Human placenta is a complex and heterogeneous organ interfacing between the mother and the fetus that supports fetal development. Alterations to placental structural components are associated with various pregnancy complications. To reveal the heterogeneity among various placenta cell types in normal and diseased placentas, as well as elucidate molecular interactions within a population of placental cells, a new genomics technology called single cell RNA-seq (or scRNA-seq) has been employed in the last couple of years. Here we review the principles of scRNA-seq technology, and summarize the recent human placenta studies at scRNA-seq level across gestational ages as well as in pregnancy complications, such as preterm birth and preeclampsia. We list the computational analysis platforms and resources available for the public use. Lastly, we discuss the future areas of interest for placenta single cell studies, as well as the data analytics needed to accomplish them. The stromal core contains a range of cells including macrophages , mesenchymal stromal cells, fibroblasts, and fetal endothelial cells . HofbaueOther cells may also exist at the maternal-fetal interface. Uterine natural killer cells (uNK) constitute a major lymphocyte population in the uterus during early pregnancy. Their number increases around the time of implantation and remains high during the first trimester of pregnancy. uNK cells play a role in protecting the conceptus from maternal immune rejection and have been implicated in placental vascular remodeling .The placenta plays a pivotal role in maintaining the health of both the fetus and the mother. Normal growth and development of placental structural components ensures a healthy pregnancy outcome, whereas alterations to placental structural components are associated with various pregnancy complications such as intrauterine growth restriction (IUGR), preeclampsia and preterm birth. These pregnancy complications and syndromes are associated with increased fetal and neonatal morbidity and have long-term negative impacts on the health of newborns. IUGR refers to a condition in which the fetus does not achieve the expected in utero growth potential. Fetal growth is a complex process that involves genetic and environmental factors, nutrient availability from the mother, as well as hormones and growth factors. The complications of IUGR include perinatal asphyxia, hypoglycemia, hypothermia, cerebral palsy, and pulmonary hypertension of the newborn . While the etiology of preeclampsia is not fully understood, risk factors are proposed including hypertension and kidney disease, diabetes, obesity, a history of preeclampsia, aberrant placental implantation, and environmental and genetic factors. Preeclampsia has been linked to IUGR, as both conditions are assumed to be related to the similar placental disorder due to reduced trophoblast invasion  in the urine in the mother , fluorescence-activated cell sorting (FACS), magnetic associated cell separation (MACS), and microfluidics, each with its own strengths and weaknesses.in situ, which offers the opportunity to restrict expression analysis to spatially defined subareas within the tissue. Pavličev et al. used LCM method to enrich SCTs from fresh-frozen sectioned human placentas and analyzed SCT-specific genes  human placentas, and successfully mapped the single-cell reads to the corresponding donor-specific reference of KIR alleles  tailing or by a template-switching approach, and cDNA amplification using PCR or byThere are a few next-generation sequencing platforms. Illumina sequencers are by far the most popular, with a claimed more than 70% dominance of the market, and have produced the majority of the publicly available human sequencing data. In 2015, BGI launched the BGISEQ-500 as an alternative to existing sequencing platforms, with lower sequencing cost. Sequencing performance comparison between the Illumina platform and the BGISEQ-500 showed that the two platforms have comparable sensitivity and accuracy in bulk RNA seq, small noncoding RNA seq, whole genome DNA seq, and scRNA-seq . In order to differentiate fetal and maternal origin, they only included male fetus samples. Twenty distinct cell populations (9 from villi and 11 from decidua) and uncovered previously unknown subtypes of the fibroblast-like (FB) cells were identified using unsupervised clustering and differential gene expression analyses. Gene profiling showed that all cell types identified in the villi sample expressed Y chromosome-encoded genes, indicating fetal origin of these villous cells, whereas X-linked gene XIST was only expressed in cell types identified in the decidua samples, indicative of their maternal origin. SCTs expressed genes involved in the production of gestational hormones, including CGA, CYP19A, HSD3B1, and GH2, that support the fetal and placental development. EVTs specifically express ECM genes, consistent with their role in invasion and mobility. Three villous FB subtypes were identified, which specifically express the genes associated with endothelial cell migration, angiogenesis, and the regulation of fluid homeostasis. Two decidual FB subtypes were also identified, which expressed the genes involved in cell adhesion and lipid metabolism. These observations indicate the influence of the microenvironment on FB cell fate determination. In addition, a proliferating and a resting NK subpopulation were differentiated through gene expression and gene ontology enrichment analyses. Receptor-ligand interaction analysis between and within villi and decidua showed abundantly expressed receptors and ligands for each cell type of villi and decidua, providing insights into the cell–cell interactions in the fetomaternal microenvironment.In the first-trimester, human placenta and decidua undergo dynamic changes to enable the establishment of a successful pregnancy. Four scRNA-seq studies so far have investigated the gene expression in human placentas from early pregnancy . Alignment of scRNA-seq reads with overlapping single nucleotide polymorphisms called from fetal and maternal genomic DNA showed that placental samples contained mostly fetal cells with some maternal macrophages, whereas most cells from decidual samples except a few EVTs were of maternal origin. Unsupervised clustering and differential gene expression analyses identified two clusters of decidual fibroblast cells (PV1 and PV2), three clusters of decidual stromal cells , and three subsets of decidual NK cells . The localization of decidual fibroblast cells and stromal cells was verified by immunohistochemistry as well as multiplexed single-molecule fluorescent in situ hybridization, and the presence of decidual NK cells was confirmed by flow cytometry. PV1 cells expressed high levels of genes involved in cell adhesion, while genes associated in tissue remodeling are higher in PV2 cells. All subsets of decidual NK cells express genes encoding proteins involved in anti-inflammatory roles, which suggests a ential role of these cells in regulating the extent of the trophoblast invasion. Expression analysis of the KIR gene family using the KIRid method indicates a likely function of dNK1 in the recognition and response to EVT. Trajectory modeling and pseudotemporal ordering of cells predicted two distinct trophoblastdifferentiation pathways, with the decidual EVTs being at the end of the trajectory and expressing high levels of HLA-G. In addition, they developed a statistical tool named CellPhone to identify the specific interactions between decidual NK cells and invading fetal EVTs, maternal immune and stromal cells. Using this tool, they predicted the ligand–receptor interactions that may control the differentiation processes of trophoblast cells to SCTs or EVTs  and second-trimester (24 weeks of gestation) placentas. Unsupervised clustering analysis and differential gene expression analysis of maker genes revealed 14 subtypes of placental cells which vary with the different gestational stages. Specifically, they identified three CTB subtypes  from the first-trimester placenta. These CTB subtypes have various degrees of proliferative capacity, which may serve as the pool of CTBs and progenitor cells of the STBs, respectively. CTB_8W_1 cells, with the least proliferative capacity, expressed high levels of potentially important fusion genes, indicating that they may be the fusion-competent CTB cells. They also identified five EVT subtypes . Gene ontology analysis and immunostaining revealed that EVT_8W cells with proliferative potential were localized at the proximal end of the placental cell column, while EVT_8W cells involved in receptor activity regulation and the immune response were found to be at distal end of the placental cell column. Gene ontology analysis of EVT_24W cells showed that one subtype may be associated with the response to wounding, digestion and immunomodulation, while the other subtype related to growth regulation, gonadotropin secretion and pregnancy. A pseudotime analysis of CTB_8W, EVT_8W and EVT_24W cells predicted a differentiation pathway of CTB_8W_2 cells to CTB_8W_3 cells and then to EVT_8W and EVT_24W cells, which is consistent with the study by Placental cellular lineage commitment occurs mainly during the first- and second-trimester pregnancies. The process of invasion of EVTs from anchoring villi into the uterine spiral arteries is also completed by 20 to 22 weeks of gestation in normal pregnancy  with unique crosstalk at the fetal-maternal interface. Gene ontology enrichment analysis showed that the top enriched processes include cell proliferation and adhesion in trophoblasts, cell differentiation and signal transduction in stromal fibroblasts, inflammatory responses in Hofbauer cells, and cell migration and angiogenesis in endothelial cells. Cell-type-specific upstream regulators were predicted by Ingenuity pathway analysis. In addition, a number of chemokine genes were found to have sex-differential expression in trophoblasts and Hofbauer cells, suggesting a sexual dimorphism of immune responses in the placenta , and the smaller cluster as EVT. These trophoblast clusters likely reflect the continuous differentiation process of cytotrophoblasts. The expression profiles of iVCT cells suggest that they are a very heterogeneous group and that the differentiation proceeds iVCT stage. In addition to the identification of cell clusters, they inferred the cell–cell communication network at the maternal-fetal interface using public databases of ligand-receptor relationships based on the mRNA expression. A large number of interactions were found to be between decidual cells and the SCT cells. Further, they showed that the cell-type specificity of G-protein-coupled receptor (GPCR) profiles may characterize the cell type identity. Tsang et al. analyzed single-cell transcriptomes of more than 24,000 cells isolated from full-term and second-trimester preeclamptic placentas. Clustering analysis by t-SNE identified diverse placental cell types and comparison of the ratio of transcripts carrying fetal-specific SNP alleles with those carrying maternal-specific SNP alleles showed that most clusters were of fetal origin. Gene profiling analysis showed that SCTs specifically expressed genes involved in the production of important gestational hormones, and EVTs expressed HLAs associated with maternal immune tolerance of the fetus with uNKs. In addition, they predicted the differentiation relationship of trophoblast cells using clustering and pseudotime analyses. They showed that SCTs expressed high levels of the genes involved in cell fusion and identified several genes that could serve as regulators in SCT development. Analysis of the EVT developmental pathway revealed upregulation of various extracellular enzymes associated with invasion and migration. Importantly, comparison of transcriptomic profiling of biopsies taken from different sites of the placenta showed spatial heterogeneity, consistent with previous bulk tissue transcriptomic profiling .More recently, Pique-Regi Placenta is a vascular organ with maternal and fetal origins and various cell types. The human Placenta scRNA-seq studies reviewed here reveal that vast transcriptional differences exist among different loci of placentas, between various cell types, gestational ages as well as in pregnancy diseases. These studies open up the possibilities to better understand cellular networks and pathways in cells of normal and complicated pregnancies, which could help to discover the underlying causes of adverse pregnancy outcomes.However, one needs to be cautious before attempting scRNA-seq studies using human placenta. Access to normal human placenta before delivery is challenging due to ethical concerns, limiting the kind of studies that can be conducted. Upon delivery, various factors need to be taken in consideration including hospital procedures, mode of delivery and sample sizes. Placenta grows and constantly adapts to the maternal environment to ensure a healthy pregnancy, thus static views of placenta cell proportions and gene expression are incorrect. Temporal, spatial and between individual variability have to be considered. Fresh samples are limited to deliveries and planning is critical. This also comes with challenges regarding deconvoluting disease state vs batch variations. To get around these issues, single-nucleus RNA sequencing (snRNA-seq) has arisen as an alternative to single-cell methods. snRNA-seq does not require enzymatic digestion, which reduces dissociation artifacts. In addition, unlike scRNA-seq which requires the preparation of a live single cell suspension from fresh tissue which inevitably introduces batch effects, snRNA-seq can be used for frozen tissues, enabling single-cell gene profiling of archived clinical samples simultaneously, reducing batch effect and increasing power of the study by allowing for more biological replicates   resolution of gene expression data can be spatially mapped back to the tissue at 2D level  will be very useful to delineate the coherent changes from epigenetic to transcriptomics level , R01 LM012373 and R01 LM012907 awarded by NLM, and R01 HD084633 awarded by NICHD to L X Garmire.This work was supported by grants K01ES025434 awarded by NIEHS through funds provided by the trans-NIH Big Data to Knowledge (BD2K) initiative (L X G envisioned and supervised the project. H L wrote the majority of the manuscript, with significant help from Q H, Y L and L X G.


