Stimulator of interferons genes (STING), which is crucial for the secretion of type I interferons and proinflammatory cytokines in response to cytosolic nucleic acids, plays a key role in the innate immune system. Studies have revealed the participation of the STING pathway in unregulated inflammatory processes, traumatic brain injury (TBI), spinal cord injury (SCI), subarachnoid haemorrhage (SAH) and hypoxic–ischaemic encephalopathy (HIE). STING signalling is markedly increased in CNS injury, and STING agonists might facilitate the pathogenesis of CNS injury. However, the effects of STING-regulated signalling activation in CNS injury are not well understood. Aberrant activation of STING increases inflammatory events, type I interferon responses, and cell death. cGAS is the primary pathway that induces STING activation. Herein, we provide a comprehensive review of the latest findings related to STING signalling and the cGAS–STING pathway and highlight the control mechanisms and their functions in CNS injury. Furthermore, we summarize and explore the most recent advances toward obtaining an understanding of the involvement of STING signalling in programmed cell death  during CNS injury. We also review potential therapeutic agents that are capable of regulating the cGAS–STING signalling pathway, which facilitates our understanding of cGAS–STING signalling functions in CNS injury and the potential value of this signalling pathway as a treatment target. The central nervous system (CNS), which is composed of the brain and the spinal cord, is highly sensitive to external mechanical damage. Acute CNS injury, which mainly includes traumatic brain injury (TBI), spinal cord injury (SCI), subarachnoid haemorrhage (SAH) and hypoxic–ischaemic encephalopathy (HIE), is a leading cause of death and disability worldwide –4. AcuteAs stimuli, DNA disruption, mitochondrial damage, apoptosis, exosomes, DNA viruses, retroviruses and microbes facilitate the generation of pathogen-related molecular patterns (PAMPs) and danger-related molecular patterns (DAMPs). PAMPs or DAMPs can be identified via pattern-recognition receptors (PRRs), which serve as innate cellular sensors that induce a cellular stress response. In eukaryotes, hereditary DNA substances are limited to the nucleus and mitochondria, whereas cytosolic or extracellular DNA serving as PAMPs activates DNA sensors to induce innate immune responses . To offs−/− mice exposed to CCI experience less damage than their wild-type littermates [Increasing evidence shows that neuroinflammation facilitates nerve damage in response to TBI , 46. A ptermates . Such netermates . Inhibittermates , 51. Bectermates , 53. Sintermates . Remarkatermates . In additermates . In summHIE is characterized by the accumulation of dead cells, disrupted inherent DNA metabolism, and DNA leakage into the cytosol . GamdzykSAH, which is primarily induced by ruptured intracranial aneurysms, remains a remarkable clinical challenge with prominent incidence and death rates worldwide . RecentlSCI is a destructive CNS injury involving primary and secondary injury , 63. PriVarious types of CNS injury contribute to cell death and subsequent inflammation, and oxidative stress is an important contributor to the pathophysiology of a variety of CNS injuries, including SCI, TBI, SAH and HIE , 70. OxiNo study has investigated which nerve cells play a main role in the activation of cGAS–STING pathways. Many studies of CNS injury suggest that microglia are the principal innate immune cells in the brain and the first responders to pathological insults and produce high levels of type I IFN , 75. CurIn addition to cytokine induction, STING signalling plays a role in cell death pathways, such as autophagy, necroptosis, ferroptosis and pyroptosis. Because STING is essentially involved in innate immunity, the induction of cell death might represent an ultimatum to prevent injury. However, excessive cell death results in loss of bodily function. Therefore, exploring the relationship between STING and cell death will provide more insight for the treatment of CNS injuries.Autophagy, a fundamental cellular process in eukaryotes that involves the sequestration of cytosolic constituents within double membrane-bound autophagosomes that are subsequently fused with endolysosomal vesicles, which leads to the degradation and recycling of the sequestered substrates, plays an important role in both the activation and regulation of innate and adaptive immune responses . The litNecroptosis is a form of necrotic cell death that occurs downstream of receptor-interacting protein kinase (RIPK)1 and 3 activation and disruption of the plasma membrane by the pseudokinase mixed lineage kinase like-domain (MLKL) , 90. IFN2O2) or organic peroxide (ROOH) into water or their corresponding alcohols. GSH depletion deactivates GPX4 and thus elevates the lipid ROS levels. Recent evidence demonstrates that GPX4 is needed for activation of the cGAS–STING pathway. GPX4 deficiency enhances lipid peroxidation, which promotes STING carbonylation and inhibits its translocation from the ER to the Golgi complex [Ferroptosis is a newly discovered form of programmed cell death that results from the accumulation of iron-dependent lipid peroxide, and the term ‘ferroptosis’ was first used by Stockwell et al. in 2012 . Ferropt complex . Overall complex –99, the The term pyroptosis was first observed in macrophages that underwent unique caspase-1 programmed cell death following exposure to Salmonella . In brieLysosomes act as cell recycling centres and are filled with numerous hydrolytic enzymes that can degrade most cell macromolecules. The permeabilization of the lysosomal membrane and the subsequent leakage of the lysosomal content into the cytoplasm results in cell death. One study has revealed that STING signalling leads to the deceleration of lysosomal digestion by affecting the regulation of the lysosomal pH. Continuous STING pathway activation contributes to termination of the autophagic flux by perturbing lysosomal digestion . The autNo study has defined the interrelationship among cell death pathways acting downstream of STING signalling. Based on the aforementioned research, we assume that lysosomes may be an important link among autophagy, pyroptosis, ferroptosis and necroptosis acting downstream of STING signalling. Further studies are needed to determine whether this mechanism occurs in CNS injury.Sterile inflammation induced by the cGAS–STING pathway plays an important role in CNS injury. Thus, inhibitors of the cGAS–STING pathway may represent potential targets in the treatment of CNS injury. Previous studies have indicated that damage to cells may abnormally discharge DNA from the cytoplasm and that cGAS catalyses the production of 2′,3′-cGAMP by recognizing DNA from the cytoplasm. Subsequently, 2′,3′-cGAMP transmits signals to downstream STING, which induces transcription factors such as IRF3 and NF-κB to translocate into the nucleus and express inflammatory factors such as IFN. This process can activate both innate immunity and adaptive immunity . TherefoTTAGGG, abrogates the activation of cytosolic nucleic acid-sensing cGAS and the AIM inflammasome by binding to these molecules in a manner that competes with immunostimulatory DNA and that A1515 decreases the volume of cerebral infarction and the number of dead cells by inhibiting the cGAS–STING signalling pathway in middle cerebral artery occlusion (MCAO) [−/− rats and fibroblasts from Aicardi–Goutieres syndrome (AGS) patients. However, the efficacy of these agents in CNS injuries is currently unknown and awaits further investigation [cGAS catalyses GTP and ATP to generate 2′,3′-cGAMP after recognizing endogenous and exogenous DNA, which represents a major facet of the cGAS–STING pathway. Therefore, preventing the combination of cGAS and DNA and inhibiting the catalytic activity of cGAS constitute a feasible method for blocking this pathway. By establishing methods for monitoring cGAS activity in vitro, Vincent et al. screened a compound called RU.521, which specifically inhibits the activity of cGAS by binding the catalytic sites in cGAS to influence the synthesis of 2′,3′-cGAMP . In addin (MCAO) . Interesn (MCAO) . Unfortutigation . Liu et tigation . HoweverMicronuclei , mtDNA , abnormaBy screening compounds in vitro, researchers have identified a type of nitrofuran ramification called c-176. These researchers found that c-176 inhibits the ability of STING to combine with CDN by integrating with Cys-91 sites in rat STING, which blocks STING palmitoylation . This prAster tataricus, exerts STING-inhibiting activity by targeting the downstream transcription factor IRF-1 [Furthermore, some scholars have found that the invasion of viruses produces nitro-fatty acids (NO2-FAS), which combine with Cys88 and Cys91 residues in the N-terminal domain of STING through covalent bonds and thus prevent the palmitoylation of STING to inhibit its activation . Some scor IRF-1 . This coor IRF-1 . HoweverCytosolic DNA is linked to heightened inflammation, which is consistent with previous reports of increased levels of proinflammatory cytokines during ageing. Thus, the clearance of cytosolic DNA may afford therapeutic benefits. One of the pathways involved in the clearance of damaged or cytosolic DNA is autophagy, and defects in autophagy can potentiate the STING pathway and promote an inflammatory phenotype , 133. AcIFN-γ can stimulate JAK/STAT signalling