Apart from its metabolic or physiological functions, melatonin has a potent cytoprotective activity in the physiological and pathological conditions. It is synthetized by the pineal gland and released into the blood circulation but particularly cerebrospinal fluid in a circadian manner. It can also easily diffuse through cellular membranes due its small size and lipophilic structure. Its cytoprotective activity has been linked to its potent free radical scavenger activity with the desirable characteristics of a clinically- reliable antioxidant. Melatonin detoxifies oxygen and nitrogen-based free radicals and oxidizing agents, including the highly toxic hydroxyl-and peroxynitrite radicals, initiating cellular damage. However, the cytoprotective activity of melatonin is complex and cannot be solely limited to its free radical scavenger activity. It regulates cellular signaling pathways through receptor– dependent and independent mechanisms. Most of these downstream molecules, such as PI3K/AKT pathway components, also contribute to the cytoprotective effects of melatonin. In this term, melatonin is a promising molecule for the treatment of neurodegenerative disorders, such as ischemic stroke, which melatonin reduces ischemic brain injury in animal models of ischemic stroke. It regulates also circadian rhythm proteins after ischemic stroke, playing roles in cellular survival. In this context, present article summarizes the possible role of melatonin in the pathophysiological events after ischemic stroke. Stroke is one of the leading causes of death and long-term disability worldwide [1] with increasing incidence rates in low and middle-income countries [2]. Disability due to a stroke incidence has significant emotional, social and economic burden on the patients and their society. It was suggested that deaths due to stroke cases will reach to 12 million and number of patients surviving from a stroke incidence will reach to about 70 million by the year 2030 [3].Stroke cases are categorized into two groups: hemorrhagic and thrombotic (or ischemic) stroke. In hemorrhagic stroke, intracerebral or subarachnoid hemorrhage occurs in the brain due to the rupture of a cerebral blood vessel, but this type of stroke is less common  worldwide . On the other hand, ischemic stroke contributes to the majority of stroke cases and occurs when a cerebral artery is occluded by thrombus or thromboembolism, meaning that a blood clot may occur and cause a block in a small cerebral artery or a blot clot formed in a large vessel lodges in a small vessel in the brain. Due to the occlusion of the cerebral artery, blood supply to a certain brain region is restricted. Therefore, this event is called ischemia or ischemic stroke. Despite being a global problem with major social and economic burden on the society, treatment strategies are limited, and prevention measures are not enough [1]. Currently, patients survived from an ischemic stroke are treated with tissue plasminogen activator (tPA) that helps to relieve the obstruction in the blood vessels [6]. However, tPA has a very short therapeutic window which makes it impossible to use in the majority of the patients because of unfavorable actions of thrombolytics. Therefore, researchers have been in the search for a treatment option that can be used in patients who were diagnosed after the end of the therapeutic window of tPA or add-on treatment with thrombolytics. In this sense, it was shown that melatonin also reverses unfavorable actions of tPA [7] and, we will focus on the novel findings for the use of melatonin in ischemic brain injury in the present article.The pathophysiologic mechanisms underlying ischemic stroke or ischemia/reperfusion injury in humans include loss of the prooxidant/antioxidant balance, excitotoxicity and related increase in intracellular Ca++ levels, dysfunction in mitochondrial processes, increased neuroinflammation and eventually apoptotic neuronal cell death [8].The studies reporting the antioxidant effects of melatonin justified its use for treatment of ischemic injury in animal models [9]. Thereafter, further research provided evidence that melatonin is able to alleviate several of the detrimental effects of stroke-related pathophysiologic processes . In addition, melatonin was shown to protect the integrity of blood-brain barrier [u291c], and functional recovery [17] with minimal, if any, side effects even in high doses [18]. Moreover, melatonin exerts neurological protection by reducing the cerebral inflammatory response, cerebral edema and brain-blood barrier permeability after ischemic stroke [19].Although melatonin can be synthesized in a number of tissues, it is believed that the main production center may be the pineal gland due to the fact that melatonin’s protective effects are severely diminished by pinealectomy as reported in experimental model of stroke [20]. Melatonin synthesis in the pineal gland occurs in a circadian manner, in which the melatonin levels peak in the night-time, whereas in the day-time melatonin production is inhibited [21]. Secreted melatonin is believed to modulate the circadian clock mechanisms through the ubiquitin-proteasome signaling pathway. Several gene transcripts [22] and proteins [23] have been identified to be expressed in a circadian fashion. Of these genes, the six core clock genes are known as Per1 and Per2, Cry1 and Cry2, Clock and Bmal1 . We have reported that melatonin regulates Bmal1 expression under normal conditions in vitro after hypoxia and in vivo after ischemic stroke [26]. Interestingly, in parallel with the highest blood concentrations of melatonin, the ischemic stroke incidence is lowest at the midnight hours in human [27]. In this term, we have indicated that tolerance to ischemic injury changes according to the time of day in which the injury occurs and the underlying mechanism of this tolerance includes circadian clock genes, specifically Bmal1, which is also regulated by the phosphorylation of AKT signaling pathway [24]. Consistently, Bmal1 expression is enhanced following melatonin treatment following ischemic brain injury in mice and this increase is blocked when the survival kinase AKT inhibitors are present [26]. In addition to the transcriptional control of Bmal1 gene, melatonin may also regulate clock genes by stabilizing the protein through the inhibition of the ubiquitin-proteasome system [28]. It has been speculated that melatonin can act as a proteasome inhibitor and we showed that melatonin inhibits the proteasome machinery by downregulating Nedd4-1 E3 ligase expression [29]. Collectively, these data suggest that melatonin may still have unelucidated roles in the body that promotes endogenous recovery