Transthyretin cardiac amyloidosis (ATTR-CA) has been traditionally considered a rare and inexorably fatal condition. ATTR-CA now is an increasingly recognised cause of heart failure and mortality worldwide with effective pharmacological treatments. Advances in non-invasive diagnosis, coupled with the development of effective treatments, have transformed the diagnosis of ATTR-CA, which is now possible without recourse to endomyocardial biopsy in around 70% of cases. Many patients are now diagnosed at an earlier stage. Echocardiography and cardiac magnetic resonance have enabled identification of patients with possible ATTR-CA and more accurate prognostic stratification. Therapies able to slow or halt ATTR-CA progression and increase survival are now available and there is also evidence that patients may benefit from specific conventional heart failure medications. A wide horizon of possibilities is unfolding and awaits discovery. During the last decade, perception of cardiac ATTR amyloidosis has been radically transformed through major diagnostic advances in cardiovascular magnetic resonance (CMR) and bone tracer scintigraphy, and the development of treatments designed to ameliorate its natural history . The floIn this issue of the Global Heart Journal, Brito et al. present the World Heart Federation Consensus on Transthyretin Amyloidosis Cardiomyopathy which provides a comprehensive review of ATTR-CM, prioritizing patients’ needs, including treatment accessibility, and the role of Patient Advocacy Groups, and highlighting the importance of multidisciplinary patient management, emphasizing the key role of specialized amyloidosis centers.To achieve this aim, a panel of 18 expert clinicians specialized in ATTR amyloidosis from 13 countries was assembled along with a representative from the Amyloidosis Alliance, a patient advocacy group. Such a breadth of expertise is required since ATTR amyloidosis is highly heterogeneous with significant variation worldwide in terms of clinical phenotype at presentation, presence and consequence of the vast number of TTR gene mutations, diagnostic pathways, outcomes and treatment strategies. Identification of patients with early ATTR-CM has afforded the opportunity to broaden our understanding of its natural history and the factors influencing its clinical course such as the fibril type (ATTRwt vs. ATTRv amyloidosis), particular TTR gene mutations , severitUntil recently, most patients with ATTR-CM were diagnosed with severe advancements already progressed throughout the disease, with characteristic echocardiographic features including severely increased wall thickness, restrictive LV filling pattern, atrial dilatation, and reduced ejection fraction . However10The use of CMR and repurposed bone scintigraphy for diagnosing ATTR-CM represents a milestone advance in the field, which has led to an exponential increase in the number of patients identified worldwide, and a much greater proportion being diagnosed at an earlier stage . Brito eATTR-CM is now treatable, making early diagnosis all the more important. Remarkably, a number of very different therapeutic strategies have been developed, with theoretical potential to be used together. Small molecule drugs that bind to circulating TTR, the liver-derived amyloid precursor protein, can enhance its stability with the aim of reducing its propensity to misfold and aggregate into ATTR amyloid fibrils. The TTR stabiliser tafamidis, was the first ever drug to be approved for any kind of amyloidosis; in the ATTR-ACT trial for ATTR-CM , treatmeA quantum leap beyond gene silencing is permanent one-shot interference of the TTR gene by means of CRISPR-Cas9  gene editing. Long applied in plant biology, current trials in ATTR amyloidosis represent its first in vivo use in humans. This treatment approach achieves near sustained up to 95% knockdown of TTR serum concentrations in patients with hereditary ATTR-PN , and it These remarkable advances in therapy for ATTR-CM have between them shed substantial light on the continuously dynamic nature of cardiac amyloidosis, i.e. the interplay between amyloid production and amyloid clearance 2223, bot22


Sarcoidosis is a systemic inflammatory disease histologically defined by the non-caseation granulomas formation in different organs, most commonly lungs, liver, skin, gastrointestinal system, eyes, neurologic and cardiac systemWe report the case of a 42-year-old Gilaks woman who presented with myelopathy with characteristic MRI finding called trident sign. By finding this view in axial spinal Magnetic Resonance Imaging (MRI) imaging, a systemic evaluation was performed on the patient, which led to the diagnosis of cardiac involvement in Sarcoidosis with the specific appearance of this disease in cardiac MRI despite the negative Fluorodeoxyglucose (FDG)-positron emission tomography (PET) scan.Sometimes characteristic findings such as the trident sign prompt the physician to high suspicion and wide evaluation of the patient to reveal important organ involvement that changes the treatment decision and saves the patient. Sarcoidosis is a systemic inflammatory disease histologically defined by the non-caseation granulomas formation of subsequent tissue scarring in different organs, most commonly lungs, liver, skin, gastrointestinal system, eyes, neurologic and cardiac system. According to the diagnostic criteria of neuro-sarcoidosis (NS), pathologic confirmation in the nervous system is needed for a definitive diagnosis and patients with a probable diagnosis need a systemic pathologic confirmation (tissue outside the nervous system). For a possible diagnosis in the absence of pathological findings, we need a set of findings that highly suggest the disease on imaging or other para-clinical tests . AlthougConsidering the difficulty of finding proper and adequate tissue for biopsy in the heart and brain, identifying specific findings in non-invasive methods such as MRI can be very helpful in distinguishing this disease from other differential diagnoses .A 42-year-old Gilaks female patient presented with mild lower limb weakness. She had hypokalemia in the initial lab tests, so the patient's weakness was attributed to hypokalemia in several visits and finally, she was diagnosed with renal tubular acidosis.After three months, the patient's weakness increased and the symptoms of urinary frequency and urgency along with dysesthesia and pain were added. In the examination, she had progressive spastic paraparesis with hyperreflexia and impaired position sense. She was able to walk with help. After brain and cervical Magnetic Resonance Imaging (MRI) without gadolinium, she was admitted and treated with steroid pulse (1000 mg methylprednisolone) with the diagnosis of longitudinally extensive transverse myelitis (LETM) with normal brain MRI. AQP4 antibody test was borderline by enzyme-linked immunoassay (ELIZA) method . Due to the incomplete response to steroids, the steroid pulse was repeated three weeks later, but she developed covid-19, her weakness worsened, and then she was unable to walk. The patient was referred to Sina Hospital for further evaluation with a possible diagnosis of Neuromyelitis Optica Spectrum Disorder (NMOSD). MRI of the brain and spinal cord was performed again with gadolinium, which showed LETM with dorsal subpial longitudinal enhancement with trident sign on axial cuts , and poor R Wave progression Fig.Fig. 2ElCardiac biomarkers like highly sensitive troponin and Pro–B-type Natriuretic Peptide (Pro-BNP) were in the normal ranges High-sensitivity Troponin (HsTrop<0.0 ng/ml) and (Pro_BNP <100 pg/mL).Echocardiography findings revealed mild dilated LV (Left Ventricle) but moderate LV systolic dysfunction with positive regional wall motion abnormality in the territories of the anterior wall including the base of anterior, anteroseptal, anteroapical, apical, septoapical, and thinning and fibrotic changes of myocardial wall in these regions. Also, neither LV clot, significant valvular regurgitation, mass nor vegetation were found in transthoracic echocardiography. The right ventricle wall had normal size with preserved systolic dysfunction. Diastolic dysfunction was registered in grade III. Mild pulmonary hypertension was detected for this patient with Pulmonary Arterial Pressure (PAP) 38mmHg. There was no pericardial effusion or changes in the pericardium matrix. The Inferior Vena Cava (IVC) flow and size were normal Table .Table 1EThe cardiac team had planned for both cardiac MRI and coronary angiography. Coronary angiography from the radial artery was unsuccessful and the patient did not allow to do it via femoral site.So, we had to confirm the etiology of ischemia by myocardial perfusion scan. Myocardial Perfusion Imaging Single-Photon Emission Computed Tomography (MPI-SPECT), as illustrated in Fig has reveA cardiac MRI was performed at Rajaee Heart Center, Tehran, Iran. 4-chamber Cine images demonstrate global severe hypo-kinesia associated with septal wall dys-synchrony more prominent in the basal segment as shown in Figures Short axis STIR images shows multifocal edematous changes in anterior, lateral and inferior walls of LV basal to mid segments which are compatible with Sarcoid granulomas Fig. .Short axis late gadolinium enhancement images demonstrate a large burden of myocardial loss and thinning in the basal LV segment with intact endocardium and also patchy mid mural to subepicardial enhancement in mid part of the septum Fig. .24 ECG Holter monitoring showed only rare PAC  and PVC (Premature Ventricular Complex) without any episode of VT, or advanced block or pause. Furthermore, we have decided to treat the patient with carvedilol, spironolactone, atorvastatin, and aspirin and follow her with symptoms and serial echocardiography and electrocardiogram.After cardiac evaluation, the patient was diagnosed with sarcoidosis with two organs involvement and infliximab was started for the patient because of very specific imaging findings in both cardiac and spinal cord MRI. Two months after infliximab she was able to walk with a walker about 20 meters.Sarcoidosis is a multisystem cell-mediated inflammatory disorder in which the formation of non- or limited necrotizing epithelioid cell granuloma due to unknown antigenic causes is pathologic hallmark of the disease . NervousMyelopathy caused by sarcoidosis, could be in the differential diagnosis of other inflammatory diseases such as NMOSD . LongituThe same type of involvement is usually seen in NS patients with spinal cord myelopathy, but some key diagnostic findings in clinical and MRI can differentiate between these two diseases .Usually, the onset of paraparesis in sarcoidosis patients is gradual and chronic as in our case, and its onset is less acute and subacute , 6. SubpTherefore, in suspicious cases, investigation of systemic signs and symptoms in clinical and para-clinical workup can help to accurately diagnose these cases , 5.Interpretation of the ELISA Aquaporin-4- IgG (AQp4) antibody test should be done with caution because of high false positive results in medium or low titers and the positivity of this test should be confirmed by the CBA method. One case was previously reported with a misdiagnosis due to the ELISA test . Lung tiIt is possible to identify asymptomatic occult granulomatous inflammation by using fluorodeoxyglucose positron emission tomography (FDG-PET), which shows increased glucose uptake of active inflammation . TherefoSystemic sarcoidosis has cardiac involvement in up to 25% of patients' autopsy. Cardiac involvement portends a worse prognosis . CS can The sensitivity of endomyocardial biopsy is hindered by the myocardial tissue's patchy involvement, thereby limiting its effectiveness as a diagnostic method. Therefore, it is recommended to perform a cardiac MRI or FDG PET scan .According to the diagnostic criteria of CS in addition to histological or clinical diagnosis of extra-cardiac sarcoidosis, two or more of the five major criteria below or one of the major criteria and ≥ two of the minor criteria are satisfied to confirm probable CS . High-grade AV block or fatal ventricular arrhythmiaBasal thinning of the ventricular septum or abnormal ventricular wall anatomyAbnormally high uptake with 67Ga citrate or 18F-FDG-PETDepressed ejection fraction of the LV (< 50%)Delayed enhancement on gadolinium-enhanced MRIMinor criteria includeECG: ventricular arrhythmias, BBB, axis deviation, or abnormal Q wavesPerfusion defects by myocardial perfusion scintigraphyEMB : monocyte infiltration and moderate or severe myocardial interstitial fibrosisMajor cardiac involvement criteria include:In our patient, there are two major criteria of reduced Left Ventricular Ejection Fraction (LVEF) and delayed enhancement on gadolinium-enhanced MRI and also two minor criteria of LAHB in electrocardiogram and perfusion defects by myocardial perfusion scintigraphy. So, the satisfied CS was confirmed for her. As it is described in our patient, STIR images show multifocal edematous changes in the anterior, lateral and the inferior walls of LV basal compatible with sarcoid granulomas.Because of the high basal glucose metabolism of the cardiac tissue, the FDG-PET scan may have a diagnostic pitfall in showing inflammatory areas with a high metabolism. Therefore, the preparation of the patient before the FDG-PET scans with different methods such as a proper diet can reduce this defect in the evaluation of cardiac sarcoidosis , 10.Deciding the type and duration of treatment in NS and CS patients is currently challenging , 12.In patients with spinal sarcoidosis or symptomatic cardiac involvement, treatment with oral steroids and simultaneous initiation of an immunosuppressive drug such as Methotrexate Azathioprine or Mycophenolate Mofetil is recommended. In refractory cases, it is recommended to start infliximab but its use is contraindicated in cases of heart failure , 12.This case report indicated that Sarcoidosis may present with variety of signs and symptoms associated with wide range of radiological findings which needs to be promptly recognized and carefully treated.