pathways to enhance the inflammatory response. IFN-γ plays an important role in subsequent CNS injury. The suppression of downstream IFN-γ has also been explored as a therapeutic option. JAK1/2 inhibitors are neuroinflammation inhibitors, including ruxolitinib, which is widely used for various autoimmune/autoinflammatory conditions. Research has shown that ruxolitinib attenuates inflammatory responses by selectively inhibiting the JAK/STAT signalling pathway in IFN-γ-stimulated microglia, which leads to reductions in neuronal cell death and the inhibition of glial scar formation . HoweverCNS injury and its devastating consequences continue to challenge clinicians. Therefore, understanding the molecular basis of CNS injury may be beneficial for improving neuronal and glial survival and attenuating neurological deficits. Recent studies on STING signalling in the brain and spinal cord have increased our understanding of the role of this pathway in neural injury and neural innate immunity as well as in inflammation-mediated neurodegeneration.Studies on the temporal pattern of STING activation in CNS injury are crucial and are beneficial for obtaining an understanding of STING and selecting appropriate time points for STING regulation after CNS injury. However, the results from these studies are not understood. As observed in a previous study using a mouse model, STING expression was increased at 2 h and at 24 h after TBI, and its expression at 24 h was higher than that at 2 h . In the STING activation occurs in response to a wide array of stressors, ranging from viral infection to ER and mitochondrial stress, which suggests that it is a major player in a number of neuropathologies. Because both beneficial and detrimental effects of STING having been reported, the targeting of this pathway is complex. In this review, we detail the current knowledge of the STING pathway and its protective and detrimental activity in acute CNS pathologies. In addition, we outline several potential links that warrant further investigation of the potential causal link between cGAS–STING and programmed cell death in CNS injury. We also explore potential drugs for the inhibition of STING activity and thus the treatment of CNS injury.Based on these conclusions, we make some suggestions concerning future studies: (a) the correlation among the STING pathway and autophagy, necroptosis, ferroptosis, pyroptosis and lysosomal cell death in CNS injury should be comprehensively investigated; (b) it is essential to elucidate which type of programmed cell death plays the dominant role in CNS injury; (c) the regulatory mechanisms of the STING pathway in CNS injury, particularly methods to regulate the expression levels of programmed cell death-associated genes, should be explored; and (d) the relationship between miRNAs and the STING pathway is now well understood. Some research has found that miRNAs regulate the STING pathway , 142. FoAlthough the STING pathway plays important roles in different types of programmed cell death, we stress that the complexity of CNS injury cannot be reduced to a single pathophysiological mechanism or to the inhibition or activation of a single molecular pathway. Therefore, specific combination therapies targeting the cGAS–STING pathway may represent a promising strategy for the treatment of CNS injury. Furthermore, the translation of the current findings to the postinjury administration of drugs that block or activate the cGAS–STING pathway needs further effort. In conclusion, this review can help elucidate the functions of the STING pathway in the pathological process of CNS injury. Thoughtful consideration and more detailed findings concerning the roles of the STING pathway will contribute to improving our understanding of CNS injuries of unknown aetiology in the near future.


Vascular aging is an inevitable process with advancing age, which plays a crucial role in the pathogenesis of cardiovascular and microvascular diseases. Diabetic retinopathy (DR) and age-related macular degeneration (AMD), characterized by microvascular dysfunction, are the common causes of irreversible blindness worldwide, however there is still a lack of effective therapeutic strategies for rescuing the visual function. In order to develop novel treatments, it is essential to illuminate the pathological mechanisms underlying the vascular aging during DR and AMD progression. In this review, we have summarized the recent discoveries of the effects of oxidative stress and epigenetics on microvascular degeneration, which could provide potential therapeutic targets for DR and AMD. Vascular aging is an irreversible pathophysiological process involving the whole body of the elder. It is regarded as the common character in age-related cardiovascular and microvascular diseases, such as atherosclerosis, hypertension, cerebrovascular pathologies and retinal vascular diseases  is most prevalent with a large afflicted population  and a decrease in mtDNA transcription, compared with the vessels from age-matched non-DR donors. Hyperglycemia upregulated the expression of DNA methyltransferase DNMT1 and mtDNA methylation in human retinal endothelial cells, leading to mitochondrial damage as well as cell apoptosis  at SOD2 promotor. Moreover, the upregulation of SUV420h2, one of the prime enzymes for the trimethylation of histone H4K20, was detected in diabetic retinas. To explore whether histone methylation was correlated with antioxidant defects in the process of vascular aging in DR, SUV420h2 knockdown was performed in retinal endothelial cells followed by HG treatment. Deprivation of SUV420h2 normalized SOD expression through inhibiting histone methylation, which indicated the epigenetic regulation of endothelial antioxidative defense (Zhong and Kowluru To date, clinical pharmacological treatment for DR and AMD mainly focuses on anti-VEGF therapies, which targets at the inhibition of NV at advanced stage of the diseases. However, not all patients respond optimally, and there are potential adverse effects arisen from frequent intravitreal injections, such as the risk of endophthalmitis and retinal detachment (Gahlaut et al.


During seed development, the endoplasmic reticulum (ER) takes care of the synthesis and structural maturation of very high amounts of storage proteins in a relatively short time. The ER must thus adjust its extension and machinery to optimize this process. The major signaling mechanism to maintain ER homeostasis is the unfolded protein response (UPR). Both storage proteins that assemble into ER-connected protein bodies and those that are delivered to protein storage vacuoles stimulate the UPR, but its extent and features are specific for the different storage protein classes and even for individual members of each class. Furthermore, evidence exists for anticipatory UPR directly connected to the development of storage seed cells and for selective degradation of certain storage proteins soon after their synthesis, whose signaling details are however still largely unknown. All these events are discussed, also in the light of known features of mammalian UPR. In a relatively short time, seeds accumulate large amounts of storage proteins, destined to provide an early source of amino acids at the beginning of germination. In major crops, from around 7% (rice) up to more than 35% (soybean) of dry seed weight is constituted by proteins, mostly represented by storage proteins, encoded in each species by around a couple of dozens genes or less . All stovia a mechanism called regulated Ire1-dependent decay (RIDD). When BiP is released from bZIP28, this membrane-bound transcription factor traffics from the ER to the Golgi complex. Here, specific proteases release its cytosolic, active domain, which enters the nucleus and activates UPR-responsive genes , a network of signaling pathways with the main function of maintaining ER homeostasis . The firve genes . A thirdve genes , which hve genes .An increase in BiP expression is a landmark of UPR, first observed in rapidly growing cancer cells that undergo glucose depletion, hampering cotranslational protein glycosylation in the ER , and theThe following paragraph will therefore introduce the different seed storage protein classes.Cucurbita sp), in which the storage proteins form ER-localized accretions that are then directly delivered to storage vacuoles, but also in these cases the proteins remain soluble in aqueous buffers (Triticum aestivum), they can be delivered to protein storage vacuoles via autophagy-like processes  embryo maturation, decreasing at late stages of seed development, thus correlating with the synthesis of soluble vacuolar storage proteins (Oryza sativa) has five BiP genes; BIP1, the only one expressed to high levels in seeds, also increases its expression at the beginning of endosperm development, and BiP protein accumulation in this case persists until the end of development (Phaseolus vulgaris) cotyledons, newly synthesized, unassembled polypeptides of the 7S globulin phaseolin can be co-immunorecipitated with BiP, whereas assembled trimers still present in the ER do not interact with the chaperone  vegetative tissues, 16γz assembles into thread-like electron-dense structures that markedly enlarge the ER lumen, being unable to form homotypic PBs. 16γz is partially soluble also in the absence of reducing agents, but becomes fully insoluble when co-expressed with 27γz, supporting the hypothesis that 16γz establishes the contact between the outer layer and the inner core of the maize PB , the ER-resident HSP90 chaperone endoplasmin, and protein disulfide isomerase 1–2, all of which are folding helpers known to be induced by UPR (TUNICAMYCIN INDUCED PROTEIN 1 (TIN1) were also specifically induced by 16γz; TIN1 was previously identified as the second most enhanced transcript after BIP3 upon treatment with the N-glycosylation inhibitor tunicamycin, a typical UPR-inducing chemical , of several, albeit not all, folding factor genes, including OsBiP2, OsBiP3, and OsBiP5, and cell death genes expected to be under UPR control, thus indicating a major role of BiP1 in the synthesis of rice storage proteins and the expected role of UPR as a mechanism activated by low availability of BiP  and α-globulin, although its PB-forming prolamins are also relatively abundant . This haprolamin ; it alsoy of BiP .OsERdj7 expression during endosperm development induced UPR and specifically reduced the accumulation of 10 and 16 kDa oryzins as well as of α-globulin  are co-chaperones that participate in BiP activity, stimulating its ATP-dependent substrate interactions and in certain cases also directly binding unfolded BiP clients . Rice haglobulin . 