systems.The experimental models in rodents focusing on the antioxidant effect of melatonin in stroke pathophysiology mainly involved in the ischemia/reperfusion. In this model, generally transiently middle cerebral artery occlusion (MCAo) is performed and melatonin is usually administered either at the onset of ischemia or reperfusion [8]. In the ischemia/reperfusion injury, a blood vessel is obstructed and blood supply to a certain brain region is restricted, causing immediate apoptotic and necrotic cell death in the ischemic core. The surrounding tissue, called the penumbra, has relatively higher levels of blood supply compared to the ischemic core, however, apoptotic cell death through the complex interplay of several mechanisms can be observed in the penumbra even several days after ischemic injury [8]. In order to resupply the ischemic brain tissue with oxygen, hyperbaric (HBO) and normobaric oxygen (NBO) treatments gained considerable interest due to the possibility of oxygen to diffuse through the blood-brain barrier to reach the injured brain tissue. However, conflicting literature exists for HBO treatment in transient or permanent MCAO models possibly because of increased free radical production upon effect of oxygen [30]. Even detrimental effects of HBO treatment were reported [31], but it should be kept in mind that the underlying reason for conflicting results may be partly because of the different timing of HBO treatment. Conversely, NBO therapy is an inexpensive and easy-to-access strategy that can be administered by simple facemasks. The favorable effects of NBO on the infarct volume and cell death were demonstrated in different models of brain injury [32–35]. However, the NBO treatment in these studies is usually started during the ischemia period or immediately at the onset of reperfusion. Although NBO can be beneficial in these models, this therapy can be only translated to patients who were admitted to hospitals with stroke symptoms that last for less than 12 h. In the light of these results, our group investigated the use of NBO treatment during reperfusion [12]. We also evaluated the combination of melatonin with NBO treatment. Our results indicated that melatonin potentiated the protective effect of NBO therapy in terms of infarct volume, brain swelling, neurological deficit score and DNA fragmentation [12].Even though the most important step in rescuing the cells’ from apoptotic death in the penumbra is to remove the obstruction and resupply the blood flow (i.e. reperfusion), this results in oxidative damage due to the excess production of free radicals. The cells of the central nervous system (CNS) are already exposed to high amounts of free radicals in the physiological state. The adult human brain uses about 20% of the total oxygen intake even though it weighs only about 2% of the total body weight [36]. Not only brain produces the highest number of free radicals compared to any other organ in the body, but also it has high levels of polyunsaturated fatty acids which make it more prone to oxidative stress. Under normal conditions, the production of free radicals and antioxidant enzymes are kept in a delicate balance. If an imbalance occurs in favor of oxidants as a result of brain injury, the ability of the CNS cells to neutralize oxidants using endogenous antioxidant systems is overwhelmed and eventually inflammation and apoptotic cell death are observed. It is well-known that the free radicals including reactive oxygen species (ROS) and reactive nitrogen species (RNS), including superoxide anions (O2•−), hydrogen peroxide (H2O2), hydroxyl radicals (HO•) nitric oxide (•NO) and peroxynitrite (ONOO−) are mainly produced during the reperfusion phase of an ischemic stroke. The sources of these free radicals are diverse. For instance, nitric oxide is a signaling molecule that is involved in vasodilatation, neurotransmission and blood pressure maintenance and is synthesized by NO synthase (NOS) enzymes mainly in a calcium-dependent manner [37]. Three types of NOS enzymes were characterized as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) in the central nervous system [38]. During ischemic brain injury, excess nitric oxide is generated, which in turn results in lipid peroxidation, energy depletion, and formation of highly neurotoxic oxidizing agent ONOO− [39]. Although the nitric oxide synthesized by the eNOS at the early phases of ischemic stroke is believed to provide protective effects, NO synthesis by iNOS and nNOS exacerbates the injury by activating the inflammatory mechanisms [40].It has become clear that excessive free radical generation is a critical pathophysiological step in ischemic injury and regulates several other steps. This prompted researchers to investigate the possible use of chemical or biological molecules to reduce the generation or accumulation of free radicals. We and others have tested several antioxidant molecules, such as glutathione [41], vitamin E [42], vitamin C [43] or melatonin [20]. To the best of our knowledge, melatonin is the only antioxidant whose metabolites also have antioxidant capacities. In fact, the direct and indirect capacity of melatonin and its metabolites  to scavenge free radicals is called the “antioxidant cascade” [44]. As the members of the antioxidant cascade of melatonin are all free radical scavengers, the detoxification capacity of melatonin was predicted to be up to 10 times more than any other antioxidant molecule [45]. Therefore, antioxidant capacity of melatonin has been studied in many ischemia/reperfusion injury models, including brain, kidney or heart. In fact, melatonin can interact with and detoxify the free radicals by donating an electron or a hydrogen atom in the injury models. In addition to its free radical scavenger activity, melatonin was also shown to upregulate antioxidant enzymes, such as superoxide dismutase (SOD), catalase or glutathione reductase (GR) [46]. Our group evaluated the effect of melatonin on the production of nitric oxide and our results demonstrate that melatonin treatment significantly downregulates nNOS and iNOS after ischemic brain injury .Considering the higher melatonin concentrations compared to blood levels were measured in rat liver mitochondria [48], it is not surprising to assume that high melatonin levels are required to protect the mitochondrial DNA from the continuous production of reactive oxygen species by the oxidative phosphorylation [49]. Because of its lipophilic structure, melatonin can easily pass through the biological membranes and accumulate in organelles, such as mitochondria and nuclei. Since the discovery of melatonin in bacteria and chloroplasts,it has been speculated that