Melatonin (N-acetyl-5-methoxytryptamine) is a multifunctional hormone that is naturally produced from tryptophan and released rhythmically throughout the night by the pineal gland to regulate sleep–wake cycles . MelatonWe should note that melatonin exerts other relevant cellular functions given its antioxidant and anti-inflammatory potential as well as its ability to regulate cell death processes . Therefo2+-calmodulin in the hippocampus of mice injected with doses of melatonin that mediate antidepressant-like effects. Interestingly, these authors also show that calmodulin adopts different conformations during its interaction with melatonin, depending on the aqueous or lipid nature of the microenvironment, and with opposite effects on the CaMKII activity [Melatonin mainly mediates its effects through its MT1 and MT2 membrane receptors, which belong to the superfamily of G protein-coupled receptors . Interesactivity .Yoo and Joo (2023) provide an update regarding other relevant intracellular targets of melatonin. In particular, CHOP , GRP78/BiP (78-kDa Glucose-Regulated Protein/Binding immunoglobulin Protein), and PERK (Protein kinase R-like Endoplasmic Reticulum Kinase) have also been considered as potential intracellular targets for melatonin. These proteins are mediators of the endoplasmic reticulum’s stress responses, which are commonly activated in some neurological diseases, such as Alzheimer’s disease and Parkinson’s disease. Thus, melatonin might be protective for these neurodegenerative conditions by regulating endoplasmic reticulum stress via these specific cellular targets [It is of paramount importance to note that melatonin levels and its receptors significantly and gradually decline in several organs over the life-span ,10, whicThe therapeutic potential of melatonin acquires special relevance in the fight against common metabolic disorders, such as obesity . The stuThe anticancer properties of melatonin have also been extensively studied in recent years. The study of Wand and collaborators (2022) provides a comprehensive review of the updated potential benefits and the limitations of melatonin as a preventive and therapeutic agent for cancer treatment in its initiation, promotion, and progression phases, either alone or in combination with other anticancer drugs . ParticuThe therapeutic applications of melatonin in clinical practice are incredibly varied, even including improvements in assisted reproductive technology (ART) outcomes . In thisThe application of machine data learning technology in biomedicine is currently in full growth. For instance, we recently identified prognostic networks by applying this technology that are key for unraveling the biological mechanisms of sarcopenia . InteresRecently, we have demonstrated that melatonin induces a recovery in the functionality of adult hippocampal neurogenesis in aged and neurodegenerative brains of mice with accelerated senescence . In thisWe have recently revised benefits of the neurogenic potential of melatonin for treating neurological and neuropsychiatric disorders under which processes related to adult brain neurogenesis are affected . In partIn summary, this Special Issue contains nine papers, including five original research articles and five systematic review articles. All of them provide important data related to the benefits of melatonin in several common pathological conditions, including diabetic neuropathy and retinopathy, cancer, neurodegenerative diseases, and fertility problems as well as under local and systemic inflammatory conditions. Melatonin plays a key role in different cellular signaling pathways, such as cell differentiation processes and gene expression, and it modulates different physiological mechanisms  with high efficacy and low toxicity. Therefore, is paramount to rigorously determine those basis mechanisms that melatonin may modulate in order to support the utility of melatonin in the therapy of these different pathological conditions in clinical practice.


The coronavirus disease 2019 pandemic demonstrated broad utility of pathogen sequencing with rapid methodological progress alongside global distribution of sequencing infrastructure. This review considers implications for now moving clinical metagenomics into routine service, with respiratory metagenomics as the exemplar use-case.Respiratory metagenomic workflows have completed proof-of-concept, providing organism identification and many genotypic antimicrobial resistance determinants from clinical samples in <6 h. This enables rapid escalation or de-escalation of empiric therapy for patient benefit and reducing selection of antimicrobial resistance, with genomic-typing available in the same time-frame. Attention is now focussed on demonstrating clinical, health-economic, accreditation, and regulatory requirements. More fundamentally, pathogen sequencing challenges the traditional culture-orientated time frame of microbiology laboratories, which through automation and centralisation risks becoming increasingly separated from the clinical setting. It presents an alternative future where infection experts are brought together around a single genetic output in an acute timeframe, aligning the microbiology target operating model with the wider human genomic and digital strategy.Pathogen sequencing is a transformational proposition for microbiology laboratories and their infectious diseases, infection control, and public health partners. Healthcare systems that link output from routine clinical metagenomic sequencing, with pandemic and antimicrobial resistance surveillance, will create valuable tools for protecting their population against future infectious diseases threats. Clinicians review many sources of information when assessing patients with an acute infectious disease, which for the purposes of this review is on the adult or paediatric intensive care unit (ICU) with a suspected severe lower respiratory tract infection (LRTI). There are multiple viral, bacterial, and fungal pathogens, some with potential for transmission to family, recent community contacts and now healthcare staff, and there may be antimicrobial resistance which complicates treatment choices, particularly if the LRTI was acquired in hospital. There is also the rare but not discountable possibility of novel pathogens including with pandemic potential as happened with Middle East Respiratory Syndrome Coronavirus (MERS-CoV) on our own ICU in 2012 .Respiratory and other samples are tested, each for a selected range of pathogens and often in different laboratories that provide results back from within an hour, to a few hours, the next day or many days, and weeks later. Data from biochemistry, haematology, imaging, history, examination, and physiological parameters are available that day, but the identity and antimicrobial resistance (AMR) of causative pathogens are not. Consequently, treatment, infection control and public health decisions are made empirically at different times based on the likelihood and consequence of different pathogens. A total of 25–47% of community LRTI cases remain undiagnosed after all conventional testing [The clinical metagenomics proposition in an acute care setting is to sequence nucleic acid from all organisms in a patient sample, to provide all required pathogen information ideally on the day of sample collection. Rapid turnaround time is important but shared by other molecular tests, however it is the comprehensive pathogen-agnostic capability that is the unique aspect setting metagenomics apart from all other tests. The ability to identify conventional agents and their AMR profile, alongside the unexpected, unrequested, hard to detect or novel pathogens, and therefore conversely a more dependable “rule-out” result. Application to respiratory infection has potential for greatest impact, with data from a single test used for patient treatment, antimicrobial stewardship, hospital infection control, public health surveillance and pandemic preparedness ,7.This opportunity was clearly articulated before the pandemic along with equally matched technical and implementation challenges ,9, such Pneumocystis jirovecci in non-human immunodeficiency virus immunosuppressed patients [Trophorema whipplei[With these technical advances have come increasing case reports and series identifying unusual or unexpected pathogens –28, comppatients ,38, but  whipplei. These w whipplei.Given these recent developments this review considers an end-state where every patient having a deep respiratory sample submitted to the diagnostic laboratory has first-line metagenomic testing as standard of care. Culture and targeted molecular tests continue for selected cases or as reflex tests, rather than the other way round, which is the current generally accepted position ,41. TargMycobacterium tuberculosis or Chlamydia psittacci. Indeed, targeted approaches for some respiratory pathogens such as M. tuberculosis when their presence is specifically sought [Clinical input is needed to define user requirements to develop clear target product profiles that will inform late-stage methodology refinement . It is ny sought , which iThe content and time of report production needs consideration. Accurate organism identification is possible from sequencing <0.1% of the genome after a few minutes so there may be benefit producing organism profile or “No (significant) organisms detected” reports first . AlternaImproving diagnostic pathways will require improvement to the pre-analytic and post-analytic stages in addition to the testing methodology itself. The UK standards for microbiology investigations for blood cultures has set new targets for time to analysis of <4 h alongside bigger blood volumes to increase sensitivity . SimilarClinical reporting will need re-evaluation with turnaround times for validation or auto-release rather than batch reporting. At least initially, interpretation may be needed at the bed-side or at daily hybrid MDTs with clinical microbiology, infectious diseases, radiologists, antimicrobial pharmacists and intensivists present. This will be an important opportunity to achieve better consensus about aetiology and diagnosis of LRTI, particularly ventilator-associated pneumonia (VAP), which has suffered from longstanding uncertainty and controversy ,54 and dProviding evidence of combined clinical and health-economic benefits at a system level will be an important driver for RMg adoption and re-imbursement but will likely come from a range of sources and trials that capture the different benefits. They will need to assess (1) Immediate patient benefit, commencing appropriate antimicrobials when organisms are identified predicted to have intrinsic or acquired resistance to empiric therapy. The frequency will depend on prevalence of endemic AMR, local guidelines and individual risk assessment made by attending clinician. (2) AMR benefit, targeting or stopping antimicrobials by identifying organisms predicted to be susceptible to a narrower antimicrobial spectrum or reporting no organisms. Almost all antimicrobial prescribing includes some retrospectively definable over-treatment, so measures of safe achievable reduction are required for clinical trials. (3) Wider patient and organisational benefit through faster identification of organisms with infection control significance prompting interventions to prevent transmission or further cases. (4) Public