13 kDa globulin , the picDuring B cell differentiation into plasma cells, the actual start of IgM synthesis that drives UPR is preceded by an “anticipating” ER expansion and folding helpers increase . A similOsHRD3, which is part of the ubiquitin–ligase complex involved in ERAD, caused impairment of general protein polyubiquitination, resulting in the induction of OsbZIP50 mRNA splicing and enhanced transcription of several ER folding helper genes, indicating UPR activation  induced the synthesis of cytosolic heat shock proteins as indicated by pulse-chase radioactive protein labeling, but decreased general protein synthesis, including those of BiP and endoplasmin  share a high degree of homology with those of common bean, but they are more extensively N-glycosylated and abundant. Tunicamycin treatment of common bean cotyledons does not affect traffic of the unglycosylated storage proteins to storage vacuoles (The phaseolin and lectin storage proteins of Lima bean (vacuoles , but simvacuoles . Also, svacuoles . AltogetZmbZIP60 in response to heat stress compared to lines used in temperate climates (Whether the protein folding machinery and UPR sensors can be manipulated with the aim of improving seed protein quality and quantity constitutes an important challenge, also in light of the use of seeds for the production of recombinant pharmaceuticals . As it hclimates . In bothAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.Supported by the projects CNR-DISBA Cambiamenti Climatici 7009-FOE 2019 and PORFESR 2014-2020 Reg. Lombardia sPATIALS3 1 176485.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ideal drug carriers should be stable in biological environments but eventually release their drug load once inside the targeted cells. These two aspects can be in contrast with each other, thus they need to be carefully tuned in order to achieve the desired properties for specific applications. Quantifying drug release profiles in biological environments or inside cells can be highly challenging, and standard methods to determine drug release kinetics in many cases cannot be applied to complex biological environments or cells. Within this context, the present work combined kinetic studies by flow cytometry with aging experiments in biological fluids and size-exclusion chromatography to determine drug release profiles in biological environments and inside cells. To this purpose, anionic and zwitterionic liposomes were used as model nanomedicines. By changing lipid composition, liposome stability in serum and intracellular release kinetics could be tuned and formulations with very different properties could be obtained. The methods presented can be used to characterize liposome release profiles in complex biological media, as well as inside cells. In this way, liposome composition can be tuned in order to achieve formulations with optimal balance between stability and release kinetics for specific applications. Liposomes are one of the most clinically established therapeutic delivery platforms in nanomedicine ,2,3. SinIt is generally believed that drug release from liposomes following endocytosis occurs either through the fusion between liposomes and the endosomal membrane ,11,12,13Despite the importance of determining such parameters, no standardized methods are available yet for this purpose ,24. DialWithin this context, in the present study, flow cytometry analysis, imaging by fluorescence microscopy, aging experiments in biological media and size-exclusion chromatography have been combined in order to determine the release behavior of liposomes upon exposure to complex biological fluids and inside cells. An anionic liposome (DOPG liposome) and a zwitterionic liposome (DOPC liposome) showing very different physicochemical properties and cellular uptake behavior were used as liposome models, and sulforhodamine B (SRB), a membrane impermeable fluorophore, was used to mimic hydrophilic drugs entrapped in the inner aqueous volume of liposomes and to quantify liposome release behavior . After eLipids including 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG) and cholesterol were purchased from Avanti Polar Lipids, Inc., Alabaster, AL, USA. DOPC liposomes composed of DOPC and cholesterol at a molar ratio of 2:1 and DOPG liposomes composed of DOPG and cholesterol at a 2:1 molar ratio were both prepared by thin-film hydration followed by freeze–thaw cycles and extrusion. Briefly, 10 mg lipid mixture was dissolved in chloroform, and the organic solvent was evaporated with dry nitrogen for 30 min, followed by further removal under vacuum overnight. Lipid films were rehydrated with 1 mL 25 mM sulforhodamine B (SRB) in PBS followed by 8 freeze–thaw cycles using liquid nitrogen and a 37 °C water bath and extrusion 21 times through a 0.1 µm polycarbonate membrane using an Avanti Mini-Extruder (Avanti Polar Lipids) in order to obtain small unilamellar liposomes. Zeba Spin Desalting Columns  were used to remove excess free SRB, and the obtained liposomes were stored at 4 °C and used up to 1 month after preparation.The concentration of DOPC or DOPG after extrusion and free dye removal was quantified via the Stewart assay, which allows the quantification of the concentration of phospholipids . For thiv/v) (Sigma Aldrich), and their fluorescence was measured using a ThermoMAX microplate reader , with excitation at 550 nm and emission at 600 nm (negligible fluorescence was detected with these settings for triton solutions at this concentration). Addition of 0.5% (v/v)) triton has been shown to completely disassemble liposomes in dispersions at 2 mg/mL lipid , ta, ta24], iposomes A, the ef3 strongly decrease uptake . The much lower uptake efficiency of free SRB and the strong effect of sodium azide on the uptake of aged liposomes, together, confirmed that, despite the leakage upon interaction with serum, the fluorescence detected in cells exposed to aged liposomes came primarily from the active uptake of the residual SRB loaded inside the liposomes rather than the uptake of the leaked SRB.Overall, even though the fluorescence decrease observed in cells exposed to aged DOPG dispersions was rather strong A, the upFinally, given that the data of Optimal liposomal formulations should retain their drug load in blood and as they distribute in the body and then release it once at their target. Very stable formulations could ensure no drug is lost during delivery but may show limited release at the target. Thus, liposome stability and release properties can be contrasting and need to be tuned in order to optimize formulations with the required delivery and release properties. Classic in vitro release studies with methods such as dialysis or centrifugation may be hard to apply to complex biological environments, and methods to determine liposome stability in complex biological media and intracellular release kinetics are highly sought. Herein, we showed that the aging of liposome dispersions and size-exclusion chromatography, combined with uptake kinetics and pulse and chase experiments by flow cytometry, can be used for this purpose. The methods presented allow addressing at least in part some of the limits of simpler release studies, in order to characterize liposome stability in complex biological fluids, as well as to determine release kinetics inside cells. Similar approaches can also be used for nanomedicine formulations other than liposomes.Thus, the stability and release properties of DOPG and DOPC liposomes encapsulating comparable amounts of SRB were compared. The DOPG liposomes were able to deliver very high amounts of SRB in short time inside cells; however, the high uptake efficiency and fast release properties inside cells were also accompanied by a substantial loss of the SRB load outside cells upon interaction with serum and corona formation. On the contrary, DOPC uptake efficiency was much lower and the release more gradual and sustained over time, and this formulation also showed higher stability in biological conditions when exposed to serum. Depending on the application and drug load, liposome formulations and other nanomedicines can be tuned to achieve the required balance between stability in serum and drug release at the target.