melatonin synthesis can also occur in the mitochondria [50]. Furthermore, enzymes  required for the synthesis of melatonin were detected in oocyte mitochondria [51]. These results strongly suggest that melatonin synthesized in the mitochondria is used as direct free radical scavenger and indirect antioxidant enzyme regulator in this organelle. Moreover, in a recent study, mitochondrial melatonin was shown to be secreted into the cytosol where it can bind to the receptors on the surface of the mitochondria [52]. It is proposed that this feature gives another advantage to melatonin in its role in mitochondrial homeostasis. This involves the restoration of the activity and expression of complexes I and IV which are decreased during ischemic injury. This in turn, reduces the electron leakage and prevents further damage to the organelle.In addition, increased free Ca++ levels following ischemic brain injury are also responsible for mitochondrial dysfunction and formation of further free radicals. If these toxic effects are not neutralized, ATP production would be severely affected. Intriguingly, Xu et al. implicated that melatonin can control free Ca++ movement in the cytoplasm and protects the homeostasis of mitochondria [53]. Loss of blood supply during ischemic brain injury depletes the cellular energy and results in the release of glutamate neurotransmitter into the extracellular space [8]. As a result, the transmembrane glutamate receptors  and several channel and transporter proteins  are overactivated. Activation of these receptors is followed by an excessive Ca++ influx, either due to the release from mitochondria and endoplasmic reticulum or due to activation of plasma membrane proteins and lead to apoptotic cell death [54]. Of these receptors, overactivation of postsynaptic NMDA receptors by glutamate results in increased Ca++ load in the cytosol and mitochondria [55]. In this term, we used a combination treatment with an NMDA receptor inhibitor, memantine, and melatonin in a MCAo model and our results indicate that administration of melatonin/memantine combination significantly reduces the infarct volume, while improving the vascular leakage [56]. Furthermore, electrophysiological studies revealed that melatonin depresses NMDA receptor activity in the brain, possibly due to reduced nNOS activity .In an attempt to rescue from Ca++ overload, the cells in the injured brain tissue try to reduce the high cytosolic Ca++ levels by storing Ca++ ions into mitochondria or endoplasmic reticulum. However, when Ca++ levels are increased in the mitochondrial matrix, free radical production is enhanced. Increased free radical accumulation disrupts mitochondrial membrane, results in permeabilization and depolarization followed by the release ofproapoptotic molecules, such as cytochrome c and apoptosis inducing factor (AIF) into the cytosol [u2934]. Andrabi et al. suggested that melatonin inhibits the cytochrome c release and decreases DNA damage in transient MCAO in rats [60]. Our results proved that melatonin improves neuronal survival by caspase-3 inhibition [16]. In addition, we showed that melatonin inhibits antiapoptotic Bcl-xL, while promoting the expression of proapoptotic Bax [12]. Downregulation of survival kinases in the injured brain tissue also contributes to apoptotic cell death. It is well-known that ischemia/reperfusion inhibits PI3K/AKT pathway. Our planar immunoassay analyses revealed that melatonin treatment results in the increased AKT phosphorylation especially at the Thr308 site of the activation loop via PDK1 and PTEN as well as decreased GSK-3α/β, and p53 phosphorylations [19], suggesting that neuroprotective activity of melatonin directly involves the activation of survival signaling pathways. Additionally, we have observed that melatonin treatment phosphorylates AMPKα, which is particularly activated by the reduced intracellular energy. AMPKα drives the cell to a catabolic state which this molecule mobilizes alternative energy sources, such as fatty acid oxidation, in order to supply ATP in the condition of ischemic stroke, suggesting also that melatonin may activate alternative collateral survival pathways [19].It has been shown that one of the early mechanisms of ischemic injury is the release of inflammatory cytokines, such as IL1β, IL6 and TNFα [61]. Following an ischemic attack in the brain, the free radicals are generated in high amounts. This excessive free radical production especially during the reperfusion phase also contributes to the disruption of the integrity of the blood-brain barrier and stimulates the infiltration of lymphocytes, neutrophils, monocytes, T cells, and macrophages to the injury site [62]. Simultaneously, resident microglial cells are activated, change their morphology (deramification) and start to release proinflammatory cytokines. Microglial activation following ischemia also results in increased proliferation and accumulation in the penumbra region. Microglial activation seems to have a dual role in the pathophysiology of stroke. During the course of ischemia/reperfusion, microglial cells are believed to switch from an antiinflammatory state to proinflammatory phenotype [63]. Melatonin treatment was shown to inhibit the proinflammatory shift of microglial cells through the regulation of SIRT1 and STAT3 . Moreover, TLR4 activates NF-κB after stroke, resulting in the secretion of inflammatory molecules . Melatonin decreases the secretion of inflammatory mediators by downregulating NF-κB, while promoting Nrf2 upregulation [66]. Studies indicated increased melatonin levels can be observed as early as in the first 10 minutes of intraperitoneal or subcutaneous application, indicating that melatonin can easily pass through the blood-brain barrier [67]. Melatonin is also able to diffuse through cellular and organelle membranes due its small size and lipophilic structure [68]. On the other hand, melatonin has two G protein-coupled transmembrane receptors; MT1 and MT2 (earlier names Mel1a and Mel1b) which are ubiquitously found in almost all cells in the body [69]. It has been proposed that melatonin exerts its neuroprotective effects on the brain by both receptor-independent or receptor dependent mechanisms [70]. Models of acute and chronic ischemia have been used to investigate the role of MT1/MT2 receptors in ischemic brain injury by using melatonin receptor agonists, such as ramelteon , agomelatine [u2937] or Neu-P11 [u2938] and whether it is involved in melatonin’s antioxidant, antiapoptotic or antiinflammatory activity should be further investigated. Interestingly, MT3 knockout mice were less susceptible to menadione toxicity [80], suggesting that the inhibition of this receptor