health benefit, identifying organisms or variants with wider public health significance including increasing community epidemiological trends, new or re-emerging infections.These benefits will be captured in target product profiles  that speFinally, there is the challenge with RMg presented as a hypothesis-free test not restricting itself to a predefined list of pathogens, with utility for the unexpected, the un-requested, the unknown or novel agent ,7. TheseMost pathogen sequencing is currently performed in academic or public health laboratories but will need to be performed in diagnostic laboratories for routine service. Indeed, there may be advantages moving the dominant sequence capability into the clinical laboratory , with daEstablishing pathogen sequencing within the microbiology laboratory has implications for the target operating model, which in many countries is for consolidation including to off-site facilities, utilising increased automation of culture-based core laboratory workflows. This is anticipated to enhance investment in molecular diagnostics and innovation, while improving quality and turnaround times ; howeverEvery laboratory network will have different factors to consider in placing sequencing capability alongside relevant clinical and scientific expertise, ensuring efficient sample transport, clinical reporting and data infrastructure. For some it may be considered a disruptive additional change overlaid on existing plans. For all, it will require investment that is probably beyond the diagnostic laboratory budget or strategic remit and will need leadership from the healthcare system embedding infectious diseases within its wider genomic, artificial intelligence, and digital strategy ,63. It cThis review summarises the dramatic progress in pathogen sequencing during the COVID-19 pandemic, which served as a reminder that serious emerging and re-emerging infections are frequent events, exacerbated by global warming, biodiversity loss, international trade and travel . When coNone.JE received funding from the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and STtThomas’ National Health Service (NHS) Foundation Trust and Kings College London, the programme of Infection and Immunity (RJ112/N027) and the Guy's and St Thomas’ Fund grant TCF 190910. JE receives research funding from Oxford Nanopore Technologies under a collaboration agreement with Guy's & St Thomas’ Hospital.JE is employed part-time by Oxford Nanopore Technologies


Erythroblasts possess unique characteristics as they undergo differentiation from hematopoietic stem cells. During terminal erythropoiesis, these cells incorporate large amounts of iron in order to generate hemoglobin and ultimately undergo enucleation to become mature red blood cells, ultimately delivering oxygen in the circulation. Thus, erythropoiesis is a finely tuned, multifaceted process requiring numerous properly timed physiological events to maintain efficient production of 2 million red blood cells per second in steady state. Iron is required for normal functioning in all human cells, the erythropoietic compartment consuming the majority in light of the high iron requirements for hemoglobin synthesis. Recent evidence regarding the crosstalk between erythropoiesis and iron metabolism sheds light on the regulation of iron availability by erythroblasts and the consequences of insufficient as well as excess iron on erythroid lineage proliferation and differentiation. In addition, significant progress has been made in our understanding of dysregulated iron metabolism in various congenital and acquired malignant and non-malignant diseases. Finally, we report several actual as well as theoretical opportunities for translating the recently acquired robust mechanistic understanding of iron metabolism regulation to improve management of patients with disordered erythropoiesis, such as anemia of chronic inflammation, β-thalassemia, polycythemia vera, and myelodysplastic syndromes. Iron is an essential element for almost every organism on earth. Iron can donate and accept electrons from various substrates due to its unique oxidation-reduction properties, making it an important cofactor in mammalian cells. Despite its abundance in the Earth’s crust, high oxygen availability in the atmosphere leads to iron oxidation and the formation of poorly soluble ferric iron. As a consequence, iron-dependent organisms like mammals have evolved complex mechanisms to conserve and recycle iron, preventing its loss and enabling its enhanced absorption during a deficit or in times of increased demand. The physiological status of iron in biological systems can be delineated into three categories: iron coordinated by protein side chains, iron complexed within the porphyrin ring of heme, and iron within iron-sulfur clusters. Outside of these contexts, iron displays promiscuous reactivity that can damage cells and tissues poorly equipped for handling what is termed ‘labile iron.’ Extensive coordination is also required in multicellular organisms for movement of iron between organs, in and out of cells, and between compartments within cells to effectively avoid inadequate iron supply that may limit systemic functioning and excess iron that may be toxic to cells and tissues. Thus, dysregulated iron homeostasis can manifest as total body iron deficit (iron deficiency) or excess (iron overload), as well as iron maldistribution among tissues in which individual organs may be iron-deficient while other iron-overloaded. Such iron disorders may be caused by genetic lesions that directly impair iron regulation or conditions that impact iron regulation indirectly.Although all cells in mammalian systems require small amounts of iron, systemic iron homeostasis is mainly regulated by the specific compartments involved in iron absorption, transport, storage and recycling, and high-level utilization. These include duodenal enterocytes; serum transferrin-bound iron; hepatocytes and macrophages in the liver and spleen; and erythroid precursors in the bone marrow, respectively. The largest of these compartments is the erythron, comprising the majority of total body iron in adult humans, mainly contained within hemoglobin inside erythroblasts and ultimately red blood cells (RBCs). Said another way, erythropoiesis, even at steady state, consumes most of the transferrin bound iron in circulation. Furthermore, stimulation of increased erythropoiesis  requires enhanced iron availability to keep pace with acutely increased hemoglobin synthesis within erythroblasts in the bone marrow. A large body of observational work amassed in the last >50 y suggested the presence of a direct ‘erythroid regulator’ of iron metabolism to enable this crosstalk. The last few decades have provided a series of substantial advances, enabling significant progress in our understanding of iron metabolism regulation; normal, stress, and ineffective erythropoiesis; and the crosstalk between them. In addition, the development of novel tools, both experimental methods and models of disease, has furthered and provided additional opportunities to better understand these pathways, robustly confirming and extending previously held assumptions and conjectures. This article aims to bring together our collective understanding of what has been learned and developed to study iron metabolism and erythropoiesis in health and disease.Erythropoiesis is a continuous process required for making new RBCs in order to replace the senescent RBCs lost at the end of their lifecycle. To determine how many RBCs are required, the bone marrow relies on the kidney in which interstitial fibroblasts in the renal medulla sense hypoxia, leading to the hypoxia-inducible factor 2 (HIF-2) mediated production and excretion of EPO. EPO binding to EPO receptor on erythroid precursors in the bone marrow in turn induces their survival, cell division, and differentiation to ultimately enucleate, producing reticulocytes that mature to RBCs in the circulation. Hemoglobin synthesis in the developing erythroblasts requires iron-containing heme.The first wave of erythropoiesis starts as early as embryonic day 7.5 (E7.5) in mouse  and 3 wkDefinitive erythropoiesis is a multi-step, complex process that can be divided into three maturational stages, namely early-stage erythropoiesis, terminal erythroid differentiation, and reticulocyte maturation . Early-sTerminal erythroid differentiation is a process by which proerythroblasts (Pro) differentiate sequentially to basophilic (Baso), polychromatic (Poly), and orthochromatic (Ortho) erythroblasts that expel their nuclei to become reticulocytes . During Enucleation is a distinctive feature of mammalian erythropoiesis. Given the physiological significance of enucleation in generating highly deformable RBCs for effective oxygen delivery, understanding the mechanistic basis of enucleation has been an active area of investigation. Cytoskeleton proteins such as actin , tubulinReticulocyte maturation is the final step of erythropoiesis. During this process, a series of major changes occur. These include membrane surface area loss via membrane vesiculation , organelErythropoiesis is tightly regulated by multiple soluble factors. To determine how many RBCs are required, the bone marrow relies on the kidney in which interstitial fibroblasts in the renal medulla sense hypoxia, leading to the HIF-2-mediated production and excretion of EPO. Very recent preliminary data using single-cell RNA and transposase-accessible chromatic (ATAC) sequencing to molecularly identify EPO-producing cells indicate that a distinct population of renal stromal cells, termed Norn cells, are the main source of EPO production in mice and humans . EPO binSimilar to EPO, iron is also essential for erythropoiesis. Hemoglobin synthesis in the developing erythroblasts requires iron-containing heme. While EPO allows survival of erythroid progenitors and early-stage erythroblasts by activating transcription of anti-apoptotic genes via the EPO/EPOR/JAK2/STAT5 signaling pathway , iron moBecause of its low bioavailability, complex living organisms have developed sophisticated mechanisms to obtain, distribute, and sequester iron that have also enabled competition for iron with pathogens and prevention of iron excess. In a seminal discovery more than two decades ago, the peptide hormone hepcidin, secreted primarily by hepatocytes, has been shown to be the principal regulator of iron homeostasis , modulatHepcidin:ferroportin binding leads to both occlusion of the ferroportin channel  and indub (DCYTB), and the basolateral exporter ferroportin  . NTBI, iAs mentioned, iron is required for homeostatic function in all cells, essential for the production of heme and iron-sulfur clusters, themselves components of proteins/enzymes involved in respiration, nucleic acid replication and repair, metabolic reactions, and host defense. Specifically, iron is necessary for enzymatic reactions in the electron transport chain and the tricarboxylic acid cycle, and iron participates in reactions catalyzed by microsomal cytochromes involved in the detoxification of drugs and other foreign substances. Despite broad functioning in physiological processes, the majority of iron functions as an oxygen carrier in the heme groups of hemoglobin and myoglobin molecules. Because iron can be highly toxic to cells, cellular iron trafficking requires deliberate coordination