Almost half of under-five deaths occur during the neonatal period. Delivery with a skilled attendant, adherence to essential newborn care (ENC) and postnatal care (PNC) standards, and immediate treatment of infections are essential to improve neonatal survival. This article uses Demographic and Health Survey data from 45 low- and middle-income countries to assess 1) levels of ENC and PNC that mothers and newborns receive and how this differs by place of delivery and 2) levels of and sources for care-seeking for neonates sick with fever. For five of the ten ENC and PNC indicators assessed, less than two-thirds of mothers and newborns received care in alignment with global standards. Adherence is higher in private facilities than public facilities for all indicators other than immediate breastfeeding and skin-to-skin contact. Except for immediate breastfeeding, adherence is lowest for newborns born at home with a skilled birth attendant (SBA). Socioeconomic disparities exist in access to skilled delivery and adherence to ENC and PNC, with the largest disparities among newborns delivered at home with a SBA. Private provider adherence to ENC and PNC standards was relatively high for newborns from the wealthiest families, indicating that meeting recommended guidelines is achievable. On average across the 45 countries, half of caregivers for neonates with fever sought care outside the home and 45 percent of those sought care from the private sector. There were substantial socioeconomic disparities in care-seeking for fever, but illness prevalence and sources of care seeking were consistent across wealth quintiles. Closing inequities in neonatal care and care seeking and ensuring that all families, including the poorest, can access high quality maternal and newborn care is crucial to ensure equity and accelerate reductions in neonatal and child mortality. Despite considerable improvements in child survival over the past 20 years, 2.6 million neonates still die every year in their first month of life . Almost Neonatal mortality can be reduced by providing skilled care at birth, quality essential newborn care (ENC), postnatal care (PNC), and immediate treatment for small and sick newborns –5. LevelAlso critical for neonatal survival is early detection of and care seeking for illnesses . In partThe public and private sectors are both important sources for reproductive, maternal, and child health services –20, yet https://dhsprogram.com/Methodology/.We analyzed data from recent Demographic and Health Surveys (DHS)  conducteOur analysis answers the following research questions:What percentage of mothers and newborns receive essential newborn and postnatal care in accordance with global guidelines, and how does the quality of care they receive vary by sector? Further, within the same delivery source category , are there disparities in care by socioeconomic status and/or maternal age?When neonates are sick with fever, what percentage of their caregivers seek advice or treatment outside the home and from where? How do care-seeking patterns for sick newborns compare to those for older infants and children, and are there disparities by socioeconomic status?To identify disparities in access to and quality of care, we examined all indicators by socioeconomic status using DHS’s asset-based wealth quintiles and compare levels of care sought and received by caregivers and newborns from the poorest 20 percent of the population to the wealthiest 20 percent of the population in each country. In addition, to further investigate equity, we examined all indicators related to the first research question by three categories of maternal age: 15–19, 20–24, and 25–49, to test the hypothesis that adolescent and younger mothers may receive poorer quality care. In addition, we disaggregated all ENC and PNC indicators by babies’ birthweight (<2.5 grams) and by survival status at the time of survey. However, we did not find substantial differences in birthweight or by survival status within the same source category, so we have not included these findings in the results.To examine how care-seeking patterns may change as children get older, we compared results from neonates in their first month of life in the second research question to several older age categories: young infants 30–59 days, infants 2–11 months, and children 12–59 months. DHS surveys have always measured child age in months, and in recent surveys began measuring child age in days. To standardize across surveys with and without detailed age data, we have defined the first month of life to equal 30 days.To produce the regional and all-country averages, we pooled data across the 45 DHS surveys and multiplied survey weights by a country-specific constant so that each country contributes equally to regional and all-country estimates. We conducted logistic regressions with robust standard errors to determine if differences in results across sectors, maternal age groups, and socioeconomic status categories are statistically significant, and we only comment on differences when they are significant at the p<0.05 level.To answer our first research question, we draw on data reported by women about their most recent births and care they and their infant received (or did not receive). We exclude births that occurred more than 2 years before the survey to limit potential recall bias.6,12,13, the DHS, beginning with the DHS7 round implemented in approximately 2016, added to the questionnaire standardized indicators on whether neonates received immediate skin-to-skin contact, a postnatal care check before facility discharge, and four components of a postnatal care check within two days after delivery. These indicators are described further in As noted in 14We measure adherence to ENC standards among all births that are presumably attended by a health care worker: those delivered in public facilities, private facilities, or at home with a skilled attendant. We exclude home births without a skilled attendant, as there is already substantial evidence that these births result in poorer maternal and neonatal outcomes.To facilitate comparisons across sectors, we also limit some indicators to vaginal births, as we found that births in private facilities are more likely to be caesarian (C-section) births than those in public facilities (25% versus 11%) and are associated with longer health facility stays and lower levels of immediate breastfeeding and skin-to-skin contact. Please see We standardized delivery sources into four categories: 1) Public sector; 2) Private sector; 3) Home births attended by a skilled birth attendant (SBA); and 4) Home births unattended or attended by someone unskilled such as a traditional birth attendant, family member, or friend. Health facilities are disaggregated into public and private sector. We further disaggregate public facilities into hospitals and health centers  and private facilities into for-profit and non-for-profit facilities .To examine our second research question, we analyze data from mothers who gave birth in the month before the survey and therefore had a child less than 1 month old at the time of the survey. We examine the prevalence of fever among neonates in the two weeks preceding the survey, as fever is a symptom used to classify PSBI in neonates. Among neonates sick with fever, we examine the prevalence of out-of-home care seeking; and among neonates for whom treatment was sought, the source of that treatment. We compare the two-week fever prevalence, care seeking, and source patterns among neonates to those of older children.Sources for sick neonatal care varied by country. We standardized these into three categories: 1) Public sector including public facilities  and community health workers; 2) Private sector including private facilities  and private retail outlets ; and 3) Other including traditional healers, friends, and family.We conducted a secondary analysis of DHS data, which is publicly-available, de-identified data that been reviewed by the ICF Institutional Review Board and typically by a review board in the host country, as well. Therefore, further ethical review was not necessary. The informed consent statement for each country survey is in each country’s respective final report. Additional details on the DHS’s ethical review procedures can be found here.To answer the first research question, we analyzed DHS survey data from 289,729 mothers who gave birth in the two years preceding the survey. To answer the second research question, we analyzed DHS survey data from 8,231 mothers who gave birth in the month before the survey and therefore had a neonate less than one month old. In pooled analysis across the 45 countries analyzed, 57 percent of women delivered in a public facility , 12 percent in a private facility (10 percent in a for-profit facility and 2 percent in a non-for-profit facility), 4 percent at home with a SBA, and 26 percent at home without a SBA.Delivery sources varied substantially by country . In NigeWe examine breastfeeding within the first hour of life only among neonates born vaginally, as immediate breastfeeding rates were substantially lower among neonates delivered by C-section  than if they were born at home with a SBA or in a private facility . Differences by source were largest in the Asian countries analyzed: 76 percent of newborns in public facilities were immediately breastfed compared to 64 percent of newborns in private facilities. In both sub-Saharan African regions, the lowest levels of breastfeeding within the first hour were seen among home births with SBAs.Our analysis of immediate skin-to-skin contact is limited to neonates born vaginally, as skin-to-skin contact levels were substantially lower among women who had a C-section  than those born in a public (62 percent) or private facility (59 percent). This pattern was fairly consistent across regions but was particularly pronounced in West and Central African countries where 20 percent of children born at home with a SBA received skin-to-skin contact compared with approximately half of those born in a facility. There was one deviation to this pattern by region: in Asian countries, the level of skin-to-skin contact was lower among neonates born in the private sector (59 percent) and at home (53 percent) compared to those born in a public facility (75 percent).This indicator also excludes mothers and neonates delivered by C-section, as C-sections were associated with a longer health facility stay . In contrast, 90 percent or more of mothers and newborns remained in the facility for 24+ hours in Malawi, Namibia, Myanmar, the Maldives, and the Philippines.In the all-country pooled estimate, newborns delivered in private facilities were more likely than those delivered in public facilities to stay in the facility for 24+ hours (63 percent versus 60 percent). There are also sectoral differences in the percentage of mothers and newborns who stay in the facility for 24+ hours by region. In the East and Southern Africa region, the private sector had a higher level of adherence to the 24+ hour stay guideline than the public sector (68 versus 63 percent). In Asian countries analyzed, the reverse was true: 66 percent of mothers who delivered in the public sector stayed for 24+ hours compared to 61 percent of those in the private sector.On average across all countries, 24 percent of newborns received a PNC check within the first hour of life and 66 percent received a PNC check within the first day (24 hours) of life. These indicators were consistent across the