may have protective effects. Moreover, knockdown of MT3 by RNA interference in vitro resulted in enhanced expression of antioxidant enzymes [81], while overexpression of MT3 resulted in excessive production of reactive oxygen species [82]. It was shown that melatonin is able to inhibit this receptor at nanomolar levels in which antioxidant effects were documented [83]. Therefore, it is tempting to speculate that MT3 inhibition by melatonin is involved in melatonin’s protective effects on the pathophysiological outcomes of brain injury not only by increasing the expression of antioxidant enzymes, but also by providing resistance to oxidative stress.Moreover, the nuclear receptors RZR/ROR from the retinoic acid receptor superfamily were proposed as nuclear binding sites for melatonin [84]. RZR/ROR expression was demonstrated in the brain, and in the pineal gland [69]. ROR receptors were shown to induce the expression of several clock genes, including Clock, Cry or Bmal1 [85–87] by binding to retinoic acid-related orphan receptor response elements (ROREs) in the promoter region. In parallel with these data, cyclic expression of ROR mRNAs were noted in different tissues, suggesting a circadian function possibly under the control of melatonin. However, whether these receptors play a part in the protective mechanisms induced by melatonin should be further investigated.Recently, noncoding RNAs including microRNAs, long noncoding RNAs, and circular RNAs have gained considerable attention as regulatory molecules. Recent studies indicated that circular RNAs (circRNAs) are found abundantly in the brain and are involved in the embryonic development [88]. CircRNAs are enriched in specific brain regions such as cerebellum, cortex, striatum, olfactory bulbs, and hippocampus. Since they are circular, these types of noncoding RNAs are more resistant to digestion. CircRNAs can be made from introns, coding or noncoding exons, or from both exons and introns by a process called “back-splicing” [89]. Zhang et al. suggested that because the circRNAs are formed by the circularization of skipped exons, formation of circRNA can cause the downregulation of its parental gene by using up the pre-mRNA molecules [90]. In addition to their regulatory roles in the brain, circRNAs are also associated with neurological disorders, including, but not limited to, ischemia/reperfusion injury, traumatic brain injury, and Alzheimer’s disorder [91]. In a study performed in acute ischemic stroke patients to profile the changes in circRNA, 3 circRNAs have been proposed as diagnostic and predictive biomarkers for stroke [92]. Significantly altered circRNAs in a transient MCAO model in C57BL/6J mice were characterized, and bioinformatics data suggested that all these circRNAs possess binding sites for microRNAs [93]. However, the exact pathophysiological mechanisms that they have a role in are not fully elucidated. It has been predicted that melatonin may be involved in the regulation of circRNAs as the circRNA prolife in the pineal gland has been altered in a mouse model of Alzheimer’s disease [94]. In fact, a recent study reported that melatonin exerts protective effects through the regulation of CircRIC3/miR-204-5p/DPP4 signaling in calcific aortic valve disease [95]. Therefore, we hypothesized that melatonin also regulates circRNAs in ischemia/reperfusion injury, however, these circRNAs should be further investigated in future studies. Moreover, we reported that lower endogenous melatonin concentrations were associated with increased injury after transient ischemic stroke in pinealectomized rats and when those deprived animals were given exogenous melatonin, injury size could be reduced [20]. It has been shown that melatonin levels are reduced in elderly compared to young adults; therefore, aged population is more prone to serious ischemic injury [96]. In addition, accumulation of other risk factors, such as cardiovascular disorders, diabetes, obesity or hypertension with ageing increases the risk of ischemic injury incidence . In animal models, we demonstrated that prophylactic uses, and delayed uses of melatonin successfully protects the brain from the ischemic injury [u2960]. In conclusion, prophylactic doses could be considered in the elder population in order to compensate for the reduced melatonin levels due to the calcification of pineal gland and to promote the endogenous repair mechanisms against stroke or other neurodegenerative diseases.


The largest subtype of breast cancer is characterized by the expression and activity of the estrogen receptor alpha . Although several effective therapies have significantly improved survival, the adaptability of cancer cells means that patients frequently stop responding or develop resistance to endocrine treatment. ER does not function in isolation and multiple associating factors have been reported to play a role in regulating the estrogen-driven transcriptional program. This review focuses on the dynamic interplay between some of these factors which co-occupy ER-bound regulatory elements, their contribution to estrogen signaling, and their possible therapeutic applications. Furthermore, the review illustrates how some ER association partners can influence and reprogram the genomic distribution of the estrogen receptor. As this dynamic ER activity enables cancer cell adaptability and impacts the clinical outcome, defining how this plasticity is determined is fundamental to our understanding of the mechanisms of disease progression. Despite substantial achievements in our understanding of breast cancer and the advancement in treatment options, it currently represents the most common cancer in women worldwide  , hormonede novo resistance and stop responding to standard-of-care treatments  primarily localize away from transcription start sites to cis-regulatory regions called enhancers . These rImportantly, enhancers can stimulate promoter activity over long genomic distances, being located anywhere between 1 kb to more than 1 Mb away from the target gene. With up to 400 000 putative enhancers proposed in the mouse and human genomes , it has The chromatin looping model has been informed by the advent of chromatin conformation capture–based technologies . TogetheUnderstanding and defining enhancer function and its role in 3D genome organization continue to represent exciting areas of intense research, with a number of excellent reviews summarizing current knowledge in the field and discussing alternatives to the chromatin looping model , 58-65. One of the main players in ER biology is represented by the pioneer transcription factor Forkhead Box A1 (FOXA1). The main feature of pioneer factors is their ability to