to enable its safe utilization. Here, we focus on specific cell types that are central to systemic iron metabolism.Duodenal enterocytes: The primary site of dietary iron absorption involves enterocytes within duodenal villi. These polarized cells are in contact with the gut lumen and dietary contents on their apical side and with blood in circulation on their basolateral side (3+) to ferrous (Fe2+) state  state . Iron ths export . Finallys export .Reticuloendothelial macrophages: At the end of their 120-day life cycle, RBC recycling by macrophages in the spleen and liver supports the recovery of iron for further systemic use , primarily BMP6, BMP2, and/or their heterodimers via BMP receptors, and transferrin-bound iron via TFR1 and TFR2, leading to signaling to induce hepcidin expression  leads to increased iron absorption, and the last few decades have provided a more robust mechanistic understanding of how iron availability is regulated by erythropoiesis. For the purposes of this article, we will discuss several aspects of this crosstalk, including how iron is taken up and chaperoned in erythroblasts, how erythropoiesis modulates iron metabolism directly and indirectly, and how iron metabolism itself impacts erythropoiesis.Pcbp1 knockout mice, with microcytosis and anemia, demonstrate that iron delivery to ferritin is required for normal erythropoiesis  underlying erythropoiesis-mediated regulation of hepcidin required separating how EPO, hypoxia, anemia, reticulocytosis, and erythropoiesis itself are individually involved. Prior experiments demonstrate that phlebotomy, EPO administration, and hemolysis all resulted in decreased hepcidin expression . AdditioSuch an erythroid factor secreted by erythroid precursors, functioning as a hormone to distally suppress hepcidin expression in the liver, was predicted several decades prior to its recent discovery. Although multiple factors correlate with pathologically expanded or ineffective erythropoiesis, they do not support physiological regulation of iron by erythropoiesis. For example, although circulating growth differentiation factor 15 (GDF15) increases in patients with some congenital and acquired anemias and inversely correlates with hepcidin , levels ERFE is expressed in bone marrow erythroblasts  provided a mechanism for the physiological regulation of hepcidin in the absence of chronic disease . ERFE isroblasts . As Erfel period , ERFE ex-/- mice  after ph-/- mice , increas-/- mice .Anemia as a result of systemic iron deficiency is the most common cause of anemia worldwide. There is great consensus that iron deficiency inhibits the production of heme and hemoglobin but is erroneously synonymous with the resultant anemia. However, decreased heme and hemoglobin production in iron-deficient conditions contributes to decreased mean corpuscular volume (MCV) and hemoglobin (MCH). Conversely, disease states of excess iron are often associated with higher MCV and MCH as a functional utilization of iron within a non-toxic compartment . Anemia,Tfr2-deficient hematopoietic cells demonstrate increased EPO sensitivity, including erythrocytosis and activation of the JAK2-STAT5 and AKT pathways . In addition, iron refractory iron deficiency anemia (IRIDA) is an interesting albeit rare form of IDA discussed here. In a subsequent section below, we will also discuss iron metabolism in the context of ineffective erythropoiesis in iron loading anemias, namely β-thalassemias and MDS.ACI is also termed anemia of chronic disease; while iron deficiency results in sometimes severe (i.e. hemoglobin 5–7 g/dL) microcytic, hypochromic anemia, ACI is typically a milder normocytic normochromic hypoproliferative anemia (i.e. hemoglobin 8–10 g/dL) and is considered the second most frequent anemia in the world, after IDA . Both prSalmonella and others) await additional clarification   . TMPRSS6he liver  and encohe liver . Matripthe liver , and TMPchanisms . As a coTMPRSS6 mutations have been reported in IRIDA with most variants unique to individual families , very low MCV (45–65 fL) and transferrin saturation (<5%), suppressed oral iron absorption, and abnormal iron utilization in response to parenteral iron. Surprisingly, infants with IRIDA demonstrate normal birth weights, normal growth and development, without cognitive concerns on long-term follow-up. Because these patients are generally healthy, anemia diagnosis is made via routine screening conducted in the first few years of life . Close tfamilies , and somrameters . Taken tIneffective erythropoiesis can be defined as the diminished production of enucleated RBCs despite an increase in the number of erythroid precursors . When thExpansion in the number of erythroid precursors leads to increased ERFE production and consequent hepcidin suppression . HypoxiaNovel drugs and genetic approaches are now being translated to improve the quality of life in β-thalassemia (and other) patients or even cure them. Although a full description of these therapeutics is beyond the scope of the current review, these drugs can be broadly classified based on their mechanism of action as shown in Beyond the inherited forms of iron-loading anemias, that is, β-thalassemia, MDS is an acquired form of ineffective erythropoiesis associated with iron overload. MDS is a heterogeneous group of bone marrow stem cell disorders; several subtypes of MDS are characterized by ineffective erythropoiesis, leading to blood cytopenias and increased incidence of transformation to acute myeloid leukemia . The majThe TELESTO trial was designed and executed to test whether iron chelation provides clinical benefit in iron-overloaded lower risk MDS patients . The resSF3B1) mutations in hematopoietic stem and progenitor cells is a hallmark of this disease, inducing aberrant splicing of genes involved in heme biosynthesis and mitochondrial iron transport, leading to the abnormal deposition of iron in erythroblasts, and resulting in dysfunctional hemoglobin synthesis and formation of ringed sideroblasts  and European Medicines Agency (EMA)-approved for very low- to intermediate-risk MDS with ringed sideroblasts. Luspatercept is a modified activin receptor IIB ligand trap, a member of the transforming growth factor-β (TGF-β) superfamily. While this agent was studied in multiple different MDS subgroups of patients in a phase II trial, its efficacy was most robust in patients with MDS with ringed sideroblasts  and led subtypes . A more JAK2 mutations in their stem cells, namely JAK2V617F on exon 14 (95% of PV patients) or mutations in exon 12 of the JAK2 gene (2–3% of PV patients) (Polycythemia vera (PV), one of the chronic myeloproliferative neoplasms, is a clonal hematopoietic stem cell disorder driven by EPO hypersensitive signaling via the JAK2-STAT5 pathway, resulting in excess proliferation of erythroid precursors . The vasatients) . PV patiatients) , and thiatients) . Repeateatients) . Recent atients) .We previously demonstrate that PV, compared to secondary forms of erythrocytosis, is associated with relative suppression of hepcidin, potentially due to more expanded erythropoiesis and iron depletion . In addiErfe deletion in Jak2V617F mice did not alter hepcidin levels or disease severity, resulting in stably elevated hematocrits and RBC counts. Using a human hepatocyte cell line, HepG2 cells, the authors further explored the hypothesis that PV-associated inflammation leads to hepcidin upregulation, demonstrating that the increased hepcidin expression was induced by plasma from PV patients but not plasma from normal controls and was normalized by blocking IL-6 binding to its receptor  in phlebotomy-requiring PV patients demonstrate a virtual elimination of phlebotomy requirements, control of RBC count, increase in systemic iron stores, and a potential decrease in systemic symptoms . A dramaJak2V617F (PV mouse model) and Hbb3/+  mice, macrophage depletion normalized erythropoiesis (Erythropoiesis occurs at the erythroblastic island (EBI) that is composed of a central macrophage surrounded by developing erythroid cells  and granopoiesis . Togetheopoiesis  and thatopoiesis . Supportopoiesis . In addiopoiesis . Finallyopoiesis .+EpoR+ and F4/80+EpoR- macrophages  critical for DNA degradation of the engulfed nuclei  and CD71. Within the Kit+ fraction, the level of CD71 expression was used to separate BFU-E (CD7110%low) and CFU-E (CD7120%high) with more than 90% purity  enabled stage-specific purification of murine erythroblasts with more than 90% purity . Under phiband3neg), early Baso (α4 integrinhiband3low), late Baso (α4 integrinhiband 3med), Poly (α4 integrinmedband3med), and Ortho (α4 integrinlowband 3hi) erythroblasts , reticulocyte (high forward scatter SYTO16-), and extruded nuclei (low forward scatter SYTO16+) are thus identified   . Evidenc–50 ppm) . FinallyTaken together, a great deal is currently known about the physiology of erythropoiesis and iron metabolism as well as the pathophysiology of diseases in which these biological systems are dysregulated. Despite this, significant unknowns remain, both regarding the mechanisms of normal function and their disordered regulation in disease; we thus include here an incomplete list to guide the next generation of targeted inquiry along these lines:A mechanistic molecular understanding of ineffective erythropoiesis and a more complete delineation of how EPO responsiveness is modulated in both physiological and pathological conditions remains elusive. This direction of investigation could yield novel therapeutic development for a variety of diseases associated with anemia, for example, anemia of chronic inflammation and in renal failure.The role of and purpose for iron and heme export from erythroblasts  is counterintuitive and incompletely understood.Whether and how iron-specific proteins  in non-hepatocyte cells regulate the crosstalk between iron metabolism, immunity, and erythropoiesis is not well understood. Such exploration may yield novel therapeutic targets for a variety of disorders.Whether and how EPOR expression outside of erythroid lineage cells regulate the crosstalk between iron metabolism and erythropoiesis remains to be more completely elucidated.How iron deficiency anemia exerts and influences platelet production remains largely unknown despite the long-standing clinical recognition of thrombocytosis as a common co-occurrence. Such mechanisms could potentially be exploited in designing novel treatments for thrombocytopenia.Genetic factors most likely contribute to heterogeneity in the human response to iron deficiency. Why do individuals with the same degree of iron deficiency show differences in the extent of anemia and of thrombocytosis? Understanding such factors may enable a more personalized approach toward iron-related therapies.A novel mechanism for cell death, ferroptosis, was recently discovered, an iron-mediated mechanism leading to lipid peroxidation and cell death that is being targeted as a therapeutic approach in various cancers. Understanding the role of ferroptosis as it pertains to iron trafficking in erythroblasts may shed light on iron-loading anemias associated with ineffective erythropoiesis .Given the critical role of EBI macrophages in supporting erythropoiesis, understanding whether and how EPO/EPOR signaling in these cells further enables coordination of erythropoiesis and enucleation in normal and disordered erythropoiesis is important.