Chemotherapy agents used in the standard treatments for many types of cancer are neurotoxic and can lead to lasting sensory and motor symptoms that compromise day-to-day movement functions in cancer survivors. To date, the details of movement disorders associated with chemotherapy are known largely through self-reported symptoms and functional limitations. There are few quantitative studies of specific movement deficits, limiting our understanding of dysfunction, as well as effective assessments and interventions. The aim of this narrative review is to consolidate the current understanding of sensorimotor disabilities based on quantitative measures in cancer survivors who received chemotherapy. We performed literature searches on PubMed and found 32 relevant movement studies. We categorized these studies into three themes based on the movement deficits investigated: (1) balance and postural control; (2) gait function; (3) upper limb function. This literature suggests that cancer survivors have increased postural sway, more conservative gait patterns, and suboptimal hand function compared to healthy individuals. More studies are needed that use objective measures of sensorimotor function to better characterize movement disabilities and investigate the underlying causes, as required for developing targeted assessments and interventions. By updating our understanding of movement impairments in this population, we identify significant gaps in knowledge that will help guide the direction of future research. Chemotherapy agents used in the standard treatments for many types of cancer—including platinum compounds, taxanes, and vinca alkaloids—exhibit neurotoxic adverse effects. Depending on individual compounds, chemotherapy can damage the nervous system via various mechanisms  . These aThe prevalence of CIN varies from 19% to more than 85%, with the highest reported for platinum compounds 70–100%) and taxanes 11–87%) . Althoug0–100% an–87% . AlDescriptions of movement dysfunction associated with chemotherapy have come largely from self-reported symptoms and functional limitations, with few quantitative evaluations of movement function. Patient-reported outcome measures are the common clinical tools for assessing chemotherapy-induced neurotoxicity . These mWith the rising number of long-term survivors of cancer , there iWe performed a literature search on 5/15/2020 in PubMed, with a combination of search terms including derivations related to movement deficits  and the disease . Six hundred and eighty-six articles were identified from the search. Articles were included if they met all of the following inclusion criteria: (1) published within 2000–2020; (2) human subjects of any age, any cancer type; 3) most of the participants had received or were receiving neurotoxic chemotherapy, including platinum compounds, taxanes, and vinca alkaloids; (4) provided quantitative and instrumented assessments of movement deficits; (5) published in English. Articles were excluded if they (1) were a review or abstract; (2) assessed movement deficits only based on patient-reported outcome measures, functional outcome measures, or electrophysiological methods. Sixteen articles were selected after reviewing the titles and abstracts. We then used the ‘Similar Articles’ feature of Pubmed and identified 127 additional articles using Kneis et al. 2016  as the s most of Thirty-two movement studies related to chemotherapy-induced neurotoxicity were identified. All of the reviewed movement studies focused on one of the three areas of movement function: (1) balance and postural control; (2) gait function; (3) upper limb function; therefore, we organized the results and discussion using these three themes.Postural imbalance is one of the most common movement dysfunctions reported by cancer survivors. The ability to maintain postural balance, therefore, is an area commonly investigated in cancer survivors. Maintaining postural balance is a complex process involving various components of postural control, including a neural representation of body segments and position of the center of gravity, multisensory inputs that monitor the orientation and stability of body segments, and reactive or anticipatory responses for balance recovery after perturbations or postural stabilization during voluntary actions . Most ofThese existing spontaneous sway studies suggest that there is excessive postural sway in cancer survivors, especially in the ML direction, but the factors underlying the amplified postural sway in this population have yet to be clarified. One hypothesis is that excessive postural sway is caused by the peripheral sensory neuropathy associated with CIN. This hypothesis is well motivated as the somatosensory system contributes more to postural stability than the visual and vestibular systems . The perThe correlational studies, however, do not suffice to conclude a causal relationship between peripheral sensory deficits and excessive postural sway or rule out other contributing factors. In fact, McCrary et al. 2019 found that cancer patients, regardless of sensory symptoms, had greater postural sway compared to age-matched normative values. Among the five factors contributing to increased postural sway , only two were related to peripheral sensory deficits . These rPostural balance depends on the integration of sensory inputs from the somatosensory, visual, and vestibular systems to elicit appropriate motor responses . AlthougIn summary, studies of sensory integration have revealed that cancer survivors underutilize somatosensory feedback for postural control, likely due to CIN-related somatosensory deficits. As a compensatory strategy, cancer survivors increase the weight of the visual and vestibular systems, but the summarized evidence indicates that this strategy compensates incompletely for the deficits in the somatosensory system during static standing. The extent to which the visual and vestibular dysfunction contribute to postural instability remains unclear, as few of the reviewed postural studies performed rigorous tests of these systems. Likewise, few studies performed detailed assessments of the motor system. Future studies should consider how the CIN-related motor function changes  affect postural stability. Kneis et al. 2020 presented a useful paradigm for investigating sensory integration strategies adapted by cancer survivors. However, the study was based on a small sample with severe balance deficits, so the conclusion cannot be extrapolated to cancer patients with different levels of CIN severity. The sample also consisted of mixed cancer types and treatments; whether there are cancer type-related, treatment-related differences, or interaction effects  remains Falls are common in cancer survivors. It is estimated that about 30% of cancer survivors fall every year , and indSimilar to postural instability, this impaired gait pattern was shown to be associated with CIN-related neuropathy. Winters-Stone et al. 2017 found a significant association between lower walking speed and increasing numbness/tingling and discomfort in feet . Zahiri Cancer survivors with CIN demonstrated conservative gait patterns characterized by slower gait speed, shorter step length, longer double support time, and greater ML gait variability. These altered gait patterns have been linked to somatosensory deficits associated with CIN , 49, butIn summary, current evidence on the underlying causes of gait abnormality in cancer survivors remains limited. Musculoskeletal deficits at the ankles, including reduced range of motion and strength and increased muscular co-contraction, contribute to altered gait patterns in pediatric cancer survivors, but further kinematic and kinetic gait analyses are warranted to determine if similar musculoskeletal changes occur in adult cancer survivors. Future gait studies should also investigate how chemotherapy-related changes in the central nervous system  contribute to gait impairments.CIN-induced sensorimotor dysfunction not only contributes significantly to balance and gait dysfunction in cancer survivors but also plays a significant role in upper extremity dysfunction. Particularly, cancer survivors with CIN report difficulties with skilled hand function such as typing, writing, and buttoning a shirt , 65–68, In summary, these two studies provide preliminary evidence of suboptimal hand function linked to CIN-related sensory and motor disturbances. However, since thumb-index grasp and handwriting only represent parts of skilled hand function, further studies are needed to investigate other skilled hand function and manual dexterity  and how they are affected by CIN. Furthermore, neither study considered the compensatory effect of vision on task performance, thus potentially misidentifying the functional significance of CIN-induced sensory and motor dysfunction. Although incorporating vision is more functionally relevant and takes hand-eye-coordination into account, identifying the relative contribution of sensory and motor dysfunction independent of vision can be useful for identifying targets of intervention.Chemotherapy-induced neurotoxicity produces unique sensory and motor symptoms that contribute to dysfunction in postural control, gait, and upper limb function. While further research is warranted to fully characterize CIN movement dysfunction and its underlying causes, researchers should also consider other side effects of cancer and treatments, including fatigue, cognitive changes, and pain, when designing future studies. Cancer-related fatigue is common, with most studies reporting prevalence rates above 60% . Cancer In summary, individuals with cancer might present with other side effects add to the CIN-induced sensory and motor symptoms. Side effects like fatigue, cognitive dysfunction, and pain could complicate the interpretation of movement dysfunction. Researchers should consider monitoring these side effects, if not controlling for them when investigating movement dysfunction linked to CIN.Motivated by improving the management of chemotherapy-related movement dysfunction, this literature review evaluated 32 studies and consolidated the knowledge of common movement disabilities in cancer survivors who received chemotherapy. Overall, cancer survivors with chemotherapy-induced neurotoxicity have been shown to present with increased postural sway, conservative gait patterns, and suboptimal hand function, but the current understanding of CIN-related movement function changes is far from comprehensive.We identified a number of areas where more information is needed. Cancer survivors with CIN report a wide range of dysfunction in gross mobility  and fine motor skills  –68, 77. There are currently no effective treatments for CIN. Many early reports suggest a possible beneficial effect of exercise In summary, we identify significant knowledge gaps in CIN-related movement dysfunction and recommend frameworks for future clinical studies. Filling these gaps will help improve the clinical understanding of CIN-related movement dysfunction and guide the development of targeted assessments and treatments.