access their binding motifs in closed chromatin, thereby promoting a local chromatin decompaction which enables the binding of canonical TFs and subsequent lineage-specific patterns of active enhancers and unique transcriptional programs. Despite discussions of whether steroid receptors can reprogram a pioneer TF at discrete subsets of genomic loci , 94, it Alongside FOXA1, several coactivators are found at ER-bound enhancer elements, including the steroid receptor coactivators SRC1/2/3  , 69, thec-fos and c-jun gene induction despite enhanced histone acetylation on those targets, indicating that the active and dynamic turnover between acetylation and deacetylation is required for robust gene expression  complex, nuclear receptor corepressor (NCoR), or silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) , 76 alsopression .Interestingly, recent mass spectrometry–based absolute quantification work revealed that, on average, corepressors are ~ 100 times more abundant than coactivators, meaning that, overall, the nuclear environment is highly repressive . This abConsistent with their fundamental role in transcriptional control and genome integrity, members of the ATP-dependent chromatin remodeling complexes are frequently mutated at the somatic or germline level across cancer types . For exaTwo recent papers reveal that loss of ARID1A promotes endocrine therapy resistance , 78. In While the differences between the 2 studies underline the complexity of disentangling the change in the enhancer landscape upon BAF impairment, both papers link ARID1A mutations to changes in ER function and treatment response. These studies also provide a feasible and actionable strategy for the clinic, first as an opportunity to stratify patients based on ARID1A aberrations, with patients harboring mutant ARID1A more likely to relapse on endocrine therapy. Second, these studies highlight the need to explore the vulnerabilities or synthetic lethality opportunities arising from these alterations, such as an increased dependency on BRD4 or the identification of ARID1B as a vulnerability in ARID1A-mutant cancers , 124.While defined as a transcriptional repressor, the NuRD complex can also bind accessible regions such as active enhancers and promoters. The cistrome of various NuRD subunits, including the ATPase protein Chromodomain helicase DNA-binding protein 4 (CHD4), largely overlaps with the profile of the BAF catalytic subunit BRG1 in a variety of cell types . In addiMultiple links between NuRD and ER biology were proposed , 80. ForWhile missense loss-of-function mutations which affect the ATPase domain of CHD4 are considered a driver in endometrial carcinoma , 136, suPolycomb group proteins are essential regulators of gene expression, acting as transcriptional repressors of developmental regulatory loci such as the Hox cluster . Largelyde novo (not marked with high levels of H3K27ac prior to estrogen stimulation) enhancers. The observation that chromatin accessibility at estrogen-stimulated genes is dynamic and cyclical in a manner that seems dependent on RING1B raises the possibility that this Polycomb factor plays a role in resetting the ER-occupied enhancer chromatin landscape to allow the cell to be adaptive and responsive to environmental stimuli, although mechanistic insight into this will require additional work. How depletion of RING1B impairs accessibility specifically at these de novo enhancers and whether this is a cause or instead a consequence of the reduced ER binding seen following Polycomb knockdown requires further exploration. While the E3 ligase activity of RING1B appears to be important, it remains unclear whether this is due to impaired ubiquitination of its histone target (H2AK119) or loss of activity toward a nonhistone target, perhaps a TF such as ER. Unlike RING1B which seems required for estrogen-induced gene expression, another E3 ubiquitin ligase complex consisting of RNF20/RNF40 was shown to repress estrogen-inducible enhancers in a manner dependent on its activity toward the histone substrate H2B, underlining the complex chromatin landscape characterizing these regulatory elements  , 90. At elements .In addition to RING1B, other Polycomb group proteins were linked to ER signaling, with both the PRC1 component B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and the PRC2 catalytic subunit Enhancer of zeste homolog 2 (EZH2) reported to contribute to tamoxifen resistance , 92. WhiFor a TF such as ER, there are many potential binding sites throughout the genome, which can be attained either directly via its own DNA-binding domain, or indirectly, via recruitment by other factors. However, only a minority of these binding sites are occupied at any given time. The vast repertoire of possibilities enables plasticity in terms of a TF cistrome leading to highly adaptable transcriptional programs. While adaptability allows cells to quickly respond to environmental stimuli, it can also be associated with disease transition, providing a proliferative benefit to a handful of cells leading to unregulated growth. Understanding the constraints on adaptability is an exciting therapeutic avenue, as it may provide an opportunity to define new ways to limit tumor growth. Dissecting the identity and expression levels of ER interaction partners is crucial to building our understanding of how such a dynamic ER cistrome is determined.The ER cistrome is reprogrammed both during the transition from normal mammary epithelial tissue to primary tumor and also when endocrine resistance develops. Due to a lack of good models and scarcity of material, the role of ER in the early events of tumor development remain poorly explored. However, the chromatin binding profile of ER is different in normal mammary epithelial cells compared with ER+ tumor specimens, a differential cistrome which translates into distinct gene expression patterns . It is nMultiple studies implicate an altered FOXA1 activity in mediating endocrine resistance and resulting poor clinical outcome , 163. ERAlong these lines, recent evidence indicates that activating the progesterone receptor (PR) in ER+, PR+ breast cancer drives a unique transcription program that is associated with a good clinical outcome . In partFor an efficient and sustained antiproliferative effect, an approach that considers not just ER in isolation but also its varying nuclear environment and interactome repertoire is likely to be required. For maximum antagonist activity (loss of estrogen-driven gene expression and cell proliferation), drugs which achieve highest ER degradation have been assumed to be best. However, recent work suggests that estrogen receptor degradation may partially be a consequence