three regions examined.By source, neonates born at home with a SBA were far less likely to receive a PNC check within their first 1 or 24 hours. Ten percent of mothers who gave birth at home with a SBA reported a PNC check for their newborn within one hour of delivery. In comparison, 24 and 29 percent of mothers who delivered in the public and private sectors, respectively, reported a PNC check within one hour of delivery.Within 24 hours of birth, 32 percent of newborns delivered at home with a SBA received a PNC check. The level was substantially higher in the public (67 percent) and private sectors (76 percent). Among neonates born in a private facility, a PNC check within 24 hours was more common in Asian countries analyzed (80 percent) and least common in East and Southern African countries analyzed (69 percent). Among neonates born at home with a SBA, a PNC check within 24 hours was also much more common in the Asia region (38 percent) compared with both sub-Saharan Africa regions (24 percent). This indicator did not vary regionally for neonates born in the public sector.On average across 21 countries in our analysis with available data, 65 percent of neonates who were delivered in a public or private facility received a pre-discharge postnatal check. This indicator was highest in Asian countries analyzed (74 percent), lower in West and Central African countries (67 percent), and lowest in East and Southern African countries (58 percent).Neonates born in a private facility were more likely to receive a pre-discharge postnatal check than those born in a public facility (72 versus 64 percent). This disparity was most pronounced in West and Central Africa (65 percent private versus 56 percent public) and less so in East and Southern Africa (72 percent private and 66 percent public) and Asia (76 percent private and 72 percent public). Ethiopia had the lowest level of pre-discharge PNC checks across sectors– 40 percent in the private sector and 35 percent in the public sector. South Africa was an outlier compared to its regional average, with a high level of pre-discharge postnatal checks in private (99 percent) and public (95 percent) facilities.This indicator examines if the neonate and mother received the following four components of postnatal care within two days after delivery: 1) neonate’s cord examined, 2) neonate’s temperature taken, 3) mother was counseled on breastfeeding, and 4) breastfeeding was observed. As above, data are only available for 21 of the countries included in this analysis. On average across these 21 countries, more than half of mothers and neonates received each check , but fewer received all 4 PNC content components (38 percent). As with other ENC indicators, the proportion of neonates who received all 4 PNC components was lowest among analyzed countries in West and Central Africa (26 percent), higher among countries in East and Southern Africa (39 percent), and highest in Asia (45 percent).Neonates born in a private facility were slightly more likely to receive all four PNC content components than those born in public facilities (38 versus 41 percent). Those born at home with a SBA were at a much greater disadvantage: just over one in four (28 percent) received all four components. This pattern concealed regional differences. In East and Southern African countries, the public and private sectors performed equally (40 percent). However, in Asia, neonates born in the public sector were slightly more likely to receive all four PNC content components (47 versus 43 percent). The converse is true in West and Central Africa, where one third of neonates born in the private sector compared to one-fourth of neonates born in the public sector received all four PNC content components. Neonates born at home with a SBA were much less likely to receive all four PNC components in the African countries analyzed (20 percent) than in the Asian countries analyzed (37 percent).When examining each PNC content component individually, neonates born at home with a SBA were at a disadvantage compared to those born in the public or private sectors. The public and private sectors performed similarly for breastfeeding observation, but private facility health workers were more likely than public facility health workers to examine the neonate’s cord (68 versus 56 percent), take the neonate’s temperature (63 versus 54 percent), and counsel the mother on breastfeeding (61 versus 58 percent).Across the ENC and PNC indicators, there were differences in adherence to global quality standards within the public sector by type of public facility: hospitals versus health centers, clinics, or posts. Neonates born in public hospitals were more likely than neonates born in public health centers to receive skin-to-skin contact (70 versus 57 percent), 4 PNC content components (48 versus 31 percent), a PNC visit before facility discharge (84 versus 70 percent), and to stay in the facility for >24 hours (68 versus 52 percent). These disparities were largely consistent across regions with one exception: in Asian countries analyzed, neonates born in a public health center were more likely than those born in a hospital to receive skin-to-skin contact (79 versus 73 percent). Interestingly, the pattern was also flipped in all regions for immediate breastfeeding: neonates born in a public health center were more likely to be immediately breastfed than those born in a public hospital (74 versus 68 percent).Within the private sector, we compared ENC and PNC indicators between private non-for-profit versus private for-profit facilities. Few neonates (2 percent) were born in private not-for-profit facilities overall. On average across countries, neonates born in a not-for-profit private facility were more likely than those born in a for-profit facility to receive immediate skin-to-skin contact (68 versus 57 percent) or immediate breastfeeding (74 versus 63 percent). In the East and Southern African countries analyzed, neonates born in not-for-profit facilities were more likely to receive all 4 PNC content components (49 percent) than those in for-profit facilities (34 percent) but this difference was not apparent in the other regions. We saw the reverse pattern in the Asian region for the percentage who received a PNC check before facility discharge (91 percent for newborns in for-profit versus 75 percent for newborns delivered in not-for-profit facilities).Place of delivery varied substantially by wealth quintile. The wealthiest mothers were more likely than the poorest to deliver in a health facility (92 versus 51 percent). The wealthiest mothers were more likely than the poorest to deliver in a public facility (65 percent versus 46 percent) or a private facility (27 percent versus 5 percent). In contrast, the poorest were more likely to deliver at home without a SBA (44 versus 6 percent). Delivery at home with a SBA was low for both the poorest (5 percent) and wealthiest (3 percent).When examining the ENC and PNC indicators by wealth quintile, Among neonates born at home with a SBA, there were disparities of 10 or more percentage points between newborns from the wealthiest and poorest households for every indicator except for immediate breastfeeding. For example, receipt of all 4 PNC content components was more than twice as common among wealthiest than poorest families (47 versus 22 percent), a disparity of 25 percentage points.Among neonates born in the public sector, there was a socioeconomic disparity in the percentage who received immediate skin-to-skin contact  and who stayed in the facility for >24 hours after birth . Note that the latter disparity in length of facility stay favored neonates from the poorest households rather than those from the wealthiest households. This latter disparity in length of facility stay was not seen for neonates born in the private sector.Finally, in the private sector there were socioeconomic disparities in receipt of all four PNC content components , receipt of immediate skin-to-skin contact , and receipt of a PNC check before facility discharge . Except for skin-to-skin contact, these disparities were not seen for neonates born in the public sector. Note that there were socioeconomic disparities in receipt of skin-to-skin contact for neonates across all three places of birth—home, public facilities, and private facilities.When we examined delivery, PNC, and ENC indicators by maternal age categories , there were fewer disparities than for socioeconomic groups. Levels of delivery at home and in the public and private sectors were very similar for mothers of all age groups . The priFor neonates born in the public sector, the largest disparity by maternal age is a five percentage point difference in levels of immediate breastfeeding between mothers age 15–19 and 25+ (68 versus 73 percent). Among newborns delivered at home with a SBA, there are disparities of 5 or 6 percentage points for three indicators: 1) immediate skin-to-skin contact, 2) temperature taken, and 3) all four PNC content components completed.Most of these disparities are much smaller than those seen between newborns from the poorest and wealthiest households who were delivered at home with a SBA.Seeking treatment for sick neonates is key to enhancing neonatal health and survival. We investigate the care-seeking levels and sources for care of newborns sick with fever as a tracer indicator for PSBI in neonates. We begin by examining the prevalence of fever in newborns compared to older infants and children.On average across the 45 countries analyzed, 4 percent of neonates experienced fever in the 2 weeks prior to the survey . The twoThe prevalence of fever increased once children pass neonatal age, as shown in Among caregivers of neonates sick with fever, just over half (51 percent) sought advice or treatment outside of the home. Caregivers in Asian countries analyzed were more likely to seek care for their sick neonates (66 percent) than those in West and Central African countries (47 percent) or those in East and Southern African countries analyzed (46 percent).Care seeking for fever was higher for post-neonatal infants than for neonates. As shown in Among caregivers who sought advice or treatment outside the home for their neonates with fever, 54 percent used public facilities, 45 percent used private sources , and 1 percent went to other sources such as family or friends. Less than one percent of caregivers reported using community health workers for sick neonatal care.These source patterns differed by region, shown in Though the two-week fever prevalence changed by age, Among neonates and on average across countries, there was not a socioeconomic disparity in prevalence of fever . In contWhen comparing care-seeking sources for neonates from the poorest and wealthiest households, the biggest difference was in use of public sources. Caregivers from the wealthiest households were more likely than those from the poorest households to use public sources for sick neonates (69 versus 27 percent). Sixty six percent of caregivers from the poorest households and 31 percent from the wealthiest went to private sector sources for their sick neonates. While use of private sector sources is higher among the poorest than wealthiest families, this difference is not statistically significant, and these results should be interpreted with caution as the sample size is less than 50 observations.Delivery with a skilled attendant, adherence to ENC and PNC standards, and immediate quality treatment of sick neonates are all essential components