Aptamers are a novel technology enabling the continuous measurement of analytes in blood and other body compartments, without the need for repeated sampling and the associated reagent costs of traditional antibody-based methodologies. Aptamers are short single-stranded synthetic RNA or DNA that recognise and bind to specific targets. The conformational changes that can occur upon aptamer–ligand binding are transformed into chemical, fluorescent, colour changes and other readouts. Aptamers have been developed to detect and measure a variety of targets in vitro and in vivo. Gonadotropin-releasing hormone (GnRH) is a pulsatile hypothalamic hormone that is essential for normal fertility but difficult to measure in the peripheral circulation. However, pulsatile GnRH release results in pulsatile luteinizing hormone (LH) release from the pituitary gland. As such, LH pulsatility is the clinical gold standard method to determine GnRH pulsatility in humans. Aptamers have recently been shown to successfully bind to and measure GnRH and LH, and this review will focus on this specific area. However, due to the adaptability of aptamers, and their suitability for incorporation into portable devices, aptamer-based technology is likely to be used more widely in the future.  Gonadotropin-releasing hormone (GnRH) is decapeptide secreted into the hypophyseal portal system by specialised hypothalamic neurons, to reach its site of action at the pituitary gland. GnRH acts on pituitary gonadotropes to stimulate the production and secretion of gonadotropins (luteinizing hormone (LH) and follicle stimulating hormone (FSH)) . LH and GnRH, LH and FSH all exhibit pulsatile secretory patterns, which are pivotal to their biological functions , includiGnRH and LH pulsatility change during different reproductive stages and at different phases of the menstrual cycle. Pre-pubertally, LH levels are low and very few LH pulses are detectable. However, as puberty progresses, LH pulse frequency increases in response to increased GnRH pulsatility . In adulANOS1 gene variations in Kallman’s Syndrome)  are more difficult to select appropriate aptamers for ,92. As G15 molecules, which then require post-SELEX optimisation [There are currently no standardized aptamer selection protocols for a given target with over 20 different types of SELEX procedures described in the literature . Additiomisation ,68,96,97Aptamer development is a rapidly expanding field with numerous potential research, diagnostic and therapeutic applications. Due to the many advantages of aptamers over traditional antibody-based detection and quantification methods, adoption of aptamer technology into routine use in research and healthcare settings may occur in the near future as technical challenges are overcome. Aptamer-based technology has the potential to be a powerful research tool, as well as to enable real-time, continuous, low cost ambulatory LH pulsatility assessments in clinical settings. This could transform clinical practice by providing more accurate diagnosis of reproductive disorders (currently based on single timepoint blood tests that do not assess LH pulsatility), and thus improve the management of these conditions.


While hemorrhage is commonly encountered in various intracranial tumors, it is relatively rare in benign meningiomas. We present an interesting case report of a 75-year-old male who fell during intoxication, sustaining right frontal cranial trauma, coincidentally directly overlying a previously undiagnosed right frontal meningioma. He experienced an acute neurological decline and was found to have an intracranial hematoma, causing significant mass effect and herniation. Based on the radiographic appearance, an underlying neoplasm with significant edema was suspected. Intraoperatively, the hematoma and mass were successfully evacuated, and post-operative pathology confirmed the presence of a World Health Organization Grade I meningioma with a microcystic and angiomatous pattern. We speculate on the mechanism of injury and hemorrhage in this patient through review of the literature and discussion of medical and pathological risk factors. Meningiomas are neoplasms theorized to arise from the arachnoid cap cells, which surround and support the brain and spinal cord. Though comprising almost one-fifth of all primary intracranial tumors, intratumoral hemorrhage associated with meningioma is markedly rare . For comIn this report, we describe a unique case of an intratumoral hemorrhage within a frontal parafalcine meningioma, arising from a fall producing a direct frontal impact. To our knowledge, this is the first report of an intratumoral hemorrhage caused by direct impact trauma.Clinical presentationA 75-year-old intoxicated male with a past medical history of diabetes mellitus, hypertension, and alcohol use disorder presented to the hospital after an unwitnessed fall down one flight of stairs. Initially, the patient was measured as a Glasgow Coma Scale (GCS) score of 15 at the scene, but the patient declined neurologically to a GCS of 12 at presentation to our facility. Notably, he was drowsy, disoriented with unintelligible speech, and demonstrated left hemiplegia. Computed tomography (CT) of the head without contrast revealed a large right frontal, extra-axial, hemorrhage resulting in early uncal and subfalcine herniation (Figure After imaging was obtained, the patient’s level of consciousness continued to decline, requiring endotracheal intubation for airway protection. He was treated with hyperosmolar therapy en route to the operating room for emergent decompression.Intra-operative findingsThe patient was positioned using a Mayfield horseshoe device. A standard reverse question mark incision was made and a right frontal craniotomy was performed, followed by a curvilinear durotomy. A right frontal hematoma was immediately visible on the surface with gross necrotic and hemorrhagic debris evident upon entry. During evacuation of the interior of the hematoma, a thick fibrous surrounding capsule was encountered, and specimens were sent for histopathologic analysis. Additionally, a traumatic laceration of the anterior portion of the superior sagittal sinus and an associated draining vein were encountered, which necessitated repair due to ongoing hemorrhage throughout the evacuation. Once hemostasis was achieved, closure was performed in a standard multi-layer fashion without further event.Post-operative courseThe patient was taken to the intensive care unit in stable condition. Post-operative CT head without contrast demonstrated successful resection of the lesion and related hemorrhage Figure . Post-opThe patient described in this case report exhibits several proposed risk factors for hemorrhage including trauma, hypertension, diabetes mellitus, hematologic dyscrasia secondary to alcohol use disorder, convexity tumor location, and angiomatous histopathology. Given the evidence of a frontal subgaleal hematoma signifying the exact location of impact, and the directly underlying hematoma, we speculate that the opposing acceleration/deceleration forces and the tethering of the dense meningioma mass sheared bridging and intratumoral vessels, causing the hemorrhage. The intraoperative presence of the sagittal sinus and bridging vein laceration support this this hypothesis. Given the microcystic angiomatous pattern of the meningioma, and other pre-existing risk factors, the bleeding was perpetuated.While the meningioma itself was moderate in size, and the degree of trauma was otherwise considered mild, the combination of hemorrhage in the setting of significant pre-existing vasogenic edema caused rapid progression towards brain herniation. The phenomena described here is an excellent manifestation of the Monro-Kellie hypothesis, which suggests a rapid rise in intracranial pressure at a point where autoregulation has been exhausted. In this case, neither the tumor nor the trauma by itself would have been expected to cause such a severe clinical presentation due to regulatory mechanisms. However, the combination of tumor mass effect, pre-existing vasogenic edema, traumatic brain injury, and intratumoral hemorrhage likely overwhelmed regulatory capacity, thus causing rapid neurological decline.It is important to note that only one case report attributes the etiology of their hemorrhagic meningioma to minor head trauma whereas the majority of studies emphasize nontraumatic risk factors. Thus, while listed as a potential risk factor in several studies, trauma has since been considered a “trivial” etiology . The patWithin the literature, most of the hemorrhagic meningiomas presented with either an extratumoral component only or combined extra-and intratumoral hemorrhage ,9. ExampThe radiographic pattern of hemorrhage on initial presentation is important to scrutinize to maintain an accurate differential diagnosis and avoid delayed treatment. Early diagnosis and appropriate treatment strategy can decrease the mortality rate as low as 13.9% for hemorrhagic meningiomas . When siThe pathology ultimately resulted as a WHO Grade I meningioma. Interestingly, the histology of this case featured angiomatous morphology, a variant which makes up 2% of all meningiomas and is defined as having meningothelial cells wrapped around blood vessels in greater than 50% of the total tumor area . An incrMeningiomas may clinically manifest by acute intratumoral hemorrhage in the setting of moderate head trauma, particularly when the traumatic force is adjacent to the tumor location. Associations with alcoholism, proximity to dural venous sinuses, and angiomatous pathology may increase risk and extent of hemorrhage. In cases where emergent decompression precludes obtaining pre-operative MR analysis, careful scrutiny of CT imaging for characteristics of underlying neoplasm and generous hematoma evacuation are warranted. Given the increased rate of mortality in the literature, earlier diagnosis and definitive surgery is crucial.