of ER antagonism  and thatIt remains unclear how reduced nuclear mobility is linked to the generation of a transcriptionally inert ER complex on target enhancers and subsequent degradation of the estrogen receptor. However, prior to ER degradation, SERDs induce ER association with chromatin in a manner comparable to estrogen stimulation . This raBinding of ER is highly dynamic, with a residence time in the range of seconds , 174. InUnderstanding whether ER binding is cyclical or sustained could reveal the mechanism involved in shuttling ER off chromatin, a mechanism which could be exploited to prevent transcription of ER-regulated genes driving cell proliferation such as MYC or CCND1. Furthermore, FOXA1 binding is not cyclical, suggesting this shuttling mechanism could be ER specific. It would also be interesting to test whether cycling is observed for any of the ER-activating mutants . As these variants are characterized by a differential interactome compared with wild-type ER , 181, thDespite significant progress in our understanding of the factors involved in the ER signaling pathway and their role in enhancer functionality in breast cancer cells, it is important to consider that, experimentally, most assays were performed on a population of cells. This represents a pooled measure and therefore likely neglects the gene/locus-specific dynamics of the ER-associated enhancer regulation. Implementation of CRISPR-Cas9 based approaches to target and annotate single functional enhancer elements , 183 andFurthermore, in vivo evidence of how some of these key regulators control enhancers and mediate disease progression is still lacking, which renders it difficult to assess their clinical relevance. One of the greatest challenges for future epigenetic studies remains obtaining fresh/frozen matched primary and metastatic tumor material. This requirement is necessary for understanding the evolution of drug resistance or the development of metastasis, both scenarios representing the poorest outcome in HR+ patients. The ability to carry out chromatin occupancy analyses on primary tumor material and biopsies , 185 andThese technical advancements need to be accompanied by changes in our appreciation of how to tackle chromatin regulatory factors therapeutically. The majority of epigenetic players that are altered in cancer show context-dependent functions and can either limit or enhance cell growth depending on the tissue (influenced by the driving TF and the stoichiometry of associated cofactors in that tissue), disease stage (early versus advanced disease) or dose  . While t


Alongside positive and negative symptomatology, deficits in working memory, attention, selective learning processes, and executive function have been widely documented in schizophrenia spectrum psychosis. These cognitive abnormalities are strongly associated with impairment across multiple function domains and are generally treatment-resistant. The DTNBP1 (dystrobrevin-binding protein-1) gene, encoding dysbindin, is considered a risk factor for schizophrenia and is associated with variation in cognitive function in both clinical and nonclinical samples. Downregulation of DTNBP1 expression in dorsolateral prefrontal cortex and hippocampal formation of patients with schizophrenia has been suggested to serve as a primary pathophysiological process. Described as a “hub,” dysbindin is an important regulatory protein that is linked with multiple complexes in the brain and is involved in a wide variety of functions implicated in neurodevelopment and neuroplasticity. The expression pattern of the various dysbindin isoforms  changes depending upon stage of brain development, tissue areas and subcellular localizations, and can involve interaction with different protein partners. We review evidence describing how sequence variation in DTNBP1 isoforms has been differentially associated with schizophrenia-associated symptoms. We discuss results linking these isoform proteins, and their interacting molecular partners, with cognitive dysfunction in schizophrenia, including evidence from drosophila through to genetic mouse models of dysbindin function. Finally, we discuss preclinical evidence investigating the antipsychotic potential of molecules that influence dysbindin expression and functionality. These studies, and other recent work that has extended this approach to other developmental regulators, may facilitate identification of novel molecular pathways leading to improved antipsychotic treatments. Schizophrenia is a complex psychiatric disorder that is the exemplar of a broader spectrum of psychotic illness in which interactive contributions from genetic and environmental factors play critical roles in etiology and pathobiology ; hereaftSeveral authors have recommended inclusion of cognitive impairment in formal diagnostic criteria for this illness, as well as highlighting the need for research on developing new and more effective treatments to enhance cognitive abilities therein . CognitiTwin studies continue to affirm a primary role for genetic factors in the etiology of schizophrenia , in assovia the cumulative effect of multiple common alleles, a landmark genome wide association study of more than 36,000 cases and over 113,000 controls identified 108 loci for common risk variants that achieved genome-wide significance  represent an aggregate measure of genetic risk as they consider the additive effects of all significant variation across multiple genes and regulatory areas across the entire genome . The PRSde novo and inherited, make only a minor contribution to population risk variation despite larger effect sizes (Copy number variants (CNVs), both rare ct sizes . In schict sizes . Genovesct sizes .More recent hypotheses suggest that the complex genetic architecture of schizophrenia may be better explained in terms of an “omnigenic” framework. This hypothesis  posits tSchizophrenia, Cognitive Impairment, and Genetics above), and that the GWAS focus on diagnosis and symptom severity scores is likely to lead to neglect for genes specifically linked with cognitive dysfunction in schizophrenia. Additionally, the expression of dysbindin-1 and its isoforms is disrupted in brain tissue from schizophrenia patients (see Schizophrenia Pathobiology and Dysbindin Isoforms below). Importantly for issues to be considered further below, variations in DTNBP1 associated with schizophrenia (SNPs and haplotypes) are located in intron or promoter regions and almost all are located in the N-terminus of the gene , a gene that has been linked with cognitive and anatomical endophenotypes in both patients with neuropsychiatric disorders as well as nonclinical samples . An initthe gene .Several studies have investigated the role of this gene in cognitive deficits of schizophrenia. For example, DTNBP1 haplotypes are associated with greater decline in IQ  and impaDysbindin expression levels vary across neuronal populations throughout the brain and are particularly abundant in the dentate gyrus of the hippocampal formation , with dySequence variation might be a mechanism by which the function of DTNBP1 differs between schizophrenia cases and controls. In this scenario, instead of a simple incorrectly encoded protein, the amount of DTNBP1 expression may be affected. Indeed, it has been shown that variations in DTNBP1 affect its expression in the human brain .The hippocampal formation plays a fundamental role in working and episodic memory, highlighting the possible role of dysbindin in cognitive processes that are believed to be critical to schizophrenia . Talbot Similarly to the hippocampal formation, the dorsolateral prefrontal cortex (DLPFC) is crucially involved in working and episodic memory, highlighting a further possible contribution of dysbindin to cognitive deficits encountered in schizophrenia . WeickerA wealth of epidemiological, clinical, and biological evidence now indicates that the origins of schizophrenia are to be found primarily in the early phases of fetal brain development: during this period a variety of factors, mainly genetic but also including environmental adversities, interact to disrupt brain morphogenesis ; thereafRegulation of brain development involves a multitude of genetic processes and associated proteins that act and interact sequentially to determine “normal” adult cerebral structure and function. Among these, dysbindin is one important regulator that is linked with multiple complexes in the brain and numerous diverse functions implicated in neurodevelopment and neuroplasticity. However, while the different dysbindin isoform proteins might interact with different partners, the majority of studies do not provide sufficient information to confirm or refute isoform-specific processes.Dysbindin promotes activation of Akt , which mDysbindin is a stable component of the multi-subunit complex BLOC-1  . In the One dysbindin protein has been demonstrated in drosophila, known as CG6865-PA. While the amino acid sequence of its coiled-coil domain is closely related to that of all known orthologs of dysbindin, neither its N-terminal nor its C-terminal region is closely related to corresponding regions in any vertebrate dysbindin ortholog. CG6865-PA shares 28% amino acid identity with human dysbindin-1A . Drosophvia a reduction in the protein Ebony, a glia-specific beta-alanyl biogenic amine synthetase involved in metabolic degradation of biogenic amines in the nervous system. This study reveals distinct functions of dysbindin in neurons and glial cells and highlights the potential of new therapeutics for schizophrenia that target glial cells  in homozygous sdy/DBA mice without abnormalities related to basal activity levels or anxiety . These aStudies involving the sandy mouse on a C57BL6 background also show cognitive deficits. As previously shown in sdy/DBA mice , homozygvia upregulation of D2R. Consistent with a dysbindin-associated increase in D2 receptors, dysbindin deficiency was associated with an antipsychotic-dependent increase in the ratio between the D2Short (D2S) and D2Long (D2L) isoforms in the PFC of mice, which is associated with potentiation of cortical presynaptic D2R signalling  allele of DTNBP1 . While svia gene-environment interactions -epicatechin on brain dysbindin levels in the mdx mutant, a genetic mouse model of Duchenne muscular dystrophy; such treatment partially reversed genotype-dependent reductions in protein levels of the dystrophin-associated protein complex as well as dysbindin protein in PFC . It was Investigation of transcriptional responses of developing hippocampal neurons in sdy/B6 mutants revealed not only the characteristic GABAergic interneuron deficiency but also changes in expression of the cation-chloride cotransporters NKCC1 and KCC2 during hippocampal development . NKCC1 aConsistent with the observed involvement of dysbindin in hippocampal and PFC glutamatergic function, a recent study investigated the impact of pharmacological enhancement of endogenous levels of brain-derived neurotrophic factor (BDNF) on dysbindin-related reduction of presynaptic calcium levels in PFC and social recognition memory in sdy/B6 mice . SystemiSimilarly, administration of CDPPB, a positive allosteric modulator of MGluR5, restored short-term recognition and spatial memory deficits (Morris water maze) in sdy/B6 mice . Given tExpression of SREBP1, a sterol regulatory element binding protein (SREBP) that regulates the expression of genes implicated in biosynthesis of fatty acids, cholesterol, triglycerides, and phospholipids, is reduced in sdy/B6 mice and in schizophrenia patients; activation of SREBP1 and Arc, a protein implicated in memory and cognition, is reduced in sdy/B6 mice and both of these deficits were restored by treatment with clozapine, suggesting a link with cognitive dysfunction .Given the indicated role for dysbindin-1 in the pathophysiology of schizophrenia and associated cognitive impairment, it will be heuristic to search for drug targets and molecules that might influence its expression and functionality. Those studies that indicate relationships between dysbindin-1 function, neuronal and behavioral processes associated with the pathobiology of psychotic illness and responsivity to current D2R antagonist antipsychotic drugs are particularly provocative in this regard; this is because they offer the prospect of clues to identifying non-DAergic mechanisms of antipsychotic activity. Importantly, these concepts generalize beyond dysbindin-1 to other developmental regulators. For example, in addition to identifying a role for dysbindin-1A in the regulation of schizophrenia-related behavioral processes, including specialised delay/interference-dependent working memory , we haveThe issues reviewed above constitute a substantive basis for such a search. Furthermore, they offer tantalising glimpses into mechanisms and putative target sites relating to DTNBP1/dysbindin-1A and how these concepts might generalize to a broader spectrum of developmental genes and regulatory proteins implicated in the pathobiology of schizophrenia spectrum psychosis.JW, XZ, and CO’T reviewed the relevant literature and wrote this paper. JW, XZ, and CO’T revised the manuscript. All the authors listed agreed to the publication of this paper.This work was supported by Science Foundation Ireland through grant 07/IN.1/B960.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.


