoholic fatty liver disease (NAFLD) is defined as excessive lipid accumulation in the liver in the absence of secondary causes of hepatic fat accumulation such as alcohol consumption (<20 g/day in men and 10 g/day in women), viral hepatitis, use of steatogenic medication, or hereditary disorders . NAFLD iLiver biopsy is the gold standard for the diagnostic evaluation of the severity of NAFLD , but thiTo avoid such pitfalls, non-invasive approaches to assess liver fibrosis have been developed, using a combination of clinical–biochemical factors and imaging techniques. Simple non-invasive tools to predict liver fibrosis and liver-related morbidity include the NAFLD Fibrosis Score (NFS) and Fibrosis-4 (FIB-4) score that are endorsed by the European Association for the Study of the Liver (EASL), European Association for the Study of Diabetes (EASD) and European Association for the Study of Obesity (EASO) clinical practice guidelines . These sBoth epicardial fat thickness and liver steatosis can be considered as features of the metabolic syndrome in that they both correspond to the accumulation of ectopic lipids. One could therefore hypothesize their presence to be concomitant. Several studies have addressed this question ,19,20. HThe search strategy, study selection, data extraction and analysis were performed according to a predefined protocol described in the International Prospective Register of Systematic Reviews . This systematic review was conducted according to Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) guideline .We gathered all studies providing a quantitative estimate of the association between EFT and NAFLD presence and severity. The literature search was completed using Medline (1966–December 2021) and EMBASE (1980–December 2021). Within these databases, we conducted structured computerized literature searches using the following search terms: « OR ) AND  OR )». Duplicate entries were excluded. No time limit or language restrictions were applied to the search. Reference lists of retrieved articles were hand-searched to identify additional relevant articles. Two investigators (KG and LO) reviewed the titles, abstracts, and full-text articles. Discrepancies concerning study inclusion were resolved by discussion between the two authors or with a third author (FRJ). We retrieved data on patient age, gender, and on the imaging techniques used to characterize epicardial fat thickness and NAFLD. We used the Newcastle–Ottawa Scale (NOS) to rate the methodological quality of the included studies . The NOS2 value. Analyses were performed with Revman 5.3 (Cochrane Collaboration) and Stata 15 .Because of the various units of measures reported in the individual studies, the primary endpoint of the current meta-analysis was the standardized mean difference in epicardial fat between patients with or without NAFLD. We further compared epicardial fat thickness among (i) patients with severe steatosis versus those with mild–moderate steatosis and (ii) patients with advanced (F3–F4) liver fibrosis versus those with early (F1–F2) fibrosis (when results of a liver biopsy were reported). Random-effects models were used. We did meta-regression analyses to assess the relationship between the calculated effect sizes and patient blood level of aminotransferases. Statistical heterogeneity of the results was assessed with the Cochrane Q test and the IOur search strategy identified 146 unique publications, of which 121 were excluded on the basis of their title and abstract. We examined 25 full-text citations. A total of 12 studies were eventually retained in our systematic review. Overall, data on 3610 patients were gathered. The study selection process is illustrated in The presence of NAFLD was evaluated with ultrasound in nine studies. Magnetic resonance (MR)-imaging-derived proton density fat fraction, MR spectroscopy and liver biopsy were used in one study each, respectively. To quantify epicardial fat thickness, nine studies used trans-thoracic echocardiogram (TTE), one study used MR imaging and two studies used cardiac computed tomography (CT) . As for Critical appraisal of the included studies is shown in p < 0.001), suggesting that epicardial fat thickness was greater in patients with NAFLD. We observed a significant heterogeneity across studies . The primary outcome of interest of the current meta-analysis was the standardized mean difference in epicardial fat thickness between patients with or without NAFLD . A rando2 = 96%), the results indicated that patients with severe liver steatosis display a markedly higher epicardial fat thickness as compared to their leaner counterparts    . Furthergeneity) . p = 0.024). The trend was similar for alanine aminotransferase, though this meta-regression did not reach statistical significance (By using a meta-regression analysis, we found a significant correlation between the calculated effect size and patient blood level of aspartate aminotransferase (ificance A,B.In this meta-analysis of non-randomized studies the pooling data of 3610 patients, we found evidence supporting that the presence of exaggerated epicardial adipose tissue is associated with NAFLD. The vast majority of the studies included in this systematic review consistently showed that epicardial fat was more abundant in patients with NAFLD as compared to the control patients. This association is further supported by the results of two sensitivity analyses, which showed incrementally greater amounts of adipose tissue in patients with severe fatty liver infiltration, and a significant difference between patients with advanced vs. early liver fibrosis. Meta-regression analysis also revealed a significant and positive correlation between the depth of association reported by the individual studies and baseline AST levels.A single study did not find a positive association between epicardial fat and the presence of NAFLD . As mentThe interpretation of a standardized mean difference as an effect size may be challenging in clinical practice  and the A fine-tuned coordination between various organs and tissues is required for the maintenance of systemic homeostasis . The accIt is noteworthy that our work has several limitations. First, our systematic review and meta-analysis included observational studies that by definition carry systematic biases or undetected confounding factors. Such methodological issues limit us from making causal inferences on whether epicardial adipose tissue and NAFLD are both concomitant phenotypes that arise in patients with metabolic syndrome, or whether one of them precedes the other. Another limitation is that the majority of included studies used liver ultrasound to diagnose NAFLD instead of liver histology, which may have resulted in some degree of misclassification. However, liver ultrasound is the first-line diagnostic modality for NAFLD as proposed by the American College of Gastroenterology and the American Gastroenterology Association . On a siIn conclusion, while awaiting more prospective data, the currently available evidence indicates that excessive amounts of epicardial fat may be associated with NAFLD, the degree of liver steatosis, and the severity of liver fibrosis. Future research should identify reproducible thresholds of epicardial fat thickness and evaluate the cost-effectiveness of this approach. Comparing the impact of epicardial fat to that of other forms of ectopic adipose tissue would also address an important gap in the knowledge.