A clinical decision and study design investigating the level and extent of angiogenesis modulation aimed at vascular normalization without rendering tissues hypoxic is key and represents an unmet medical need. Specifically, determining the active concentration and optimal times of the administration of antiangiogenetic drugs is crucial to inhibit the growth of any microscopic residual tumor after surgical resection and in the pre-malignant and smolder neoplastic state. This review uncovers the pre-clinical translational insights crucial to overcome the caveats faced so far while employing anti-angiogenesis. This literature revision also explores how abnormalities in the tumor endothelium harm the crosstalk with an effective immune cell response, envisioning a novel combination with other anti-cancer drugs and immunomodulatory agents. These insights hold vast potential to both repress tumorigenesis and unleash an effective immune response.Cancer-associated neo vessels’ formation acts as a gatekeeper that orchestrates the entrance and egress of patrolling immune cells within the tumor milieu. This is achieved, in part, via the directed chemokines’ expression and cell adhesion molecules on the endothelial cell surface that attract and retain circulating leukocytes. The crosstalk between adaptive immune cells and the cancer endothelium is thus essential for tumor immune surveillance and the success of immune-based therapies that harness immune cells to kill tumor cells. This review will focus on the biology of the endothelium and will explore the vascular-specific molecular mediators that control the recruitment, retention, and trafficking of immune cells that are essential for effective antitumor immunity. The literature revision will also explore how abnormalities in the tumor endothelium impair crosstalk with adaptive immune cells and how targeting these abnormalities can improve the success of immune-based therapies for different malignancies, with a particular focus on the paradigmatic example represented by multiple myeloma. We also generated and provide two original bio-informatic analyses, in order to sketch the physiopathology underlying the endothelial–neoplastic interactions in an easier manner, feeding into a vicious cycle propagating disease progression and highlighting novel pathways that might be exploited therapeutically. The interface between malignant cells and neighboring vessels, both recently sprouted during angiogenesis, or resident ones, is one of the pivotal physiological events tangled in the expansion of neoplastic cells and their dissemination . Cancer Solid tumor is made up of a plethora of cell types rather than just a homogeneous mass of cancer cell, such as cancer associated fibroblasts, an heterogenous immune cell infiltrate, and the individual cells that form the blood and lymphatic vessels . The bioThe ability to develop an angiogenetic response is a property common to all tissues. Tumor angiogenesis has historically been uncovered to be one of the key hallmarks of cancer . NonetheDespite its essential role, tumor vasculature is structurally and functionally aberrant, with intercellular junctions and extracellular matrix attachments may not form normally in tumors, leading to impaired monolayer formation and barrier function. Completely chaotic loss of tight junctions between adjacent individual cells in the overlapping endothelium, with odd sprouts being cast across the lumen of tumour vasculature, would be an impediment to proper tumor immune surveillance. These abnormalities also occur at the levels of the vasculature in the individual cells directly interacting with the extracellular matrix (ECM) and the pericytes that typically wrap around the outside of the vessel, providing support and stability; nonetheless, in the tumor microenvironment, pericytes are sparse and they are loosely attached to the surface of the tumour vessels, directly contributing to some of the vascular dysfunction . ConsistThe cancer immunotherapy has revolutionized the way we treat neoplastic patients in the last years. Since the first Food and Drug Administration (FDA) and European Medicines Agency (EMA) approval of the immune checkpoint inhibitor (ICI) ipilimumab for melanoma, which targets the anti CTLA-4 checkpoint, an explosion of approval of different ICIs that target a PD1 or programmed death-ligand 1 (PDL1) for a wide range of cancer indications has been observed ,46. The Anti-vascular endothelial growth factor receptor (anti-VEGFR) pioneered the attempts to normalize tumor vasculature and restore its function, as indicated by tissue perfusion and decreasing intratumoral hypoxia, and fostered further investigations aimed at shaping the intratumoral immune cell phenotype in parallel with vascular normalization , while pNumerous cell types can be mobilized from the bone marrow and directed to the sites of new vessel formation, where they strengthen the proangiogenic effects . Among tMultiple myeloma (MM) is a clonal proliferation of malignant plasma cells (PCs) accumulating and disseminating in the bone marrow (BM) with ensuing induction of focal skeletal lesions and osteoporosis driving myeloma bone disease, anemia, renal insufficiency, hypercalcemia , higher https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE28331) [Recently, we demonstrated that bone marrow endothelial cells from both newly diagnosed (NDMM) and relapsed-refractory multiple myeloma (RRMM) patients feed into a vicious cycle orchestrated by aberrant adhesion molecules on the bone marrow endothelial cells and plasma cell surface and correlate with poor clinical prognosis ,35,88. BSE28331) . Raw datSE28331) . The metSE28331)  was usedSE28331) , “dendexSE28331) , “colorsSE28331) , and “ggSE28331) .To characterize the adhesion molecules-related angiogenic switch in more detail and to corroborate available at gene-expression level in a broader spectrum of disease phenotype, we interrogated different independent public datasets. Given that mobilization of endothelial precursors cells (EPCs) occurs at the early stages of MM progression , precediNext, determining whether MM EPCs could be distinguished from MM-cells according to the natural grouping of their gene expression profiles, we analyzed publically available data from 20 EPC and 12 MM-cell samples (GSE28331). The analyses clearly split the MM-cells and EPCs into two branches heatmap, A, accordThe K-means clustering from the above-mentioned GSE28331 dataset A showed Consequently, using several pre-clinical models ,88,102, The dysregulated endothelial–neoplastic interactions sketched by our bio-informatic investigations show, on the one hand, a vicious cycle of disease progression and, on the other hand, point out pathways of potential therapeutic interest. These gene expression profiles were observed in one model of disease, and thus must be interpreted with caution and need further validation on a broad spectrum of malignancies. Nonetheless, solid and hematological malignancies share common mechanisms involving the cross talk between the cancer endothelium and the immune microenvironment, as summarized in The lymphatic vasculature is a hierarchical network of vessels found within nearly all peripheral tissues. The main function of lymphatic vessels is to unidirectionally transport interstitial fluids proteins and leukocytes from tissue periphery to the draining lymph nodes structure . The orgTo overcome these caveats, promising new avenues have recently been optimized to study the fate of tumor infiltrating immune cell populations, cancer metastasis, migration patterns of alloreactive T cells, or the dynamics and plasticity of immune cell subsets in different scenarios such as infection, inflammation, and immunotolerance using the in vivo photoconvertible fluorescence protein “kaede” transgenic mice . The uniAcross the timeline of the development of various imaging techniques, both clinical and preclinical models greatly contributed to the imaging of tumor vasculature and microenvironment ,155,156.Sufficient oxygen pressure is required for our organs to function properly. Conversely, insufficient oxygen supply is a prominent feature in various pathological processes, including tumor development and metastasis ,177. HypThe association of cell signaling driving cellular adaptation to hypoxia prompted the investigation on targets that might halt the proliferation of hypoxic tumors if halted. The three pivotal oxygen-dependent molecular mechanism during metabolic adaptation rely on hypoxia inducible factors (HIF members), unfolded protein response (UPR) , and mamNonetheless, normalizing the tumor vasculature with the judicious use of antiangiogenics can revert this process, directing intervening in oxygen delivery ,185,186.As proangiogenic factors typically predominate, tumour perfusion and oxygenation are usually impaired; the re-establishment of physiologic equilibrium aims to vasogenic edema and interstitial pressure reduction, while enhancing the drug delivery and indirectly reducing neoplastic cells shedding and invasiveness ,187. A pCurrent advances in tumour immunotherapy consent to proficiently unleash immune effector cells . What enCritical mechanisms fostering blood and lymphatic vessels’ formation and facilitating immunosuppression throughout tumor growth and progression have been uncovered. Cancer cells grow and progress through a persistent crosstalk with the neighboring milieu. Next generation techniques sketch at a high resolution such that the new vessels’ formation and immune paresis regularly occur to fuel this vicious cycle. Consequently, state-of-the-art therapeutic strategies merging anti-angiogenic and immune-directed treatments appear to hold promise to shape the neoplastic ecosystem and boost the therapeutic efficacy.