Neurodegenerative diseases present increasing interest in clinical practice for the aging population and involve dysregulation of sleep-wake behaviors. Approximately 5.8 million adults aged 65 and older were living with Alzheimer’s disease (AD) in the United States in 2020 with increased mortality compared to the declining cardiovascular and cancer death rates. We conducted an extensive literature review to evaluate and synthesize evidence regarding the association between short sleep duration or sleep deprivation and the risk of developing all-cause dementia and Alzheimer’s disease. There are multiple mechanisms describing brain damage, such as brain hypoxia, oxidative stress, or blood-brain barrier (BBB) impairment, induced by chronic sleep restriction (CSR) and the potential correlation with future cognitive decline and dementia. More studies are necessary to identify the specific factors involved in the sleep loss-cognitive decline association that could be taken into consideration while elaborating recommendations for dementia prevention measures. According to the International Work Group, Alzheimer’s disease (AD) is conceptualized as a clinical-biological syndrome, comprising typical amnestic features, age-related cognitive and functional decline, and neuropathological biomarkers ,2. In 20Neurodegenerative diseases present increasing interest in clinical practice for the aging population and involve dysregulation of sleep-wake behaviors caused by specific pathophysiological processes within the brainstem and hypothalamus . MultiplAs reported by the CDC, short sleep duration (less than seven hours) was less prevalent among the respondents aged 65 and older (26.3%) compared with other age groups (18-64 years ranged from 32.2% to 35.6%), and there is no difference between males and females [Consequently, we conducted a literature review to evaluate and synthesize evidence regarding the association between short sleep duration or sleep deprivation and the risk of developing all-cause dementia and AD.Due to the lack of efficient treatment of dementia, the identification of modifiable risk factors has become imperative, considering the suggestive evidence of a correlation between dietary patterns, physical activity, sleep dysfunction, and cognitive impairment ,8. MultiSleep is essential for the revitalization of the functions of the body, including muscle restoration and hormonal regulation . Sleep dMechanisms triggered by sleep disruptionThe Impact of Disrupted Sleep on the HippocampusMultiple studies have shown the role of sleep in memory consolidation and enhanced hippocampus-dependent signaling . The mosThe Impact of Disrupted Sleep on the Glymphatic SystemIt was demonstrated that sleep deprivation could be responsible for increasing levels of soluble amyloid Aβ in animal and human models and for the augmentation of amyloid plaque deposition in mice models . Many inThe Impact of Brain HypoxiaSevere hypoxia involves brain tissue damage by decreasing oxidative phosphorylation and other oxygen-dependent processes caused by chronic disorders such as obstructive sleep apnea or environmental conditions . Sleep dThe Impact of Oxidative StressThe cognitive decline in Alzheimer’s disease is caused by an impaired neuroendocrine system, glucose metabolism, and oxidative stress . OxidatiSleep is a dynamic resting condition with antioxidant effects, accountable for eradicating the ROS generated during wakefulness . StudiesNight shift workers with an evident lack of sleep might develop elevated oxidative stress damage induced by an increase of ROS production and a reduction in enzymatic  and superoxide dismutase (SOD)) and nonenzymatic ferric reducing antioxidant power (FRAP) antioxidant protection . FurtherThe study of Teixeira et al.  determinCircadian Activity Rhythm DisturbancesSeveral animal models showed that the modifications of sleep-wake cycles could be linked to the increased levels of hyperphosphorylated tau proteins in the brain and, subsequently, of extracellular amyloid, the main component of Alzheimer’s disease . There iDisrupted circadian rhythm measures, including delayed timing of peak activity on wrist actigraphy, less vigorous rhythm, and lower amplitude, were determined to be predictive of the future development of mild to severe cognitive impairment in cognitively normal females . CircadiTherefore, understanding the influence of sleep and circadian disturbances in healthy middle-aged people on the pathogenesis of dementia and AD may help the identification of modifiable risk factors to delay their onset and the development of new therapies .Overexpression of OrexinsSmall groups of neurons in the lateral hypothalamic and perifornical areas produce a neuropeptide, orexin, also known as hypocretins, that are responsible for the regulation of sleep, wakefulness, feeding, and energy homeostasis -46. OverHypothetically, the typical AD sleep disturbances, including sleep fragmentation, nocturnal arousals, diurnal somnolence, and REM sleep impairment, are associated with orexinergic neurotransmission dysfunction . LiguoriBlood-Brain Barrier ImpairmentThe blood-brain barrier (BBB) consists of specialized microvessels that filter harmful substances from entering brain tissue that could affect sleep and cause sleep disorders ,52. UnfoSleep deprivation causes the disruption of circadian rhythms, as well as the production of new molecules, such as free radicals and cellular metabolites . ArousalPan and Kastin  designedSeveral studies have found that sleep deprivation is responsible for systemic low-grade inflammation, which is characterized by the release of various molecules, including chemokines, cytokines, and acute-phase proteins, affecting the brain endothelial cells .Vascular reactivity may have been reduced by chronic sleep restriction (CSR) because the BBB microvessels expressed lower levels of endothelin-1 and the inducible and endothelial forms of nitric oxide synthase . These aCSR increases COX-2-related inflammation, influences vascular reactivity, lowers glucose transporters, and impairs BBB permeability, although the BBB disruption generated by six days of CSR is restored after 24 hours of recovery sleep . SpecifiLong-term sleep deprivation can cause low-grade systemic inflammation and impaired blood-brain barrier function, including a reduction in the number of solute transporters. This may have a detrimental effect on cognitive functioning and increase the risk of neurological and neurodegenerative illnesses. Consideration should be given to the role of inflammation in the breakdown of the blood-brain barrier brought on by insufficient sleep. When examining the effects of sleep deprivation, the systemic and local effects of chronic sleep loss should be taken into account.The main mechanisms involved in the brain damage induced by sleep deprivation are brain hypoxia, impairment of the glymphatic system and brain-blood barrier, circadian rhythm disturbances, orexin overexpression, and the impact on the hippocampus. A multitude of aspects should be considered while analyzing the potential correlation between chronic sleep restriction in middle-aged individuals with future cognitive decline and dementia. Further studies are necessary to identify the specific factors involved in the sleep loss-cognitive decline association that could be taken into consideration while elaborating the recommendations for dementia prevention measures and subsequent public health policies.


Low-dose CT scan screening will be widely implemented on a large-scale base, aiming to reduce lung cancer related mortality in the high risk smoking population as already reported in multiple trials, in several countries. Recent evidence has suggested that the identification of liquid biopsy biomarkers may improve its accuracy in lung cancer early detection, reducing the false positive rate as well as overdiagnosis issues and potentially addressing one of the major obstacles in the implementation of Low-dose CT scan alone in this context. RNAs, particularly non-coding RNAs, are for sure the most studied and promising circulating biomarkers in this setting.Lung cancer represent the leading cause of cancer mortality, so several efforts have been focused on the development of a screening program. To address the issue of high overdiagnosis and false positive rates associated to LDCT-based screening, there is a need for new diagnostic biomarkers, with liquid biopsy ncRNAs detection emerging as a promising approach. In this scenario, this work provides an updated summary of the literature evidence about the role of non-coding RNAs in lung cancer screening. A literature search on PubMed was performed including studies which investigated liquid biopsy non-coding RNAs biomarker lung cancer patients and a control cohort. Micro RNAs were the most widely studied biomarkers in this setting but some preliminary evidence was found also for other non-coding RNAs, suggesting that a multi-biomarker based liquid biopsy approach could enhance their efficacy in the screening context. However, further studies are needed in order to optimize detection techniques as well as diagnostic accuracy before introducing novel biomarkers in the early diagnosis setting. Lung cancer remains nowadays the leading cause of cancer mortality accounting for 12% of overall cancer deaths worldwide. This is certainly linked to the peculiar biological behavior of this disease as well as to a significant diagnostic delay leading to advanced-stage diagnoses in about 50% of cases. For this reason, several efforts over the last years have been focused on the development of effective secondary prevention strategies, with different studies and metanalysis [In detail, the National Lung Cancer Screening Trial (NLST) and The Dutch-Belgian Randomized Lung Cancer Screening Trial (NELSON) randomized studies demonstrated a significant reduction around 20%) of lung cancer-related mortality in smoking populations undergoing LDCT as compared to either thorax RX or clinical observation 0% of lun, leadingA liquid biopsy is able to identify circulating tumor biomarkers that can be considered surrogates of the primary tumor as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA (miRNA), and exosomes. Liquid biopsies are already playing an important role in the clinical management of metastatic lung cancer patients through the evaluation of the tumor molecular profiling by ctDNA analysis, while also progressively extending to the early-stage disease in terms of minimal residual disease monitoring as well as cancer interception .The role of CTCs in lung cancer screening has been investigated in several trials since cell dissemination is a relatively early event in tumor progression. These trials showed that CTCs detected in high-risk patients are able to anticipate the diagnosis of lung cancer, even years earlier than CT scans . One of Several studies have investigated the role of CtDNA and cell-free DNA (cfDNA) in screening encountering a fundamental issue: the concentration of cfDNA correlates with the disease burden of the tumor that is very low in early-stage disease, making it difficult to isolate. Despite this limitation, different studies have investigated the role of cfDNA in early diagnosis. To distinguish tumor from non-tumor cfDNA, they have looked at its concentration, genetic changes, or methylation as possible biomarkers . In 2020Another interesting finding is the development of a blood-based multi-cancer early detection (MCED) test targeting a screening population. The test has been developed for the early detection of more than 50 types of cancer. The MCED test analyzed the methylation patterns of CtDNA and demonstrated high specificity (99.1%) and a positive predicted value of approximately 40% . Only a small fraction (approximately 3%) of the genetic transcript is able to encode proteins, while the remaining part is defined as non-coding RNAs (ncRNAs). The definition of “coding” encompasses RNAs that encode proteins from DNA-derived information, such as mRNAs. Noncoding RNAs have a different role since they act as cellular regulators of gene expression at different transcriptional, post-transcriptional, and epigenetic levels. A few exceptions to this definition include some ncRNAs binding ribosomes encoding peptides exerting a modulator function on cellular activities . It has ncRNAs can be classified into two macro-categories: housekeeping ncRNAs and regulatory ncRNAs. Housekeeping ncRNAs regulate basic cellular functions and are ubiquitously expressed. Regulatory ncRNAs play a pivotal role in gene expression regulation and protein translation both at transcriptional and post-transcriptional levels. Increasing evidence points out their role in cancer development, regulation, and growth, making them a precious tool for cancer management at different stages ,13. Among the housekeeping ncRNAs, we encounter transfer RNA and ribosomal RNA. Regulatory ncRNAs, instead, can be divided into three major groups: circular RNAs (cricRNAs), long noncoding RNAs , and small noncoding RNAs  . The ncRNAs can be found in different biofluids either as freely or encapsulated in extracellular vesicles ,14, makiTo date, among the different liquid biopsy biomarkers under clinical investigation in the lung cancer early detection setting, ncRNAs are for sure one of the most promising biomarkers to be implemented in the context of lung cancer screening. For this reason, this review will specifically focus on ncRNA role, describing biological function, available evidence, and clinical trials ongoing in this emerging setting.An extensive literature search was performed on PubMed, using as cut-off