Higher expressions of PDGF receptors were also observed in ovarian cancer patients with venous or lymphatic invasion. Furthermore, we uncovered the prognostic prediction of the PDGF family in ovarian cancer and constructed a PDGF family-based risk prognosis model with a hazard ratio of 1.932  = 1.27–2.95) and P value < 0.01 . Taken together, we demonstrated that ovarian cancers with high PDGF family expression biologically exhibit malignant progression behaviors as well as poor clinical survival, which is attributed to the activated tumor stroma in ovarian cancer.The initiation and progression of cancer depend on the genetic alterations inherent in cancer cells, coupled with the mutual interplay of cancer cells with the surrounding tumor stroma. The platelet-derived growth factor (PDGF) family, as a mesenchymal growth factor, was involved in tumor progression by affecting the surrounding tumor stroma in some cancer types. However, the association of the PDGF family with the ovarian cancer stroma remains elusive. In our study, we first explored the expression pattern of the PDGF family using RNA expression profiles from public databases. We found that the PDGF family was highly expressed in tumor stroma compared with the corresponding epithelial components of ovarian cancer. In particular, PDGF receptors were weakly expressed in ovarian cancer tissues compared with the respective normal tissues; even in tumor mass, PDGF receptors were predominantly expressed by tumor stroma rather than ovarian cancer cells. Importantly, functional enrichment analyses and correlation analyses revealed that the PDGF family was strongly associated with activated stromal scores in ovarian cancer, including higher stromal scores, enriched pathways related to the extracellular matrix (ECM) organization and remodeling, elevated cancer-associated fibroblasts (CAFs) infiltration, and increased tumor-associated macrophages (TAMs) infiltration, especially macrophage M2. Besides, the positive correlations of the PDGF family with CAFs infiltration and macrophage M2 infiltration were observed in other various cancer types. Of note, the PDGF family was also involved in tumor progression-related pathways, such as transforming growth factor  Ovarian cancer is the most aggressive and lethal gynecological malignancy at present, with a projected 5-year survival rate of less than 50% . The higIt has been extensively reported that the initiation and aggressiveness of cancer are determined by the bilateral interaction of cancer cells with the favorable tumor microenvironment (ME), mainly conferred by the tumor stroma . Tumor sα (PDGFRA) and β (PDGFRB)) . There are already several preclinical studies on tumor stroma targeting strategies, such as inhibition of TGF-β signaling, fibroblast growth factor (FGF) pathway, focal adhesion kinase (FAK), and CXC-chemokine receptor 4 [One study previously identified the PDGF signaling pathway as a powerful biomarker that significantly stratifies the survival rates of patients with ovarian cancer from the TCGA database . Given tceptor 4 .n = 111) in our studied cohort reminded us that we should acknowledge the limitations of our risk prognosis model. More prospective cohorts should be recruited to validate the robustness and stability of PDGF family-based signature to predicate survival outcomes in ovarian cancer patients.However, there still exist some limitations in our study. First, all analyses were obtained through data mining of public databases without validation through our clinical samples or fundamental experiments. Second, the specific molecular mechanism of the effect of the PDGF family on stroma activation and tumor metastasis is not available and needs in-depth exploration, which is also our follow-up work. Furthermore, the retrospective samples and the small number of cases (In conclusion, our findings reveal that ovarian cancers with high PDGF family expression biologically resemble metastatic tumors and exhibit a poor prognosis, which is attributed to the activated tumor stroma in ovarian cancers.