Dynamic measures of physical resilience—the ability to resist and recover from a challenge—may be informative of biological age far prior to overt manifestations such as age-related diseases and geriatric syndromes . If true, physical resilience at younger or middle ages may be predictive of future healthspan and lifespan, and provide a unique paradigm in which interventions targeting the fundamental biology of aging can be tested. This seminar will discuss research on the development of clinically-relevant measures of physical resilience in mice, including anesthesia, surgery, and cytotoxic drugs. It will further highlight how these measures compare between young, middle-aged, and older mice, and how mid-life resilience relates to later-life healthspan. Finally, it will provide insight into whether interventions targeting the biology of aging can modify physical resilience in mice. Part of a symposium sponsored by Epidemiology of Aging Interest Group.


Cells release nanometer-scale, lipid bilayer-enclosed biomolecular packages  into their surrounding environment. EVs are hypothesized to be intercellular communication agents that regulate physiological states by transporting biomolecules between near and distant cells. The research community has consistently advocated for the importance of RNA contents in EVs by demonstrating that: (1) EV-related RNA contents can be detected in a liquid biopsy, (2) disease states significantly alter EV-related RNA contents, and (3) sensitive and specific liquid biopsies can be implemented in precision medicine settings by measuring EV-derived RNA contents. Furthermore, EVs have medical potential beyond diagnostics. Both natural and engineered EVs are being investigated for therapeutic applications such as regenerative medicine and as drug delivery agents. This review focuses specifically on EV characterization, analysis of their RNA content, and their functional implications. The NIH extracellular RNA communication (ERC) program has catapulted human EV research from an RNA profiling standpoint by standardizing the pipeline for working with EV transcriptomics data, and creating a centralized database for the scientific community. There are currently thousands of RNA-sequencing profiles hosted on the Extracellular RNA Atlas alone (Murillo et al.,  Extracellular vesicles (EVs) are secreted, nanometer-scale genetic information carriers found in human biofluids. Aside from EVs, there are a number of other non-vesicular nanoparticles in circulation such as lipoproteins, RNA-binding proteins, and exomeres  vs. a eukaryotic cell of 4 × 10−15m3 (sphere of radius 10 μm), the volumes are different by a factor of 106 and only certain cellular sub-regions are necessarily sampled by the nature of EV biogenesis requiring a membrane. To this end, microscale sampling of membrane and cytosolic components along with stochastic variance in molecule distribution within the cell, and other biophysical and biochemical factors imply that differential composition of EVs relative to their parents is insufficient to claim organization. Active loading of EV-associated proteins is implied in limited instances, and we suspect they are those identified as biogenesis-related EV biomarkers such as CD63, CD9, CD81, Annexin A1, and TSG101  EVs  reporter suggests that a single cell can have between 1 and 15 multivesicular endosome-plasma membrane fusion events  per minute  considering variance within and between cell lines among the three human cell lines tested  embryo model system enabling in vivo live-tracking of single endogenous EVs  to address the research needs; these methods generally fall into four categories: centrifugation, chemical precipitation, microfluidics, and biochemical capture. MISEV2018 guidelines emphasize the adoption of the terms “separation” and “concentration” when discussing EV experimental methods. A separation technique selectively removes EVs from other fluid components, or one EV subtype from another . A concentration technique increases the EV concentration in the fluid and may not necessarily remove EVs from other fluid components . The MISEV2018 authors established a heuristic to conceptualize the efficacy of different methods by classifying extracellular particle recovery  and specificity  as either low, intermediate, or high efficacy. Each method has some component of separation and concentration regardless of whether the properties are explicitly stated. High-recovery and high-specificity together in a single method is unlikely to be achievable. EV size, density, surface markers, biofluid type, sample volume, product purity, and cost are all to be considered since every method has its own advantages and disadvantages  and product yield .Generally characterize that the sample contains vesicles and describe the purity of the preparation. Vesicle existence can be claimed by demonstrating that the sample contains intact lipid bilayers, that are unlikely the products of cell fragmentation. For example, the identification of at least one membrane-associated protein  and a transmembrane  or lipid-anchored  protein would be sufficient to meet this criterion. Investigate the presence and extent of non-EV contaminants e.g., lipoproteins (targets depend on EV source and preparation method).Employ a combination of single EV characterization techniques. Ideally use an optical  and a biophysical/biochemical  modality tailored to the EV product and considering the limitations and complementarities of each technique.When considering EV-associated biological functions, it is necessary to operate with the utmost detail and rigorous experimental design. Functional studies should be performed with strict biofluid fractions; select the appropriate method(s) to separate EVs and EV subtypes from non-EV contaminants. Test the purported activity with condition-matched controls  of the biofluid itself, the EV-depleted biofluid, crude EVs alone, and EV subpopulations if relevant. Evaluate the relevant molecular topologies  for membrane-bound molecules.A strategic approach to EV characterization is essential to advancing the field  is a mainstay EV separation technique. The operating principle of DUC is to apply step-wise increases in centrifugal force to a solution. The product of centrifugal force and time during each step then concentrates particles of a specific size and buoyancy. Ideally, using this principle, one can separate particles within a solution into discrete fractions. For example, a cell fraction, a large vesicle fraction, and a small vesicle fraction. Since DUC is a relatively simple and pure technique, it is the established EV isolation standard. The primary benefit of DUC is the ability to concentrate and separate EVs by simply subjecting the biofluid to high rotational speeds. Furthermore, DUC parameters such as rotor type, rotational force, solution density, and spin time can be finely adjusted to meet the needs of a specific application, for example a specific biofluid type and EV fraction. Most DUC protocols include some variation of a three-step method including a low-speed ~1,000 RCF spin to collect cells and other large particles, an intermediate-speed ~20,000 RCF spin to collect large EVs, and a high-speed ~100,000 RCF spin to collect small EVs. The most notable innovation/adoption among centrifugation-based protocols is iodixanol-based density gradient ultracentrifugation (DGUC)  which haCentrifugation-based protocols face a number of limitations that have driven innovation to create new methods:The amount of variance and lack of standardized protocols within the field presents a challenge for validating findings made between groups and a barrier to entry for new groups. Commercially available isolation kits  are one solution aimed at the reproducibility problem.DUC protocols for EV isolation are lengthy, taking at least several hours.Only a small fraction of EVs are captured by DUC, which can make investigations of rare samples difficult  which many people suggest may irreversibly damage EVs or change EV properties studied thereafter  centrifugation and take about 1 h to complete EV isolation from blood plasma with newer kits. PEG-based precipitation methods have been shown to yield hundreds of times more EVs than centrifugation and therefore can be used effectively in low sample input applications EV precipitation strategies are advantageous over ultracentrifugation because they often recover a larger fraction of EVs, and they can function with standard benchtop centrifuges. Reports describing chemical precipitation of virions and other nanoparticles by volume-excluding polymers e.g., polyethylene glycol (PEG) date as early as the 1970s. Alice Adams reported that the Epstein-Barr virus could be recovered with high yield and retained viral activity when separated directly from cell culture medium using PEG-based precipitation, while sucrose density gradient centrifugation had low recovery and substantially attenuated viral activity Adams, . The opeBiochemical capture techniques are most appropriate for investigators who seek to subset EV populations by specific membrane components and ascribe functions to those EV sub-populations . Biochemical capturing of EVs in most embodiments depends on immunoaffinity capture (IAC), where antibody-binding interactions dictate which particles become concentrated. The IAC approach uses antibodies conjugated either with the surface that the fluid sample passes over  technologies use physical properties to manipulate solutions on a small (μL-pL) scaleThe most simple manifestation of MF technology applied to EV separation/concentration is filtration. In this method, a solution is forced through a nanoporous filter material where particles larger than the filter's effective pore size are concentrated on the filter, while smaller particles pass through and remain in the filtrate solution. Particles of a specific size range can be separated from particles outside of that range by implementing a filter series where each filter has different effective pore sizes. Many groups use filtration alone or in combination with other techniques such as centrifugation or precipitation for greater power to select specific EV subsets. A device with low protein binding, track-etched polycarbonate membrane Apel,  filters,Chromatographic techniques, which can be further divided into size-exclusion and affinity modes, are widely employed for EV separation protocols and have a marked advantage over other methods we review here because they can yield very clean and highly-reproducible products. In traditional size-exclusion chromatography (SEC), a solution (the mobile phase) travels through a column packed with porous resin beads (the