date of 16 March 2023. Keywords included: noncoding RNA, lung cancer, screening. A total of 2538 articles were found and screened for eligibility, taking into account three major criteria of inclusion: (1) Single ncRNAs or ncRNA-based genomic signature; (2) biomarker analysis performed on blood or other biofluids; and (3) biomarker involvement in non-small cell lung cancer (NSCLC) screening or early diagnosis.Both prospective and retrospective studies were considered. Only studies performed on humans were included, but they could include an in vitro/in vivo validation part. Only studies that involved a control group were considered. The control group could also have other pulmonary conditions or pulmonary nodules (PNs) that were retrospectively prospectively identified as benign lesions. Published abstracts without associated full articles were excluded from the analysis. Three independent reviewers collected data from the included articles, and another one subsequently reviewed all of the information.To draw a clear overview of all clinical trials concerning LC screening that included a liquid biopsy part, we also performed research on clinicaltrials.gov using the following keywords: lung cancer and screening. A total of 601 trials were found and, among them, only those involving LDCT screening and encompassing biofluids collection for biomarkers research were included. MiRNAs are fragments of single-stranded non-coding RNA with a length of approximately 20 ribonucleotides. Since they remain stable in biofluids, unlike other free RNA molecules, they can be detected in both serum and plasma. They regulate gene expression at the post-transcriptional level and are involved in the regulation of cell proliferation and apoptosis. In fact, their targets include oncogenes and tumor suppressor genes and their dysregulation can lead to malignant cell transformation across different tumor types ,17. miRNAs are one of the pivotal biomarkers explored in phase III trials of lung cancer screening, and our literature search identified studies that used both multi-miRNA signatures (>2 miRNA) and single miRNA approach for early NSCLC detection. In detail both the miR-test, a serum-based 13 miRNA signature, and the micro-RNA signature classifier (MSC), a plasma-based 24 miRNA risk score, showed very promising data for clinical use . Overall, 32 studies evaluating the expression of multi-miRNA in early-stage NSCLC patients compared to healthy controls have been identified through our PubMed research. The identified signatures ranged from 2 to 24 miRNAs and were all validated on biological fluids that could be used for liquid biopsies purposes . Almost Sozzi G. et al.  conducteIn another prospective analysis conducted in the BIOMILD study, patients were  stratifiShun J et al.  applied The same group  screenedAnother plasma-based approach was conducted by Zheng D. et al. , who evaEVs and miRNAs were also tested in this setting by using NGS analysis . They anOther pulmonary pathological conditions, such as chronic obstructive pulmonary disease (COPD) and asthma, were included in some of the other studies and represent an interesting approach to eliminate some biases that could be created by smoking-related or pre-existing pulmonary conditions in the implementation of liquid biopsies within lung screening programs. Halvorsen A.R. et al.  used serSome of the studies listed in Other efforts of using miRNA-based liquid biopsy for lung cancer early detection were made by using single miRNAs as biomarkers such as miR-17-19 , miR-20 LncRNAs look quite promising since they have been demonstrated to be stable in biofluids ,89 and tSingle lncRNA-based studies were conducted for different lncRNAs obtaining lower performances similar to what has been observed with miRNAs until now ,107,108,Circ-RNAs can be freely detected in biofluids  as well as in exosomes , and areFor what concerns small-nuclear RNAs we found three studies that tested the differences between LC patients and controls. Köhler J. et al. , determiFor what concerns piwiRNAs we found a study by Li J. et al.  demonstrNo studies matching our inclusion criteria were found about ribosomal RNA (rRNA), transfer RNA (tRNA), and small nucleolar- RNAs (sno-RNAs) in the context of lung cancer screening.Few studies were conducted using a combined ncRNAs approach, according to our inclusion criteria. In detail Peng H et al.  construcWe also performed a study of ongoing clinical trials on clinicaltrials.gov using the keywords “lung cancer” and “screening”. The data collection was completed on 16 March 2023 and identified a total of 601 ongoing trials related to lung cancer screening. Out of the 601 clinical trials identified, we selected 55 trials incorporating liquid biopsy and the analysis of biological samples for the detection of predictive biomarkers in the setting of LC screening . The selAmong the 55 clinical trials shortlisted based on our inclusion criteria, 25 of them involved the use of chest CT or LDCT scans as a diagnostic tool for lung cancer screening. The HANSE trial (NCT04913155) also investigated other indicators such as coronary calcium score and emphysema score. One of the selected studies involved the use of chest MRI to assess the concordance of imaging features of nodules between LDCT and MRI in the study population (NCT05699213). Concluding, a small portion of these studies incorporates pulmonary function testing within their research protocols.These selected trials also involved the collection and analysis of various biological samples to identify possible biomarkers for the early detection of lung cancer. Specifically, they included blood samples, different airways samples , sputum samples, buccal swab samples, urine samples, and feces samples. Among the 55 clinical trials that met our inclusion criteria, blood samples were collected in 52 trials, but only 33 of these explicitly state the specific biomarkers that were intended to be analyzed, including miRNA, epigenetic biomarkers, circulating free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating tumor cells (CTC), Associated Macrophage-Like cells (CAMLs), exosome antigens, methylation changes in peripheral blood mononuclear cells (PBMC) and circulating tumor DNA, RNA integrity number (RIN), protein signatures, DNA methylation, whole-genome methylation, tumor antibodies, circulating nucleic acids, proteins, and genetic variation single nucleotide polymorphisms (SNPs), as well as DNA and RNA for germline analysis and whole-exome sequencing (WES). Specifically, only eight trials have a clear focus on the identification and analysis of miRNA. Moreover, 3 out of these 52 clinical trials involve the storage of blood samples in biobanks for potential future studies.A small part of the clinical trials that met our inclusion criteria have already published results. We have already discussed the results of The Multicentric Italian Lung Detection (MILD) study, a prospective randomized controlled screening trial that compared the diagnostic performance of two different LDCT screening intervals in high-risk smoking populations. After a median active screening period of 6.2 years, the MILD trial concluded that biennial LDCT screening for lung cancer in individuals with a negative baseline LDCT can achieve a comparable clinical outcome to annual LDCT screening. The study, as already said, highlights the potential of circulating miRNAs as biomarkers for cancer detection and prognosis . In this scenario, we previously illustrated also the results of the BioMILD trial  which isThe New York University Lung Cancer Biomarker Center (NYULCBC) enrolled high-risk smokers and lung cancer patients into a screening cohort and a “rule-out lung cancer” cohort with the aim of identifying and validating biomarkers for the early detection of lung cancer. The participants completed a medical and respiratory symptom questionnaire, underwent pulmonary function testing, blood sampling, chest CT, and were followed up for nodule stability. Greenberg et al.  conducteSeveral clinical trials are investigating the effectiveness of incorporating new blood tests along with LDCT for lung cancer screening. The NCT01925625 trial tested whether using the EarlyCDT-Lung test and subsequent CT scanning to identify individuals at high risk of lung cancer could reduce the incidence of patients with advanced-stage lung cancer at diagnosis compared to standard clinical practice. The EarlyCDT-Lung test used an enzyme-linked immunosorbent assay (ELISA) to measure seven distinct autoantibodies, each having specificity for different tumor-associated antigens, including p53, NY-ESO-1, CAGE, GBU4-5, HuD, MAGE A4 and SOX2. At 2 years, the test showed high specificity (90.4%) and moderate sensitivity (32.1%) with a higher number of early-stage lung cancers detected in the intervention arm. However, no significant differences were observed in lung cancer and all-cause mortality between the intervention and control groups . The stuAnother test is Lung EpiCheck , which has been designed to detect hypermethylation status across six markers that are associated with lung cancer, by using cfDNA analysis. Recently, this test has been validated in European and Chinese patients samples and has demonstrated high accuracy rates, as well as an independent predictive capability for lung cancer detection, suggesting potential utility for improving screening access and compliance among high-risk populations . In thisFurthermore, the NCT03452514 trial aims to validate the HMBDx microRNA Test by collecting blood samples from 400 individuals who are undergoing LDCT screening. The study plans to analyze microRNA signatures using a novel lung cancer test, compare the results with those obtained through CT scan findings and follow-up tests, and maintain a minimum follow-up period of 12 months post-enrollment. Lastly, the primary objective of the NCT05306288 clinical trial is to validate the DELFI-based test for detecting lung cancer among individuals eligible for routine screening, using a genome-wide analysis technique called “DNA evaluation of fragments for early interception” (DELFI) to detect abnormalities in cfDNA . ParticiGiven the elevated incidence of overdiagnosis and false positive cases associated with LDCT screening, the identification of reliable biomarkers capable of improving the diagnostic accuracy, represents an unmet need. In this scenario, ncRNAs might be a potential reliable tool to stratify populations into precise categories of lung cancer risk. To date, microRNAs are those most investigated in large prospective trials for lung cancer screening purposes. As reported in the bioMILD trial, the implementation of miRNAs in NSCLC screening can reduce false positive rates and improve diagnostic accuracy of LDCT, thus opening the way for personalized screening approaches. Furthermore, what emerged from our literature research is an extreme heterogeneity of the conducted studies using different methodologies of analyses and selecting various risk populations. This inevitably can be seen as a positive aspect, as in most of the studies presented, the results were consistent with the ability of ncRNAs to distinguish populations with lung cancer from those that were negative or might face overdiagnosis if subjected to LDCT. However, from a methodological point of view, it clearly constitutes a major issue to be addressed with further research in order to standardize a potential application of ncRNAs liquid biopsy in a real-world setting and safely implement them into our clinical practice.Methodological limitations of analysis also emerged from this wide literature search, including the heterogeneity of ncRNA detection methods used across the different studies, mostly based on q-RT-PCR, but also on NGS limited panels, digital-droplet PCR, and RNA-seq, pointed out the issue of standardization methods to make ncRNAs part of clinical practice. In fact, from a practical point of view, detecting and sequencing this genetic material might be challenging for different reasons. Next-generation sequencing (NGS) is one of the high-throughput screening methods that can be implemented more efficiently into clinical research to validate panels of ncRNAs that can be used for LC screening research programs . Some limits need also to be considered once we propose ncRNAs as a biofluid-based biomarker for LC screening but also in general for other purposes. First of all, the overall quantity of ncRNAs is generally lower in the intracellular, extracellular ambient, as well as in plasma or serum as compared to other genetic material, so it might be a challenge to detect them in patient-derived blood samples . AnotherMoreover, lung cancer heterogeneity is well-known and established across the board ,130,131,In conclusion, the implementation of ncRNAs for LC screening purposes is one of the most promising biomarkers to integrate LB in the prevention setting. For this reason, standardization of protocols for LB ncRNAs detection and further prospective clinical trials with larger cohorts are needed to validate and introduce these novel biomarkers in the clinical arena. In addition, we believe that the use of ncRNAs belonging to multiple subcategories can further improve the ability to discriminate between negative and positive subjects, and therefore using expanded ncRNA panels for LC early detection should be one of the next implementations for LB studies in this research context. To date, clinicians should carefully interpret LB results coming from the early diagnosis studies and policy-makers should push research to focus also on the implementation of liquid biopsy in the real-world setting.