of high-quality neonatal care and are critical to improve maternal and child survival. This study highlights some successes in these matters and many areas for improvement in the delivery of quality of neonatal care. This study provides novel insights into how ENC, PNC, and fever care seeking for neonates vary across sectors.In pooled analysis across the 45 countries analyzed, more than 1 in 4 deliveries occurred at home without a skilled attendant, making it difficult to obtain many ENC and PNC components as well as basic life-saving and emergency care. Among newborns delivered by a healthcare provider, we can glean information regarding quality from levels of adherence to ENC and PNC standards. Adherence to ENC and PNC standards was universally higher for births in health facilities compared to home births with a SBA with the notable exception of immediate breastfeeding. Among neonates born in public facilities, those born in a hospital were generally more likely to receive ENC and PNC than neonates born in a public health clinic, center, or post. There are positive signs in relatively high levels of adherence to immediate breastfeeding and pre-discharge postnatal care visits for facility-born neonates. However, levels of adherence to recommended PNC content were universally low, on average, as was the level of PNC checks within one hour of delivery. The public sector had significantly higher levels for immediate breastfeeding and skin-to-skin contact, while the private sector has higher levels of adherence to all other ENC and PNC indicators, with the exception of observing breastfeeding during the PNC check, which was similar in both sectors. This finding differs from studies in Ghana and Eastern Uganda that found that public sector providers performed better than private providers in provision of essential newborn care ,22.While the proportion of mothers who delivered in a health facility is lower than ideal (69 percent), it is higher than the proportion who seek care outside of their home when their neonates experienced fever (51 percent). When mothers or caregivers do leave their homes for delivery or care seeking, the private sector played a larger role for sick neonatal care (45 percent) than for delivery (18 percent).Across nearly all indicators examined—comprehensive of delivery, ENC, PNC, and sick neonatal care—there are substantial socioeconomic disparities. Neonates from the poorest households were less likely to be born in a facility and were far less likely to receive care outside the home if they experienced fever. Among neonates born at home with a skilled attendant, those from the poorest households were less likely than those from the wealthiest to receive essential newborn and postnatal care, which aligns with findings from a 2021 study on co-coverage and equity of newborn care . SocioecConversely, there were far fewer inequities by maternal age, largely indicating that adolescent and other young mothers do not receive less or poorer-quality care for their newborns as compared to older mothers. This aligns with the findings from a study of 16 low- and middle-income countries found that maternal age was a significant predictor of newborn care only in Senegal and was non-significant in all other countries . HoweverIt is challenging to ascertain quality solely from care-seeking data, as the DHS does not collect information on the provider-caregiver interaction. In addition, fever is a non-specific proxy for PSBI and could be representative of other causes of post-neonatal morbidity and mortality. Further investigation, particularly through the analysis of routine facility data and health facility assessments, is warranted to better understand the quality of sick young infant care. Finally, the quality of the findings presented is dependent on the quality of DHS data. The DHS program has rigorous quality assurance standards and aims to collect standardized data across countries through standard questionnaires and data collection training techniques. However, there may be differences in how the survey is administered across countries, which could result in modest differences in question interpretation and results.15 closing inequities in neonatal care and care seeking and ensuring that all families, including the poorest, can access high quality maternal and newborn care is crucial to continue positive trends and further accelerate reductions in neonatal and child mortality.Adherence to essential newborn and postnatal care is low across the public and private sectors and especially among home births with skilled attendants, demonstrating great need for improvements in quality of neonatal care. Adherence to global ENC and PNC standards is consistently higher in health facilities compared to home births with SBAs. In addition, adherence to these standards is higher in private facilities than public facilities, with the exception of immediate breastfeeding and skin-to-skin contact, which is more common among mothers and newborns in public facilities. Socioeconomic disparities persist in access to skilled delivery, adherence to ENC and PNC—particularly among newborns delivered at home with a skilled attendant, and in neonatal care seeking for fever. While there have been positive trends in reducing socioeconomic disparities in neonatal mortality,S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Zoonotic poxvirus infections pose significant threat to human health as we have witnessed recent spread of monkeypox. Therefore, insights into molecular mechanism behind poxvirus replication cycle are needed for the development of efficient antiviral strategies. Virion assembly is one of the key steps that determine the fate of replicating poxviruses. However, in-depth understanding of poxvirus assembly is challenging due to the complex nature of multi-step morphogenesis and heterogeneous virion structures. Despite these challenges, decades of research have revealed virion morphologies at various maturation stages, critical protein components and interactions with host cell compartments. Transmission electron microscopy has been employed as an indispensable tool for the examination of virion morphology, and more recently for the structure determination of protein complexes. In this review, we describe some of the major findings in poxvirus morphogenesis and the contributions of continuously advancing electron microscopy techniques. Poxviridae, that includes variola virus which is a causative agent for infamous smallpox disease that claimed millions of lives throughout the history. Despite complete eradication of smallpox in 1980, zoonotic infections by other poxvirus species and possible bio-terrorism have been continuous threats to human health  in the early 1930s, viruses have been the subjects of extensive investigation because the nanoscopic microbial agents other than bacteria were beyond the achievable resolution range of light microscopes, which were the only instruments available for imaging microbial entities at the time. Some of the earliest observations of viruses by TEM were pioneered by Helmut Ruska, the brother of Ernst Ruska who invented TEM, and date back to late 1930s , a group of proteins including L2, A30.5, A11, A6 and H7, demonstrated that one or more VMAPs interact for ER membrane hijacking and the separation of viral membrane from its origin (Suarez et al. Detailed molecular mechanism behind virus replication is often aided by determination of protein structures. To date, 111 VACV protein structures are available in Protein Data Bank. Determined structures include proteins that are involved in viral entrance and release, DNA replication, host immune modulation and other replicative machineries. The structures were mostly determined by x-ray crystallography whereas recent technical breakthroughs in cryo-EM single particle analysis facilitated structural insights into large complexes such as virus-encoded multi-subunit RNA polymerase complex (Grimm et al. While the structures of proteins involved in poxvirus morphogenesis are largely unknown, the structure of scaffold protein D13 has been relatively well-characterized. First two crystal structures of D13 trimer have shown double jellyroll secondary structure motif and addressed common viral lineage with NCLDVs which share similarly folded major capsid proteins (Bahar et al. In this review we have described the advancements in the understating of poxvirus morphogenesis aided by technical breakthroughs in TEM. More comprehensive reviews are available on VACV in general (Condit et al.


Breast cancer is clinically and biologically heterogeneous and is classified into different subtypes according to the molecular landscape of the tumor. Triple-negative breast cancer is a subtype associated with higher tumor aggressiveness, poor prognosis, and poor response to treatment. In metastatic breast cancer, approximately 6% to 10% of new breast cancer cases are initially staged IV (de novo metastatic disease). The number of metastatic recurrences is estimated to be 20–30% of all existing breast tumor cases, whereby the need to develop specific genetic markers to improve the prognosis of patients suffering from these deadly forms of breast cancer. As an alternative, liquid biopsy methods can minutely identify the molecular architecture of breast cancer, including aggressive forms, which provides new perspectives for more precise diagnosis and more effective therapeutics. This review aimed to summarize the current clinical evidence for the application of circulating tumor DNA in managing breast cancer by detailing the increased usefulness of this biomarker as a diagnostic, prognostic, monitoring, and surveillance marker for breast cancer. Breast cancer (BC) is a disease characterized by various clinical behaviors and biological characteristics, making the prediction and management process more challenging . SpecifiHER2 oncogene and is treated with trastuzumab, (3) Triple-negative breast cancer (TNBC)  subtype, which is associated with high mortality rates and is not responsive to some drug treatment approaches [Breast cancer (BC) is heterogenous, showing variable morphologic and biological features; thus, it has different clinical behaviors and responses to treatment . Based oproaches ,5. Nowadproaches ,7.In metastatic breast cancer, metastases are heterogeneous with various somatic mutations and molecular alterations. Consequently, it is essential and necessary to identify new biomarkers to guide treatment and improve clinical cancer management .Intratumoral heterogeneity and tumor evolution contribute to treatment failure in cancer patients, which is explained by cellular mutability, cancer cell survival, and drug selection pressures, including adaptive mutability. Thanks to important collaborative efforts of several research entities such as the Cancer Genome Atlas (TCGA), the Human Tumor Atlas Network, the Pan-Cancer Analysis of Whole Genomes Consortium, and the International Cancer Genome Consortium, this intratumoral and intertumoral heterogeneity has been elucidated through single-cell analyses of primary tumors and corresponding metastases, genomic studies of treated and untreated metastatic disease, and investigations of the tumor microenvironment .Therefore, the ideal approach to address the heterogeneity of breast tumors would be to find a method that can capture the entire genetic map of the tumor. Collecting serial biopsies of the tumors and longitudinal monitoring is essential to catch the phylogenetic progress of the