stationary phase). Small particles can enter the resin pores, and thus have a longer path to elution, while larger particles travel a more direct path outside of the beads and elute more quickly . For a pIn affinity chromatography, the mobile phase travels through a stationary matrix exhibiting an affinity for certain particle types; the interaction can be general  or specific . High affinity particles are retained non-covalently in the stationary phase upon sample application. Then, graduated elution of a solution with comparable affinity displaces adsorbed particles once the bonding interaction is overcome. Zeta-potential measurements of EVs by both resistive pulse sensing and electrophoretic mobility assays have indicated that EVs carry a negative surface charge  studies. The principle is to construct an obstacle network which selectively traps EVs while allowing undesired fractions to pass through. Then, the network integrity is dissolved or perturbed to release the trapped EVs. An early embodiment used ciliated silicon micropillars in a regularly-interspaced (0.9 μm) array with a tunable capturing range of 30–200 nm. The nanowire forest captured EV-like liposomes, while other cellular and protein debris flowed through. Then, the liposomes were released by incubating the MF channel with phosphate-buffered saline, thereby dissolving the silicon nanowires. This architecture was additionally flexible in that antibodies could be loaded on the nanowires; however, it was not demonstrated  is a promising technology which has already been used to concentrate EVs within human biofluids for RNA-seq analysis  has great potential for separating EVs from other nanoparticles though its implementation in EV research has thus far been limited. Field flow fractionation first was described in 1966  from large EVs (1–2 μm milk fat globules)  is now a mainstay for EV counting and sizing that is based on visible light microscopy. NTA devices shine a laser through the sample and record a video. Brownian motion of individual EVs, which can be inferred by the way light scatters through the sample over time, directly relates to their particle size . Therefo2 and 5 × 105 per μL based on the change in fluorescence polarization  functions by capturing EVs on an antibody microarray chip. Light is shone onto the chip and then the reflectance is measured. Interference between the reference light-field, produced by reflectance off of the silicon chip alone, and the scattered light field produced by reflectance off of the EV-bound microarray, gives a signal which corresponds to the EV size at each location on the chip  is another commonly implemented technique for EV counting, sizing, and surface charge characterization. RPS principally operates by electrophoretic translocation of particles through a conical pore; when within the pore, the particle increases electrical resistance across the pore and a current drop is detectable. The magnitude and frequency of current drops are proportional to particle volume and concentration, respectively , nucleic acid sequencing (NA-Seq), and antibody affinity-labeling techniques are all currently being implemented for high-throughput, multiplexed profiling of EV biochemistry. The advantages of EV-focused MS analysis have been made clear through a number of studies including the identification of GPC1 as a marker of pancreatic cancer-derived EVs, genetic renal disease markers in urinary EVs, and novel myokines secreted during exercise in mammals  conjugated with 1,2,4,5-tetrazine were added, the antibody-bead linkage was achieved by spontaneous cycloaddition, and the EV-antibody-MNP product was formed. The sample was finally washed and analyzed for protein content by nuclear magnetic resonance using a microcoil on the device. The NMR output corresponded to EV protein concentration and type. Proteins were detected with orders of magnitude higher sensitivity than traditional approaches, including flow cytometry, ELISA, and nanoparticle tracking analysis. Additionally, the NMR signature of four EV-associated proteins sensitively and specifically identified glioblastoma vs. healthy patients . For representative examples, see Ratajczak et al.  and ValaExtracellular RNAs in human serum are present in ng/mL concentrations and predominantly in the 20–200 nt size range  include riboswitch systems, ribozyme activities, cis-regulatory mRNA sequence presentation or concealment, and trans-regulatory behaviors, which are all driven by mRNA folding and/or processing  are the most heavily studied and strongly characterized extracellular RNA biotype contained within EVs. Hundreds of miRNA species can be detected in a given biofluid and there is longitudinal evidence that each fluid type has a distinct miRNA composition which can be changed by health status including pregnancy, urothelial cancers, neurological disorders, diet, and exercise  are a key component of EV-derived RNAs, taking large fractions of the RNA in most biofluids tested  are one of the most abundant RNA components of EVs, and specifically can dominate within blood  and circular RNA (circRNA) species are generally present to lesser extents in circulating biofluids . An RNA-seq study with 22 subjects identified 21 micro-RNA candidates that showed significant differential expression between those with beneficial vs. adverse remodeling outcomes. Top pathways targeted by these micro-RNAs include immune cell signaling, fibrosis, and apoptosis, indicating that specific inflammatory states lead to positive or negative resolutions of myocardial infarction (Danielson et al., in vitro by way of EVs (Balaj et al., The physiological role of exogenous EVs is being newly investigated. EVs are secreted by most organisms and accordingly, given the extensive population of humans by microbial species and viruses, we can predict a substantial number of exogenous EVs to exist in human biofluids. A large proportion of circulating RNAs, including miRNAs, are contributed by the microbiome. In some EV preparations, 75% of the total RNA reads originate exogenously (Wang et al., in vivo, and prodrug administration led to tumor death when at least 1% of the tumor cells in a given tumor received the minicircle DNA. Dysregulated EV signaling has also been implicated in tumor immunology (Ricklefs et al., EVs are particularly interesting for novel therapeutic strategies. Because EVs have a suspected mechanism for targeting and delivering their cargoes to certain cell populations (Hyenne et al., There are a number of clinical scenarios when it may be desirable to attain the genetic information of a developing fetus, such as when the pregnancy is at-risk for yielding a trisomy or other chromosomal abnormalitiesDeep and high-coverage sequencing studies of the fetal exome and genome have demonstrated the potential of extracellular RNA to make clinical assessments of the fetal genome at various time points (Fan et al., There are a number of online public databases which host EV-related gene expression data and serve as a reference for the research community . VesicleTo address the ERC program goal of generating an exRNA reference catalog, 2,270 extracellular small RNA-seq and 3,039 qPCR profiles across 19 studies and 23 health states were compiled in the ERC Consortium data repository, the exRNA Atlas (Murillo et al., In this review we have provided historical context for EVs and their rise to prominence in biomedical research, reviewed EV biogenesis and fates with perspective on functional outcomes, and identified some of the latest experimental approaches for preparing and analyzing EVs with a focus on clinical applications. We also discussed the physiology and biochemical content of EVs with a focus on RNA biotypes, and provided an overview of online databases hosting EV-related experimental findings. The research community has consistently advocated for the importance of EVs especially with regard to their RNA contents. EVs are hypothesized to act as intercellular communication agents that functionally transfer biological information between cells and tissues. EVs protect RNAs from degradation in the extracellular environment. Furthermore, RNAs contained within EVs encode biological state-specific information, and RNA-containing EVs can be easily and frequently collected in a clinical setting and analyzed. In the future, it is possible to envision noninvasive EV-based diagnostics enabling precision health applications–where sampling an individual over time is beneficial to gain insight into the evolution of a dynamic health or disease state (Chen et al., As we indicated throughout this review, there is a concerted effort toward single-EV characterization and a number of projects under the ERC commonfund have recently shifted to new stagesEV wrote and reviewed the manuscript and generated the figures. GM conceived of and supervised the project, reviewed, and edited the manuscript. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.EV declares equity ownership in Gilead Sciences, Inc., an American biotechnology company. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neutrophils are key immune cells that participate in host defense through a variety of mechanisms including phagocytosis, the generation of reactive oxygen species (ROS), release of granular contents, and the formation of neutrophil extracellular traps (NETs). The capacity of neutrophils to orchestrate inflammatory and immune responses is dependent on their release of neutrophil-derived molecules, including cytokines, alarmins, and NETs, as well as their capacity to interact with and direct other innate and adaptive immune cells. Over the past decade, the field of neutrophil biology has exploded, with remarkable discoveries highlighting neutrophils as indispensable players in immune regulation. While neutrophils are recognized to be prominent phagocytes involved in the clearance of pathogens and cell debris, they are beginning to emerge as essential communicators crucial in shaping immune response.In this Research Topic, a series of articles provides comprehensive insights into the current view of neutrophil biology, highlighting neutrophil interactions with platelets, infectious agents, and tumor cells, as well as neutrophil generation of inflammatory mediators such as NETs.Wirestam et al., we are reminded of the devastating role of neutrophils in rheumatic diseases, including systemic lupus erythematosus (SLE) and anti-phospholipid syndrome (APS) wherein neutrophils recognize circulating immune complexes promoting ROS-dependent NET formation. Intriguingly, SLE patients have impaired NOX2, relying on mitochondrial ROS for NET formation. This results in concomitant mitochondrial extrusion