During the last decade, blood sampling of cancer patients aimed at analyzing the presence of cells, membrane-bound vesicles, or molecules released by primary tumors or metastatic growths emerged as an alternative to traditional tissue biopsies. The advent of this minimally invasive approach, known as blood-based liquid biopsy, began to play a pivotal role in the management of diverse cancers, establishing itself as a vital component of precision medicine. Here, we discuss three blood-based liquid biopsies, namely circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and tumor-derived exosomes, as they relate to prostate cancer (PCa) management. The advances achieved in the molecular characterization of these types of liquid biopsies and their potential to predict recurrence, improve responses to certain treatments, and evaluate prognosis, in PCa patients, are highlighted herein. While there is currently full clinical validation for only one CTC-based and one ctDNA-based liquid biopsy for patients with metastatic castration-resistant PCa, the adoption of additional methods is anticipated as they undergo standardization and achieve analytical and clinical validation. Advantages and disadvantages of different blood-based liquid biopsy approaches in the context of PCa are outlined herein, while also considering potential synergies through combinatory strategies. Worldwide, prostate cancer (PCa) ranks as one of the most frequently diagnosed cancers among males, contributing significantly to cancer-related mortality . In the Although prostate biopsy stands as the singular gold standard procedure for a definitive diagnosis of PCa, it provides a snapshot of limited locations within the primary tumor at a specific time, rather than capturing the heterogeneity present in different areas of the neoplasm . Moreoveliquid biopsies, has emerged as an alternative for cancer researchers and oncologists to explore non-invasive techniques that may overcome some of the limitations associated with tissue biopsies. Since the growth and spread of solid tumors depend on an adequate blood supply, there has been a growing focus on blood-based liquid biopsies, as they allow repeated sampling from patients and facilitate the monitoring of cancer progression and the assessment of response to treatment in real time [Over the last decade, the analysis of various patient-derived biological fluids, commonly referred to as eal time . Furthereal time , 7, blooIn this research perspective, we present a comprehensive overview of the recent advances related to the clinical significance of blood-based liquid biopsies in PCa, with a primary emphasis placed on key biomarkers such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes.Cells sloughed off from invasive primary tumors that enter the venous circulation are referred to as CTCs and represent a transitional phase in the progression towards metastasis, as only a few of those cells survive and are able to extravasate in distant organs . CTCs ciThe isolation of CTCs poses technical challenges due to their extremely low concentration in circulation, with an estimated ratio of approximately one CTC per billion normal blood cells in patients with advanced cancer . More or® system  stands as the sole technology cleared by the U.S. Food and Drug Administration (FDA) for the isolation, identification, and enumeration of CTCs from patients with PCa [® system emerged as an independent and superior predictor of OS in metastatic castration-resistant prostate cancer (mCRPC) patients compared to prostate serum antigen (PSA) levels, with a cut-off level of 5 or more CTCs per 7.5 mL of blood consistently associated with reduced OS [® system as a biomarker of disease progression in mCRPC patients [To date, the CellSearchwith PCa . This auwith PCa . CTC couduced OS , 17. Addpatients –20.® PC1 , a microfluidic platform that enables the capture and retrieval of intact single CTCs and CTC clusters, based on the less deformable nature and larger size of CTCs compared to other blood cells, regardless of their expression of epithelial, mesenchymal, or other cell surface markers [® was FDA-cleared for use in the isolation of circulating metastatic breast cancer cells for liquid biopsy testing [During the malignant progression of carcinomas, including PCa, neoplastic epithelial cells can undergo an epithelial-to-mesenchymal transition (EMT), which has been proposed to contribute to invasion, resistance to apoptosis, and metastatic dissemination , 22. Mor markers . Last ye testing . This sy testing , 29. Stu® for transcriptome profiling of both single and aggregate forms of epithelial and mesenchymal CTCs was confirmed in a cohort of mCRPC patients, suggesting that a carefully chosen gene panel with high expression in PCa cells and low expression in leukocytes could serve as a predictive tool for therapy response [While the enumeration of CTCs can shed light on PCa progression and patient outcomes, it is expected that the molecular characterization of these cells may uncover crucial information for a more comprehensive assessment of the tumor’s behavior and potential response to specific therapies. For instance, expression of the androgen receptor (AR) splice variant 7 (AR-V7) has been associated with reduced response to AR signaling inhibitors (ARSI) enzalutamide and abiraterone, as well as with shorter progression-free survival (PFS) and OS in patients with mCRPC , 31. Anoresponse . While tresponse . DespiteIn spite of some limitations, CTCs hold clinical significance at multiple levels in PCa management, not only offering prognostic and predictive insights but also crucial DNA, RNA, and protein data that can inform targeted approaches to address metastatic disease .The existence of cell-free circulating DNA (cfDNA), believed to be released into the bloodstream through apoptosis or necrosis, was first reported by Mandel and Metais in 1948 . DecadesPatients with solid cancers often present a very low fraction of ctDNA within the cfDNA (less than 0.1%), which poses significant technical challenges for its detection and analysis . Several™ assay, which assesses 70 genes, to test the plasma from over 3,000 patients with mCRPC [AR, TP53, and BRCA 1/2, identified using both ctDNA and tissue-based comprehensive genomic profiling (CGP) [™ as a CDx for identifying patients with mCRPC who have germline or somatic mutations in BRCA1 and/or BRCA2 genes who could benefit from for treatment with the Poly (ADP-ribose) polymerase (PARP) inhibitors olaparib and rucaparib [As of now, several ctDNA-based assays have obtained FDA approval as companion diagnostic (CDx) assays for screening specific gene alterations to guide the use of targeted therapies, mainly in certain patients with advanced non-small cell lung cancer and breast cancers , 46. As th mCRPC . They weng (CGP) . The FDAucaparib .Several studies have shown an association between elevated baseline cfDNA concentrations and ctDNA% values  and reduced PFS and OS in mCRPC patients treated with ARSI  and chemotherapy  –51. FurtAR gene amplification and inactivating mutations in tumor suppressor genes  [AR that can drive resistance to ARSI and are commonly found in mCRPC, such as copy number amplification, have also been identified in ctDNA and are associated with worse clinical outcomes [In advanced stage PCa, ctDNA presents a promising alternative to traditional tissue biomarker analysis. This is supported by studies of ctDNA and matched metastatic tissue biopsies that revealed a strong concordance in several gene alterations, including , BRCA2) , suggestoutcomes .de novo [TP53, RB1, CYLD, and AR loss) and epigenomic changes  reflective of metastatic lesions were identified in ctDNA from patients with NEPC [Analysis of ctDNA has also shown promise as a non-invasive approach for detecting neuroendocrine prostate cancer (NEPC), an aggressive PCa that can emerge from prostate adenocarcinoma as a mechanism of treatment resistance or may arise de novo , 57. Clide novo , which cde novo . Overallith NEPC . These rAlthough, unlike other blood-based liquid biopsies, ctDNA does not provide transcriptomic or proteomic information , the stuThe use of ctDNA-based assays for identifying specific actionable genomic alterations in mCRPC has been approved by the FDA as a CDx that guides treatment decisions, enabling a more personalized and targeted approach to therapy.Both normal and cancer cells can release extracellular vesicles (EVs) that are surrounded by a lipid bilayer membrane and carry a diverse cargo of DNA, RNA, lipids, or proteins . EV biog9 particles/mL of plasma), which simplifies isolation, (b) they arise from viable cells, encompassing substantial representative tissue information, and (c) their robust lipid bilayer structure that makes them stable carriers of DNA, RNA, proteins, and lipids from the cell of origin, enabling extended specimen storage and yielding enhanced testing opportunities [Exosomes have emerged as a compelling avenue for liquid biopsies due to a range of advantages: (a) their abundance in body fluids . AlthouACTN4 gene knockdown in DU 145 (a well-know PCa cell line) leads to significant decrease of in vitro invasiveness [While proteomic analyses appear to offer more advantages than transcriptomic studies, as they directly identify the presence of functional proteins rather than miRNA or upstream mRNA precursors, up to date only a few of those analyses have been performed in plasma or serum of PCa patients. For instance, in a study involving exosomes obtained from the sera of 36 PCa patients with bone metastases – comprising eight untreated individuals, eight under primary androgen deprivation therapy (ADT), and 20 with CRPC – a mass spectrometry analysis identified a total of 787 unique proteins. Among these, the expression levels of six proteins were found to be increased in CRPC patients compared to those undergoing ADT. Notably, only one protein, namely actinin-4 (ACTN4), showed significantly higher expression in CRPC than in ADT patients . This disiveness .ITIH3 expression in cancer profiling arrays containing spotted tumor cDNAs when compared with matching normal tissue [In another recently published study, an untargeted proteomics analysis was conducted from a total of 938 proteins identified on plasma exosomal proteins collected from tumor-free individuals and PCa and CRPC patients. The expression levels of specific exosomal proteins were subsequently confirmed within the three groups. This validation revealed that leucine-rich α-2 glycoprotein 1 (LRG1) and inter-α-trypsin inhibitor heavy chain H3 (ITIH3) exhibited significantly higher levels in the CRPC group compared to the PCa group . Previoul tissue . Taken t™Prostate IntelloScore (EPI) test  has been certified by the FDA to discriminate indolent from clinically significant PCa, based on the measurement in urine of three PCa-specific exosome-derived RNA biomarkers  [Besides blood samples, exosome isolation from urine can be employed to stratify patients with high-grade PCa (Grade Group 2) who are aged ≥50 years and have PSA levels between 2 and 10 ng/mL, which is considered an intermediate range that is not definitively indicative of a specific condition affecting the prostate. Indeed, the ExoDxd SPDEF) . NeverthTherefore, exosomes present a promising array of clinical applications in PCa patient management due to their inherent stability, abundant presence, and capacity to carry representative native tissue information. Hence, genomic, epigenomic, transcriptomic, and proteomic analyses of exosomes  may soonThe advent of liquid biopsies is causing a paradigm shift in the approach to diagnosing, monitoring, and treating various cancers, including PCa. This less conventional type of biopsies not only is minimally invasive as compared with traditional tissue biopsies, but also can be repeated over multiple points in time allowing for real-time monitoring of cancer progression and treatment response. This, in turn, enables us to stratify PCa patients more effectively and tailor personalized therapeutic strategies.® system (for enumeration of EpCAM-positive CTCs in CRPC) and the FoundationOne Liquid™ CDx (for profiling BRCA1 and/or BRCA2 mutations in ctDNA in mCRPC patients) stand as the sole FDA-approved or cleared blood-based liquid biopsy tests for PCa. This can be partly attributed to the stringent standards set for test approval to guarantee accuracy and reliability, and the extensive trials required for clinical validation. An illustration of the latter is the Parsortix® system, which has already been approved for use in patients with metastatic breast cancer but is still undergoing evaluation in extensive cohorts of PCa patients to ascertain its validity for clinical use in the context of this disease.Until now, the CellSearchThe aim of this Research Perspective is to provide current and pertinent information on three blood-based liquid biopsies in PCa, namely CTCs, ctDNA and exosomes. Each of them presents strengths and limitations that we summarized in Additional investigation is essential to determine the clinical validity and utility of diverse blood-derived liquid biopsies in PCa patients. Securing clinical qualification for both existing and novel tests demands not only prior confirmation of their sensitivity and specificity but also necessitates their standardization for widespread implementation and the establishment of reference values. Moreover, exploring multiparametric analysis by combining strategies for liquid biopsy from a single blood sample could be undertaken to determine if resolution can be enhanced, thereby expanding the range of clinical utility in PCa patients. We anticipate that, with the assistance of artificial intelligence based on pre-established parameters, the utilization of blood-based liquid biopsies will soon enhance the stratification of PCa patients and facilitate timely therapeutic decision-making.