tumor. However, it is not feasible to conduct such procedures through tissue biopsy, as they are highly invasive and may be incomprehensible.Progress and scientific advances in liquid biopsy have raised the hope of detecting temporal and spatial heterogeneity before and after systemic therapy, or when multiple potentially discordant metastases are present .In this review, we will focus on the study of circulating DNA as a liquid biopsy biomarker, highlighting the latest pre-analytical considerations and analytical techniques for the analysis of this biomarker. This review will also focus on the study of the clinical utility and validity of circulating tumor DNA, outlining recent advances and current challenges, particularly in aggressive breast cancer.The presence of cell-free DNA (cfDNA) in healthy people’s blood was first reported by Mandel and Metais in 1948 , but it Generally, cfDNA consists of short DNA fragments of 160–180 bp, which are the size of a single nucleosome . Based oAlthough the source of cfDNA is not completely elucidated, various studies report the origin and possible mechanisms of the release of cfDNA ,27. CellThe application and potential use of cfDNA as a biomarker achieved the best success in different clinical phases, thus making it a popular and potential target in a wide range of research areas, principally prenatal diagnosis ,36, orgaIn cancer, every tumor cell potentially releases circulating tumor DNA (ctDNA) . The groIn addition to its non-invasiveness, rapidity, and low cost, ctDNA allows for longitudinal monitoring of cancer in real time and can potentially capture tumor heterogeneity ,47. IndeIn this review, we outlined recent advances and current challenges in the study of circulating tumor DNA, particularly in breast cancer.Compared to liquid biopsy, conventional tissue biopsy presents some drawbacks: in addition to its invasiveness , it can Cell-free DNA (cfDNA) analysis, also known as liquid biopsy, created new options for non-invasive diagnosis and therapeutic monitoring, and these liquid biopsies may mirror clinically relevant genetic alterations, which occur in all tumor tissues ,52,53.Circulating tumor DNA (ctDNA) represents a substantial fraction of circulating DNA, which varies from <0.05% to 90% depending on tumor location, size, and vascularity, as well as liver and kidney clearance ,54,55. CStudying circulating tumor DNA in breast cancer by determining the heterogeneity of clinically relevant alterations has created new possibilities for non-invasive diagnosis and therapeutic monitoring, ctDNA analysis could potentially reflect the genetic alterations that occur in all breast cancer tissues ,53.The application and potential use of ctDNA as a biomarker have achieved the best results in different clinical phases, making it a popular and potential target . TherefoPre-analytical considerations are those parameters that influence the quality of cfDNA and are most likely to compromise the success of the analysis. Analytical validity refers to the ability of a test to identify variants of interest accurately and reliably; the test or analysis must be sensitive, specific, and robust. Clinical validity is explained by the ability of the test to detect the presence or absence of a disease state accurately and completely. Regarding clinical utility , it is reached when there are high levels of evidence confirming that using the test improves patient outcomes compared to not using it .The pre-analytical steps are crucial in the analysis of circulating DNA because it is a very sensitive analysis, since tumor-derived DNA is a small fraction of cfDNA, with possible contamination by leukocyte genomic DNA in the case of leukocyte degradation, which makes a downstream analysis of circulating tumor DNA mutations more difficult and the quantification of this biomarker less accurate.Plasma is the optimal specimen type for cfDNA analysis, studies showed that the amount of normal DNA derived from leukocyte lysis, which dilutes cfDNA, is much higher in serum than in plasma, this crucial step is excellent to ensure if the plasma is separated from the leukocyte fraction immediately after blood collection or the blood is collected in collection tubes containing a leukocyte stabilizer. The freezing of unfiltered whole blood should not be performed and plasma should be isolated before it is frozen. Although exposure of plasma to a single freeze-thaw cycle does not affect downstream cfDNA analysis, multiple freeze-thaw cycles may lead to nucleic acid degradation. Studies agree that storage of frozen plasma before DNA extraction does not affect further cfDNA analysis.For the analytical phase, currently, ddPCR is the most widely used method for cfDNA analysis, this technique allows precise detection of known DNA mutations with a better detection limit as well as a more accurate quantification of the ctDNA fraction, the major challenge is the high cost and a low number of installed ddPCR instruments, given that ddPCR is currently capable of analyzing only one potential mutation per reaction ,69.However, the limitation of PCR approaches is that these techniques only detect known mutations in certain genes, so patients without these mutations will be overlooked, limiting the application of this technology as a generic diagnostic technique for ctDNA analysis .On the other hand, NGS approaches cover a wider range of mutations by examining the entire gene sequences of interest. However, targeted approaches for ctDNA profiling generally sequence tens of genes to hundreds of genes or even the entire exome, a high sensitivity can then be achieved by deep sequencing of specific regions of interest that cover clinically relevant mutations. Several technologies are struggling to innovate more suitable platforms for ctDNA sequencing, notably sequencing by synthesis (SBS) from Illumina, Ion Torrent from Thermo Fisher Scientific, and nanopore sequencing from Oxford Nanopore Technologies; however, Illumina’s sequencing platform currently dominates due to its high throughput and accuracy ,71.While cfDNA NGS sequencing offers a non-invasive approach to the identification of clinically relevant somatic genomic alterations, it is probably not intended to replace the need for tumor biopsies as the gold standard for diagnosis and genotyping, but it can be used as a complementary or alternative analysis when a tissue biopsy is not possible. In addition, ctDNA analysis by NGS is limited by a relative scarcity of total cfDNA and a low fraction of ctDNA (tumor-derived cfDNA fraction), and therefore the development of methods that increase the sensitivity and specificity of cfDNA sequencing is necessary for overcoming these limitations .Several genes present a potential target for studying breast cancer, in this review, we will provide accurate and relevant information on the clinical utility of circulating DNA, we have not targeted all breast cancer genes and signaling pathways, instead we have specifically targeted the most studied genes to evaluate the clinical utility of this ctDNA.The genes studied have been listed in BRCA mutations is characterized by its aggressiveness, BRCA1 mutated breast cancer is frequently high grade and triple negative, BRCA2 related breast cancer is on average of a higher histological grade than sporadic cases [BRCA1/2 occur in approximately 3% of all sporadic breast cancers [Breast cancer with ic cases . Somatic cancers .BRCA1 gene encodes a nuclear phosphoprotein that acts as a tumor suppressor gene by maintaining genomic stability [BRCA1 genome-associated surveillance complex [The tability . The enc complex .BRCA2 gene is involved in the maintenance of genomic stability and more specifically the homologous recombination (HR) pathway that repairs double-stranded DNA breaks. BRCA2 is located on chromosome 13q12.13, it is a large gene comprising 27 exons coding for 3418 amino acids [The no acids .In The Cancer Genome Atlas breast cancer study, which performed exome sequencing of tumor and normal samples from a selected cohort of breast cancer patients, a 1/3 somatic to 2/3 germline ratio was found  and a siIn a large-scale study in the United States, Vidula et al. analyzed cDNA by NGS and demonstrated that this approach can identify three classes of clinically relevant BRCA1/2 mutations: germline mutations, somatic loss of function, and reversions. This study supports the notion that systematic analysis of cfDNA in patients with advanced cancer can help identify potential candidates for appropriate genotype-based therapy .BRCA1 and BRCA2, as well as functional studies to assess the impact of putative BRCA1/2 reversion mutations on BRCA1/2 function, the findings of this study support the utility of cfDNA sequencing to identify putative BRCA1/2 reversion mutations and facilitate patient selection for PARP inhibition therapy [In line with this, another study conducted in 2017 to analyze cfDNA by massively parallel sequencing, this study targeted all exons of 141 genes and all exons and introns of  therapy .ESR1 gene. The acquisition of alterations in this gene has been identified as an important resistance mechanism to endocrine therapy [ESR1 mutations rarely occur in primary tumors (~1%) but are relatively common (10–50%) in metastatic cancers presenting resistance to endocrine therapy, this is explained by the therapeutic pressure that stimulates the emergence of activating mutations of ESR1 in the metastatic tissue. These gene mutations are associated with shorter progression-free survival [ESR1 somatic mutations in ctDNA in order to detect the onset of this molecular resistance even before clinical progression. To predict treatment outcomes, because this may dictate patient management, including monitoring and modifications of the treatment plan, eventually this approach can be used to guide sequential treatment options in patients [ESR1 mutations in metastatic cancer has increased.Over 75% of primary breast cancers are ER-positive, for which hormone therapy is the gold standard in the treatment armamentarium ,89. The  therapy . ESR1 musurvival . Therefopatients ,71. The BOLERO-2 clinical trial  was reported in the United States, including 541 patients. ESR1 gene mutations were analyzed from cfDNA using a droplet digital polymerase chain reaction. The main result of this work was the high prevalence of ER mutations for patients with ER+ metastatic breast cancer treated by aromatase inhibitors (AIs), and the association of this gene’s mutations with more aggressive disease biology, as explained by a median overall survival of 20.73 months  for patients with ESR1 gene mutations compared with 32.1 months  for patients without mutations [In 2016, a secondary analysis of the utations .ESR1 mutations in the PBC group, whereas in the MBC group ESR1 cfDNA mutations were detected in 28.9%. The clinical impact of these mutations was significantly important: all ESR1 mutation-positive patients had resistance to previous treatments with aromatase inhibitors (AIs) compared to 71.4% of ESR1 WT patients; 85% of ESR1 mutation-positive patients had resistance to previous treatment with selective estrogen receptor modulators (SERMs) compared to 51% of ESR1 WT patients. Otherwise, cfDNA monitoring in a subgroup of 52 patients showed that loss of ESR1 mutations was associated with a longer response time [In a Japanese study in 2017, involving 86 ER+ breast cancer patients, including 69 patients diagnosed with metastatic breast cancer (MBC) and 17 patients with primary breast cancer (PBC), the results showed the