and a qualitatively distinct NETs with inflammatory oxidized mitochondrial DNA as an important cargo. Takeda et al. highlighting the important role for immune complexes, namely bactericidal/permeability-increasing protein (BPI) immune complexes, in mediating NET formation in systemic anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. The regulation and role of the main immune complex receptor, Fc gamma R, in steady state and inflammatory conditions, is comprehensively covered in an excellent review by Wang and Jönsson. Beyond immune complexes, large aggregates, including monosodium urate (MSU) crystals are known to activate macrophages and neutrophils, though the underlying signaling events have not been well-characterized. In an elegant study by Tatsiy et al., performing transcriptomic analyses, the authors demonstrate the novel finding that neutrophils, upon engulfment of MSU crystals, are programmed to attract monocytes in an NF-κB- and CCL4-dependent manner. This observation is particularly interesting as neutrophils are essential in licensing macrophages for cytokine production in atherosclerosis, indicating a pathophysiological role for their crosstalk , made the novel observation of a crosstalk between a protease, calpain, and peptidylarginine deiminase 4 (PAD4) in nuclear decondensation, with PAD4-mediated citrullination facilitating proteolytic cleavage of nuclear proteins. This observation markedly advances our understanding of the molecular events during NET formation and may allow for additional therapeutic strategies to prevent exacerbated NETosis. In the final NET-related paper included in this Research Topic, Agarwal et al. demonstrate that NETs are not only a product of neutrophil activation, but also a potent stimulus for secondary NETosis, inducing NET formation through TLR9-dependent mechanisms. This mechanism may be particularly important in post-traumatic inflammation, when NETs disrupted by mechanical strain may amplify inflammation and prevent physiological wound healing.Another study investigating the underlying requirements for NET formation . These surface markers were attributable to increased platelet-neutrophil aggregates in the blood of patients with psoriasis . The aggregates also appeared to infiltrate the skin, where platelets could be found in close proximity to both intact neutrophils and neutrophil remnants suggestive of NETs . Intriguingly, platelet depletion was protective against neutrophil infiltration and overall skin inflammation in a mouse model of psoriasis .Two interesting papers in this collection suggest a critical role for platelets as orchestrators of neutrophil recruitment to diseased organs. Psoriasis is a systemic inflammatory disease characterized by intense leukocyte infiltration into the skin. Hook et al. characterized a model of SIRS triggered by administration of zymosan. This model has previously been shown to be exacerbated by NADPH oxidase 2 (Nox2) deficiency. In this new study , Nox2-deficient mice demonstrated upregulation of platelet-derived chemokines such as CXCL4 and CXCL7 in the bronchoalveolar fluid. Neutrophils strongly expressing P-selectin glycoprotein ligand-1 were found at increased levels in the same fluid . While Nox2 deficiency may protect against NETosis in some contexts, that was not the case in this model where exaggerated NETosis appeared to be driven by platelets in a PAD4-dependent fashion . In summary, these studies shine a light on the role of platelets in recruiting neutrophils into inflamed tissues, and suggest the potential for anti-platelet agents to have efficacy in neutrophil-dependent diseases.Systemic inflammatory response syndrome (SIRS) puts many organ systems at risk including the lungs, where it may lead to acute respiratory distress syndrome. Bernson et al. extended our knowledge on the molecular basis underlying the capacity of natural killer (NK) cells to trigger neutrophil apoptosis under inflammatory conditions, by performing a comprehensive characterization of neutrophil expression of ligands for NK cell receptors. Ultimately, the authors demonstrated that inflammatory neutrophils, including either blood neutrophils stimulated in vitro or in vivo-transmigrated neutrophils, become more susceptible to NK cell-mediated apoptosis than resting neutrophils. Furthermore, their enhanced sensitivity is associated with a pronounced downregulation of HLA class I expression, which is known to release activating signaling by NK cells . Secondly, Aarts et al. compared the immunosuppressive capacity of circulating neutrophils from healthy donors (HDs) with those displayed by autologous bone marrow (BM) myeloid cell fractions at various differentiation stages. They show that, unlike “early mature” BM neutrophils, immature BM neutrophils, per se, are not efficient in suppressing T cells when activated with physiological stimuli . The authors therefore speculate that, for instance in cancer patients, functional granulocyte-myeloid-derived suppressor cells (g-MDSCs) require neutrophil differentiation from immature populations in the bloodstream, which are unable to perform any immunosuppressive activity in their own right . In another article, Oberg et al., first, exhaustively review current knowledge on the reciprocal interactions between neutrophils, tumor cells, and particularly gamma/delta T cells. They then provide data on the modulation of anti-tumor cytotoxicity of short-term expanded human gamma/delta T cell lines by autologous freshly isolated neutrophils. Their results demonstrate that, under certain circumstances, the presence of neutrophils can enhance, rather than inhibit, the killing capacity of gamma/delta T cells, by increasing their release of cytotoxic mediators . Finally, the role of neutrophils in malignancy was extensively reviewed by Lecot et al., highlighting the heterogeneity of neutrophils, and their effector functions contributing to cancer suppression as well as progression. The authors also reviewed different strategies aimed at targeting neutrophils in malignancy, ranging from neutrophil depletion to succinct targeting of recruitment or other key effector functions.A further group of articles have focused on some of the functional crosstalk that neutrophils are known to engage in with other leukocytes. Firstly, In conclusion, neutrophils are heterogeneous cells with a huge range of effector functions partaking in shaping our immune system. The current papers included in this Research Topic highlights the emerging role of neutrophils as communicators in health and disease with promises of future therapies targeting these pathways.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gene expression regulation is achieved through an intricate network of molecular interactions, in which trans-acting transcription factors (TFs) and small noncoding RNAs (sncRNAs), including microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), play a key role. Recent observations allowed postulating an interplay between TFs and sncRNAs, in that they may possibly share DNA-binding sites. The aim of this study was to analyze the complete subset of miRNA and piRNA sequences stored in the main databases in order to identify the occurrence of conserved motifs and subsequently predict a possible innovative interplay with TFs at a transcriptional level. To this aim, we adopted an original in silico workflow to search motifs and predict interactions within genome-scale regulatory networks. Our results allowed categorizing miRNA and piRNA motifs, with corresponding TFs sharing complementary DNA-binding motifs. The biological interpretation of the gene ontologies of the TFs permitted observing a selective enrichment in developmental pathways, allowing the distribution of miRNA motifs along a topological and chronological frame. In addition, piRNA motifs were categorized for the first time and revealed specific functional implications in somatic tissues. These data might pose experimental hypotheses to be tested in biological models, towards clarifying novel in gene regulatory routes.  Roughly 90% of the genome is transcribed. Protein-coding  genes account for as little as ~2% of the genome, with a much larger portion of the transcribed content being represented by noncoding RNAs (ncRNAs) ,2. SinceGene expression is regulated in a tissue-specific fashion with time-related rhythms and stimuli-responsive mechanisms. This is achieved through the fine-tuning orchestrated by a number of molecular interactors and epigenetic regulators that act at different levels. These include known DNA trans-acting elements, such as transcription factors (TFs), mostly binding to promoter elements upstream of genes, and transcriptional activators and repressors, which interact with enhancer and silencer regions, respectively. In addition, different classes of sncRNAs, including miRNAs and piRNAs, perform a dynamic epigenetic regulation and surveillance of genomic integrity and functions, acting on the genome scale and determining a dense and complex regulatory network . miRNAs are short, single-stranded noncoding RNAs (20–22 nucleotides in length) that are typically transcribed by the RNA polymerase II. miRNAs are encoded by the major regulatory gene family in eukaryotic cells and operate in multiple signaling pathways ,7. In paIncreasing evidence points towards the idea that mature miRNAs are also enriched in the nucleus, suggesting noncanonical roles, i.e., in transcriptional silencing or activation and alternative splicing ,10,11. TpiRNAs are 24–32-nucleotide-long noncoding RNAs, named after their association with the argonaute subfamily PIWI proteins, originally identified in mutants affected by asymmetric division of stem cells in the Drosophila germline . piRNAs The defective or aberrant expression of sncRNAs has been associated with several human diseases, and the interest in their biological roles has increased concurrently ,19.Advances in methods that analyze RNA populations have allowed a rapid quantitative and qualitative characterization of small RNAs at the cellular and/or tissue level. As a result, a huge quantity of “omics” data are currently available, stored in specialized databases and representing a valuable source of information which could potentially provide surprising and useful details on genome architecture and regulatory frameworks . PreciouIn this scenario, the aim of this study was to analyze the complete subset the whole sequences of miRNAs and piRNAs stored in the main databases in order to identify the occurrence of conserved motifs and subsequently predict possible specific interplay with TFs within genome-scale regulatory networks. We propose a direct interaction between the sncRNAs (either miRNAs or piRNAs) with conserved common motifs (transcription factor binding site (TFBS)) on DNA sequences. This original analytical workflow allowed identifying the occurrence of conserved motifs in both miRNAs and piRNAs and categorizing these sncRNAs based on TFBS domains. The data produced by this in silico pipeline point towards the hypothesis that miRNAs and piRNAs share DNA-binding motifs with TFs. We then propose that these sncRNAs may be categorized based on TFBS. Interestingly, the functional annotation of putative target genes allowed evidencing that these binding motifs are specifically enriched in biological networks involved in embryonic development. Indeed, the analysis allowed pointing out that different miRNA and piRNA classes, sorted by TFBS, have differential implication in biological pathways, as they are able to regulate multiple target genes sharing the same conserved TFBS. Our in silico analysis provides original predictive computational data, paving the way to further biological studies that may test novel nuclear roles of miRNAs and piRNAs in gene expression regulation.http://meme-suite.org/). The experimental pipeline consisted of three main steps, as schematized in The analysis pipeline was performed using different bioinformatics tools available online in the MEME-suite collection of motif-bases sequence analysis tools  and piRNAs  were analyzed separately.The entire subgroup of http://meme-suite.org/tools/dreme) [E-value threshold exceeded 0.05 (default threshold) [http://www.mirdb.org).In the first step, all reliable conserved motifs were categorized by searching in miRNA and piRNA sequences using the Discriminative Regular Expression Motif Elicitation (DREME) tool from MEME-suite (s/dreme) . The DREreshold) . The idehttp://meme-suite.org/tools/tomtom), which compares the newly identified motifs against a database of known motifs . JASPAR CORE is a database that contains a curated and nonredundant set of open access data collections of experimentally discovered and proven TF binding sites [p-value in the order that the queries appear in the input file. The E-value and the q-value for each match were also reported. The q-value is the minimal false discovery rate at which the observed similarity would be considered significant. Tomtom estimated q-values from all the match p-values using the Benjamini and Hochberg method. By default, significance was measured by q-value of the match [In the second step, all the obtained motifs were used as query for Tomtom, a motif comparison tool within the MEME-suite . This is a flexible, user-friendly web interface for generating hypotheses about function, through analyzing gene lists and prioritizing them based on literature-proved biological functions. GeneMANIA allowed clustering functionally related TFs, using available genomic and proteomic data  introducing weights that indicate the predictive value of each selected dataset for the query [Finally, in the third step, all lists of putative miRNAs and piRNAs that contained motif-related TFs were individually loaded in GeneMANIA  multiple testing correction  associated with each ‘function’, we selected for the analysis only GO terms with The DREAM tool  were present in more than 100 miRNAs when also considering complementary sequences. The least represented motif is AUUACUUU, which can be recognized in as few as 26 miRNAs  annotated functions  GO annotations were associated with specific developmental stages, from early embryo formation to detailed organogenetic paths . We haveIn particular, the analysis of the motifs contained in miRNAs  allowed The functional cluster ‘Stem cell homeostasis and differentiation’  was not Moreover, our analytical workflow also provided evidence of a spatial and temporal distribution for TFs sharing DNA-binding motifs with piRNAs, revealing an intricate network of multiple connections with redundant functions . As alreThe complete understanding of the complex miRNA-mediated regulatory network in cells and organisms has not been achieved yet. The selection of molecular interacting targets by miRNA has been long considered to be primarily dictated by sequences at their 5′ end . Nonetheless, distinct studies have suggested that miRNAs contain additional sequence elements that control their posttranscriptional behavior, including their subcellular localization.Indeed, it is currently confirmed that mature miRNAs reside in the nucleus, where they participate at several levels of gene expression regulation ,10,11. DThe expression enrichment of different miRNA sets in the nucleus seems to vary based on cell type, function, and activity status, or in response to environmental stimuli ,28. NumeThe noncanonical nuclear role of miRNAs in the regulation of gene expression at the transcriptional level is yet to be fully clarified. Alternative mechanisms have been proposed to date that are not necessarily mutually exclusive but rather suggest that miRNAs may intervene at several levels in the gene expression regulatory network occurring in the nucleus. Most of the described mechanisms members of the nuclear subfamily of argonaute (Ago) proteins, key components of RISC complexes, as key mediators. Such nuclear miRISC complexes may bind long noncoding RNAs (lncRNAs) by sequence complementarity and modulate their function ,12. The Converging evidence also showed that miRNAs may modify (either activate or suppress) gene transcription by interacting with chromatin, besides acting at the post-transcriptional level in the cytoplasm ,32. MatuAlternatively, miRNAs may recognize complementary sequences on nascent RNAs, in a cotranscriptional mechanism, forming double-stranded RNAs that determine the recruitment of protein complexes able to modify chromatin accessibility and thus RNAs levels ,12. Finally, a third model has been also proposed, according to which miRNA–Ago complexes directly target one of the DNA strands when the target promoter region is in an open configuration during the transcription initiation process .Although numerous studies have indicated that the seed sequence can also mediate the recognition of miRNAs’ nuclear targets, a recent study supports a model in which a miRNA can form a hybrid with promoter region to modulate transcription through its nonseed region . In thisA similar interplay with TFs has not been explored in piRNAs to date, even though increasing roles for this class of sncRNAs, further preserving genome integrity in germline cells, have been recently recognized. The mechanisms at the base of piRNA biogenesis and function have become increasingly clear , and groUnderstanding the crosstalk between sncRNAs and TFs at a cotranscriptional level could provide new clues towards the involvement of miRNAs and piRNAs in the control of specific events of gene expression regulation in the nucleus during development in humans.The computational workflow exploited in this study allowed posing an experimental hypothesis according to which conserved and recurrent sequence motifs found in miRNAs and piRNAs, complementary to transcription factor binding sites (TFBS), could influence the bond and activity of TFs on the same target genes. Our results enabled categorizing different classes of motifs, associated to TFs that have known biological roles, hence predicting the possible biological consequence of the putative miRNA nuclear localization and function. The biological interpretation of the enriched functional terms in TFs indeed allowed categorizing miRNA motifs according to their involvement in key steps of the human developmental path, suggesting that different miRNA profiles exist in different developmental stages and vary in their nuclear expression across different tissue types. The direct competition/collaboration with TFs that our data suggest might provide a finer regulatory control and could explain the prompt canalization of genetic programs to maintain and stabilize the phenotypic reproducibility of embryogenesis. This type of interaction could increase the speed and efficiency of response of embryonic cells exposed to continuous differentiation stimuli. Indeed, the established mechanisms of miRNA-mediated expression regulation, based on their biding on the 3’-UTR of target mRNAs in the cytoplasm, inevitably occurs at the post-transcriptional level, while the 5’ end DNA-binding event proposed in this model occurs during or right before transcription ,11,12. TThe results obtained in the in silico analysis of piRNA motifs yielded original data providing a model for additional roles for piRNAs in somatic cells, to be further explored in the wet lab. Indeed, piRNA motifs were also found to be associated to TFs with reproducible functions exerted in somatic tissues, including tissue-specific metabolic pathways. It has been shown that the PIWI–piRNA complex binds its genomic target in euchromatin through a nascent transcript and, in heterochromatin, predominantly through a direct piRNA–DNA interaction . AlthougThese data may also suggest a feasible way to categorize functional piRNA subclasses distributed in different tissues, on the basis of the presence of conserved motifs, that could reflect their roles in shared regulatory networks and/or developmental timeframes. Each of these families could intervene in crucial parts of the epigenetic control, maintaining genomic integrity, repressing the mobilization of transposable elements, and regulating the expression of downstream target genes via transcriptional or post-transcriptional mechanisms as already reported by two independent research teams in studies of model organisms ,46. ThesOur observations, though still preliminary, could propose novel testing hypotheses to be investigated in a biological system, towards the clarification of novel aspects of sncRNA-based epigenetic regulation of cellular functions at the organism level.Further in vitro analyses will be necessary to support at the functional level the evidence derived from this in silico approach. In particular, in-depth in vitro studies at the genome-wide level will be needed to delve into the subcellular location of each class of motif-grouped sncRNAs and to clarify their involvement in the predicted biological pathways.The extended knowledge of these novel sncRNA mechanisms of action has a deep translational relevance, considering their extensive application in “theranostics”: a differential expression analysis of these sncRNA sequence motifs could enable identifying tissue- or organ-specific biomarkers of pathway function/dysfunction. On the other hand, targeting RNA metabolism is being exploited as a strategy to recover RNA alterations in a variety of diseases, paving the way to RNA-based therapeutic strategies .