Aging is associated to progressive changes impairing fundamental cellular and tissue functions, and the relationships amongst them through the vascular and immune systems. Aging factors are key to understanding the pathophysiology of stroke since they increase its risk and worsen its functional outcome. Most currently recognised hallmarks of aging are also involved in the cerebral responses to stroke. Notably, age-associated chronic low-grade inflammation is related to innate immune responses highlighted by induction of type-I interferon. The interferon program is prominent in microglia where it interrelates cell damage, danger signals, and phagocytosis with immunometabolic disturbances and inflammation. Microglia engulfment of damaged myelin and cell debris may overwhelm the cellular capacity for waste removal inducing intracellular lipid accumulation. Acute inflammation and interferon-stimulated gene expression are also typical features of acute stroke, where danger signal recognition by microglia trigger immunometabolic alterations underscored by lipid droplet biogenesis. Aging reduces the capacity to control these responses causing increased and persistent inflammation, metabolic dysregulation, and impaired cellular waste disposal. In turn, chronic peripheral inflammation during aging induces immunosenescence further worsening stroke-induced immunodepression, thus increasing the risk of post-stroke infection. Aging also alters gut microbiota composition inducing dysbiosis. These changes are enhanced by age-related diseases, such as atherosclerosis and type-II diabetes, that further promote vascular aging, predispose to stroke, and exacerbate brain inflammation after stroke. Current advances in aging research suggest that some age-associated alterations may be reversed. Future work will unravel whether such evolving anti-aging research may enable designing strategies to improve stroke outcome in the elderly. Aging is associated to a certain degree with cognitive decline, and it is a critical risk factor for neurodegeneration . The incComplex and interrelated factors are associated to functional decline during aging. A seminal paper by López-Otín and co-workers  identifiAging promotes epigenetic changes mediated by DNA methylation, histone modification and expression of non-coding RNA. Notably, aging-induced changes in the DNA methylation pattern are used as epigenetic clocks to predict the ‘functional or biological age’, which is dependent on genetic and environmental factors, versus the ‘chronological age’ . EpigeneLoss of proteostasis and disabled macroautophagy (termed autophagy in this review) may be particularly relevant at the cellular level in long lived brain cells. Alteration of these fundamental cellular processes is expected to impair cellular waste disposal leading to the accumulation of protein aggregates and lipids. Accordingly, an autofluorescent pigment termed lipofuscin, which is composed of residual lysosome products, can be typically seen by microscopy in cells of brain tissue sections from old individuals . DysfuncFinally, aging harbours the accumulation of senescent cells, i.e. cells undergoing irreversible growth arrest and secreting pro-inflammatory cytokines and other molecules conforming the senescence-associated secretory phenotype (SASP) . This phInflammaging involves a global upregulation of innate immune mediators in the elderly , 23. TypIn the brain, aging induces a specific transcriptomic signature highlighted by increased inflammation and microglial cell priming . Stroke Following stroke, injured cells generate danger signals or damage-associated molecular patterns, including nuclear proteins, nucleic acids, heat-shock proteins, amongst others, triggering immune responses by activating pattern recognition receptors (PRRs) . MicroglStroke induces a strong activation of the type I IFN program in microglia , 36. TheThe microglia type I IFN program mediated by Stat1 transcription factor intervenes in immunometabolic changes involving the accumulation of lipid storage organelles in the cytoplasm called lipid droplets , which aOverall, stroke induces an acute transcriptional and immunometabolic program in microglia of young mice with features resembling some of the phenotypic changes displayed by microglia of aged mice under steady state. Stroke in aged mice further exacerbates this response triggering more inflammation and failing to exert an adequate regulatory control to terminate the response to the acute challenge.Age-dependent inflammation is strongly associated with defects in the immune system. The capacity to mount adaptive and innate immune responses is attenuated in the elderly due to immunosenescence that explains why old people have an increased susceptibility to infections. This immunosuppressive state generated in the elderly is most likely a counteractive response trying to restrain the chronic inflammation. Decrease in naïve T cells and increase in memory T cells as well as lower capacity to generate antibody reactions against pathogens are prominent features of immunosenescence . In factThe acute brain inflammation induced by stroke is followed by systemic immunodepression –55. The Blood factors and immune cells may influence stroke outcome by affecting the function of brain vessels. The status of the vasculature is a critical player in the individual response to aging, as postulated long ago by the English physician Thomas Sydenham in the seventeenth century, who wrote the famous quote ‘A man is as old as his arteries’. All aging factors described above affect the vasculature, as recently reviewed . The agiThe intestinal microbiota has been implicated in normal development of the brain, including the normal functioning of microglia  and the Stroke-induced dysbiosis can secondarily affect the stroke outcome by a feedback loop between brain-gut-brain bidirectional pathway. Therefore, any process that alters the intestinal microbiota is susceptible of affecting stroke outcome. In this sense several vascular risk factors have been associated to intestinal dysbiosis, including diabetes, obesity, or hypertension, as well as aging . Aging iSeveral additional co-morbidities that are commonly present in stroke patients further increase systemic inflammation. We will focus on two of the most important ones: atherosclerosis and diabetes. Overall, a common factor amongst aging, stroke, atherosclerosis and type II diabetes is the contribution of chronic inflammation to the pathophysiological process. Controlling chronic inflammation should have a positive impact on the aging process, the progression of important comorbidities, and the outcome of stroke in the elderly.Atherosclerosis is a chronic inflammatory condition more prevalent in the elderly and therefore associated with aging. Atherosclerosis increases the risk of stroke, and in turn stroke increases the progression of atherosclerosis. Both directions are mediated, at least in part, by systemic inflammation. Inflammation has a fundamental role in every step of atherosclerosis formation, from the initial points to the final complications of thrombosis. Atherosclerosis is a hardening and narrowing of arteries caused specially by the accumulation of cholesterol plaques in the inner lining of an artery, inducing inflammatory mediators, such as IL-6, and the recruitment of leukocytes to the arterial wall . IncreasThe risk of type II diabetes increases with age, affecting around 25% of the population over 65 years old. Aging is associated with an increase in abdominal obesity, a major contributor of insulin resistance, and therefore aging and type II diabetes are intricately linked. In addition, aging affects adipose tissue homeostasis and metabolic functions both of which decline with aging and obesity. Aging is associated to adipose tissue senescence, which causes defective adipogenesis, inflammation and insulin resistance . PatientFinding ways to prevent aging has been for centuries the search for the Holy Grail. However, recent solid findings suggest that some hallmarks of aging may be reversible. For instance, loss of epigenetic information is postulated as a mechanism causative of aging that can be reversed . Indeed,Other studies also suggested that age-dependent BBB dysfunction might be attenuated or reversed. For instance, inhibition of TGF-β diminished neuronal dysfunction mediated by age-associated BBB alteration . Long-teInterventions directed to influence the microbiota composition and derived metabolites may offer new therapeutic opportunities for implementing the treatment of stroke in the elderly. Notably, transplantation of fecal matter from young mice enriched in butyrate-producing bacteria to old mice can reverse at least part of the phenotypes associated with aging by improving cognitive functions . FurtherAging is associated with a plethora of alterations that increase frailty, predispose to age-related diseases, and reduce the brain resilience to injury Fig. . Not onl