absence of nse time .ESR1 gene, and the result’s findings are consistent with previous studies: ESR1 mutations were more frequently detected in circulating tumor DNA of MBC patients than in PBC patients, and the ESR1 mutation frequency in patients using aromatase inhibitors (AIs) was significantly higher than those who were not using AIs [In January 2020, a Chinese comparative study was conducted comparing 297 tumor samples of primary breast cancer patients and 43 blood samples of metastatic breast cancer (MBC) patients. Next-generation sequencing (NGS) was used by targeting the whole exon of the sing AIs .ESR1 as a pilot gene specific to MBC, they confirmed that the ESR1 gene mutation is the most frequent “metastasis-specific” mutation observed in MBC, by identifying that 100% of ESR1 mutation-positive cases are ER + and present resistance to endocrine therapy [ESR1 mutations were found to be a prognostic predictor of acquired resistance to endocrine therapy [A French large study targeting twelve genes by whole exome sequencing has identified  therapy . In 2020 therapy .Collectively, these studies demonstrate how the genetic profile of MBC is different from that of primary breast tumors. Hence, metastatic cancer profiling must become a primary step in the definition of optimal treatments for patients; thus, monitoring and surveillance studies will be critical to confirm the effectiveness after treatment. A final point regarding these findings was how easily and feasibly this biomarker could be obtained, plasma samples can be easily collected for ctDNA analysis, allowing for dynamic testing that will increase our understanding of the disease progression and the design of strategies to improve outcomes.PIK3CA gene encodes the A isoform of the catalytic subunit (p110a) of the phosphatidylinositol 3-kinases class IA. Phosphatidylinositol 3-kinases (PI3K) are involved in the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). This PI3K class has a critical role in the control of various cellular processes such as cell growth and proliferation, metabolism, and migration via the PI3K/AKT/mTOR pathway [The  pathway .PIK3CA gene, these mutations induce hyperactivation of the p110α catalytic subunit, leading to constitutive phosphorylation of the AKT and its forward effectors [In approximately 40% of HR + breast cancer cases, the most common molecular alterations are the activating mutations of the PI3K subunit in the ffectors ,97.Several preclinical studies indicate that the PIK3/AKT/mTOR pathway alterations may correlate with resistance to CDK4/6 inhibitors (CDK4/6i). Cyclin-dependent kinase 4 and 6 (CDK4/6i) inhibitors have shown clinical efficacy in ER-positive MBC, although their cytostatic effects are limited by primary and acquired resistance, putative mechanisms of resistance to CDK4/6i have been identified ,98.PIK3CA mutation in the blood at the start of CDK4/6i treatment had a significantly shorter Progression Free Survival (PFS) compared to patients without a mutation. Therefore, PI3K status should be considered as a potential predictive biomarker of CDK4/6i resistance. The integration of PI3K status assessment with other molecular information in a surveillance system can improve the accuracy of predicting Overall Survival (OS) and PFS of patients with metastatic breast cancer and may suggest the best treatment strategy. On the other hand, it should be noted that PI3K status as such cannot be the only responsible for CDK4/6i resistance, and thus, special attention should be given to all mutations that also promote the activation of the PI3K/AKT/mTOR signaling pathway [An Italian study completed in January 2021 showed that patients with a  pathway .PIK3CA gene alteration, which makes their detection particularly important in tumor genetics.In the same sense, targeted therapy drugs in the final phase of clinical trials, specifically targeting PI3K, are designed to be administered to patients whose tumor has a PIK3CA mutant tumors, confirming the results in preclinical trials [In July 2017, a Phase I study was designed to evaluate the effectiveness of TASELISIB which is a selective inhibitor of tumor growth by suppression of the PI3K pathway, the results show an increase in the antitumor activity for patients with l trials .SANDPIPER trial   evaluated the clinical benefit of TASELISIB  in combination with FULVESTRANT for Advanced Breast Cancer in a population of 516 PIK3CA mutated patients. The results showed a statistically significant improvement in progression-free survival evaluated by the investigator in the PIK3CA mutant population [In February 2021, the pulation . conducted by Juric et al. aims to evaluate, in patients with advanced ER + breast cancer, the maximum tolerated dose (MTD), safety and activity of L’ALPELISIB which is an oral specific PI3Kα inhibitor, combined with FULVESTRANT, the clinical trial results suggest that the combination of ALPELISIB and FULVESTRANT may have greater clinical activity in PIK3CA-altered tumors compared to wild-type tumors [Furthermore, an open-phase 1b study e tumors .SOLAR-1  was designed to compare ALPELISIB plus FULVESTRANT with placebo plus FULVESTRANT, this study included 572 patients of which 341 patients had confirmed tumor tissue PIK3CA mutations, the results showed a prolongation of progression-free survival in patients with PIK3CA-mutated advanced breast cancer who were treated with ALPELISIB-FULVESTRANT [PIK3CA mutations in circulating tumor DNA as part of daily clinical practice.In May 2019, a Phase 3 randomized clinical trial VESTRANT . HoweverTP53 is considered the genome gatekeeper. Several studies indicate that TP53 mutations increase the risk of cancer and, once cancer occurs, these mutations promote invasion, metastasis, and chemoresistance [TP53 (p. 53) is the most frequently mutated gene, which is mutated in approximately 80% of triple-negative breast cancer (TNBC) [Tumor suppressor sistance . In invar (TNBC) .TP53 mutations is routinely investigated in clinical practice on tumor tissues, using immunohistochemistry or sequencing techniques. However, these methods have various limitations, such as invasiveness and inability to identify tumor heterogeneity and progression, which underline the need for a more robust and sophisticated technology [TP53 gene mutations can be detected by cfDNA analysis given the ability of this biomarker to detect tumor heterogeneity, which is a limitation of tumor biopsies [The presence or absence of chnology . Currentbiopsies ,107.TP53 gene at several time points: before NCT; after 1 cycle; before surgery, and after surgery. The results indicated a decrease in ctDNA levels during NCT; however, a small decrease in ctDNA levels during NCT was significantly associated with shorter survival. This research confirmed that the high prevalence of TP53 mutations in TNBC is a potential biomarker for ctDNA monitoring during NCT, and that ctDNA may become a clinically useful prognostic tool for managing TNBC patients treated with NCT [In 2017, a French study was realized including 47 patients with non-metastatic triple-negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NCT), and the study targeted the with NCT .TP53 gene are strongly observed in breast cancer samples, this study demonstrated that TP53 pathogenic variants detection and monitoring by ctDNA analysis is recommended as a useful biomarker for predictive studies, for tumor growth monitoring and personalized treatment strategy planning [In July 2019, Savli and colleagues showed that variations in the planning .TP53 mutations in ctDNA can provide monitoring for early detection of genetic events underlying drug resistance and can also inform therapy approaches [ have exploited ctDNA profiling before and after treatment with an oral anti-HER1/HER2 tyrosine kinase inhibitor for 18 HER2-positive metastatic breast cancer patients, and they identified that mutations in TP53 and PI3K/Akt/mTOR pathway genes were strongly implicated in resistance to HER1/HER2 blocking [Screening for proaches , FEI MA blocking .p53 is both a potential prognostic and predictive biomarker and a therapeutic target for breast cancer patients, particularly for TN subtype patients.The overall clinical trials confirm that HER2) is overexpressed in 20–30% of breast cancer patients [The human epidermal growth factor receptor 2  amplification acquisition in a non-invasive manner.As targeted anti-HER2 therapies are developed, the assessment of regimens , and thiregimens . The ana was conducted to evaluate the clinical benefit rate (CBR) of NERATINIB as monotherapy through determining HER2 mutations based on circulating DNA analysis, whereby the results indicated the efficacy of NERATINIB in HER2 mutated non-amplified breast cancer and justify that cfDNA sequencing may offer a non-invasive strategy to identify patients with HER2 mutated cancers [ in 18 UK hospitals was conducted, the results confirm the clinically relevant activity of targeted therapies against rare activating mutations in breast cancer, in HER2 mutant breast cancer (ERBB2) identified by cfDNA tests, NERATINIB had comparable activity to that observed when guided by tissue tests [In 2017, a phase II study  cancers . In a siue tests .ERBB2 oncogene. This result creates opportunities for better tumor characterization, in which sequential plasma samples can be collected to represent CNVs and variants more precisely over time [In October 2020, Kleftogiannis et al. developed a new error-correcting cfDNA sequencing approach using bioinformatics strategies to identify tumor-associated genomic alterations. This assay was also performed effectively on the detection of copy number variations (CNVs) in the ver time .Breast cancer displays high levels of heterogeneity and is generally subject to clonal evolution underpinning drug therapy. Through our review, we have demonstrated that the multipoint analysis of cfDNA reflects clonal evolution and allows us to track the molecular landscapes of cancer cell growth by capturing broader molecular alterations that could affect the efficacy of targeted therapies. The shorter execution time of cfDNA analysis and its high sensitivity and specificity are key factors in providing new opportunities for adaptive personalized therapies, optimizing healthcare resources, and enabling higher treatment efficacy and lower risk side effects.The potential use of cfDNA in the management of breast cancer has been significantly improved by recent advances in molecular technologies, as digital PCR and next-generation sequencing technologies take hold, and as an understanding of the biology and clinical potential of cfDNA increases, the ultimate use of cfDNA in clinical practice seems assured.Several clinical trials in Europe, North America, and Asia are underway to evaluate the diagnostic utility of ctDNA, and hence the possibility of incorporating this biomarker into cancer monitoring. Minimally invasive methods of cancer diagnosis have potential because current tumor biopsy and medical imaging techniques that require exposure to ionizing radiation are limited to high-risk individuals and those with previously identified lesions. Alternatively, liquid biopsy-based diagnostics are adapted to repeated sampling and can potentially be used for early detection or screening of cancer.The challenge is to establish adequate and international pre-analytical conditions, to optimize state-of-the-art analytical techniques with improved sensitivity and specificity, for the purpose of avoiding missing mutations and tumor changes.