Atraumatic vertebral compression fractures (VCFs) are commonly encountered in clinical practice and often represent a diagnostic challenge. MRI plays a major role in the differential diagnosis of benign and malignant VCFs, due to its high contrast resolution and the possibility to obtain quantitative and functional data with the employment of advanced sequences. Computer-aided diagnosis systems are also applied on MRI images for this purpose, showing promising results. In this setting, aim of this pictorial review is to elucidate the role of MRI in the differential diagnosis of VCFs with a specific focus on advanced and post-processing imaging techniques. Patients with atraumatic vertebral compression fractures (VCFs) may often represent a diagnostic conundrum, with specific regard to the elderly population. Indeed, VCFs are known to be caused by a broad spectrum of conditions, both benign and malignant, thus rendering hard the differential diagnosis. Osteoporosis and metastatic disease can be considered the most common benign and malignant causes of VCFs, respectively. While patient clinical history could help raising suspicion for a particular disorder, the characterization of VCFs is usually referred to diagnostic imaging. Among the available imaging modalities, MRI is increasingly gaining relevance in this field due to its high contrast resolution and the possibility to obtain both morphologic and functional information by means of advanced sequences as well as post-processing imaging techniques.Aim of this review is to elucidate the role of MRI in the differential diagnosis of VCFs with a specific focus on advanced and post-processing imaging techniques.1 Malignant VCFs often have total replacement of the high T1 bone marrow signal intensity resulting in diffuse homogeneous low signal and showing heterogeneous enhancement after contrast administration  could lead to false-positive and negative results, respectively. Six-echo Dixon MRI sequences have also been applied showing a good diagnostic accuracy of fat fraction and fat fraction ratio in the discrimination of osteoporotic and malignant VCFs, with area under the curve values of 0.98 and 0.95, respectively.4 MRI findings of benign and malignant VCFs are summarized in Chemical shift imaging is based on the different behavior in resonance frequency of hydrogen protons contained in water and lipids, providing therefore information about their concentration. Since malignant infiltrative processes tend to replace the fatty marrow components completely, the detection of fat content within bone marrow through a drop in signal intensity greater than 35% in out of phase images was proved to be useful to differentiate benign from malignant VCFs, although with lower diagnostic accuracy compared to other imaging techniques.Water molecules diffusion is usually increased in benign fractures due to bone marrow edema, while malignant fractures show diffusion restriction related to the high cellularity.5 In this setting, ADC maps calculated with a combination of low to intermediate b values  provided the best diagnostic performance to differentiate acute benign and malignant VCFs with a cutoff ADC < 1.7×10–3 mm2/s.6Besides qualitative evaluation, diffusion-weighted imaging (DWI) can also be quantitatively assessed by calculating the apparent diffusion coefficient (ADC) value. Several MRI sequences have been tested but, unfortunately, all published studies show a remarkable overlap between benign and malignant ADC values, mostly due to the presence of intravertebral hematoma in benign lesions. However, DWI may provide beneficial information in combination with conventional imaging improving sensitivity, specificity and accuracy.7 Among semiquantitative parameters peak enhancement, steepest slope and slope values were found significantly different between pathologic and osteoporotic VCFs, while conflicting results are reported regarding the role of time intensity curves pattern to discriminate the two entities.8,9Dynamic contrast-enhanced MRI is an advanced imaging technique assessing tissue perfusion and microvasculature through the extraction of semi-quantitative and quantitative parameters that reflect kinetics and hemodynamics vasculature features.7 Quantitative parameters resulted significantly different between malignant and benign VCFs, even if the available studies differ in terms of the kinetic model applied and perfusion parameters extracted. In detail, Vp and Ktrans resulted significantly higher in malignant as compared to benign VCFs, while interstitial volume and the total extracellular volume were significantly higher in benign as compared to malignant VCFs.10,11 A multiparametric evaluation of VCFs is illustrated in Differently from semi-quantitative measurements, quantitative parameters can describe tissue hemodynamic features on a biological basis, being able to depict alterations of the microvascular structure related to tumor neoangiogenesis that could lead to increased permeability and different contrast agent transfer between vascular and extravascular spaces.in vivo non-invasive imaging technique that allows a quantitative assessment of the biochemical structure within a specific tissue. Although showing early promising results, being able to determine vertebral lipid content in healthy controls, this technique shows a high between-subject heterogeneity, influenced by several factors including local environment and surface coil.12 For these reasons, also considering the availability of more feasible and accurate diagnostic tools, magnetic resonance spectroscopy is not routinely performed in the evaluation of patients with VCFs.Magnetic resonance spectroscopy is an 18F)flu-2-deoxy-d-glucose PET/CT has been widely used for detecting vertebral metastases in oncologic patients. Although malignant fractures are expected to demonstrate different accumulation patterns and a higher 2-(18F)flu-2-deoxy-d-glucose uptake as compared to benign fractures with a reported standard uptake value (SUV) threshold ranging from 3 to 4.5, high SUV values have been found in not tumor-related VCFs during the acute phase; a 3 months follow-up is recommended in such cases to demonstrate a normalization of SUV values.2,132- of 0.92 in distinguishing benign from malignant VCFs.14 TA and ML workflow is reported in Texture analysis (TA) quantifies the heterogeneity of an image by analyzing the signal intensity through gray level value distribution, allowing for the extraction of a great number of parameters. These can be analyzed through univariate and multivariate statistical tests or can be used in combination with machine learning (ML) classifiers, enabling computers to learn how to classify data without prior explicit programming. ML and TA have been proven useful in the differential diagnosis of benign and malignant VCFs. In this setting, the employment of statistical measures of gray levels, texture features, shape factors extracted from T1 weighted MR images were employed using a ML classifier and found useful in the classification of benign and malignant VCFs with an AUC value up to 0.95.15Fractal dimension provides a way to quantify the shape complexity of an object that represents self-similarity. Several attempts have been made in order to apply fractal analysis to the evaluation of different human body organs showing fractal geometry, including bone trabeculation. One of the major attractions of fractal features is that they are less influenced by randomness and roughness of medical images and thus suitable for the evaluation of MR images in which a certain degree of noise is often encountered. Similar to TA, fractal features extracted from A checklist summarizing conventional and advanced MRI findings to be assessed in order to characterize VCFs is shown in Conventional MRI plays an important role in the differential diagnosis of benign and malignant VCFs due to its superb contrast resolution. Furthermore, the possibility to employ advanced MRI sequences as well as to combine MRI findings with metabolic imaging may enable the depiction of biological processes underlying VCFs. Finally, promising results concerning the diagnostic accuracy of post-processing imaging techniques in discriminating benign and malignant VCFs could lead to the development of dedicated computer-aided diagnosis systems.


BRCA-mutated ovarian carcinomas (OC) are associated to have better response with platinum-based chemotherapy and long-term prognosis than non-BRCA-associated OCs. In addition, these mutations are predictive factors to response to Poly(ADP-ribose) polymerase (PARP) inhibitors. Different positioning papers have addressed the clinical recommendations for BRCA testing in OC. This consensus guide represents a collection of technical recommendations to address the detection of BRCA1/2 mutations in the molecular diagnostic testing strategy for OC. Under the coordination of Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH), these recommendations have been developed by pathologists and geneticists taking into account previously published recommendations and their experience in the molecular characterization of these genes. Since the implementation of BRCA testing as a predictive factor can initiate the workflow by testing germline mutations in the blood or by testing both germline and somatic mutations in tumor tissue, distinctive features of both strategies are discussed. Additionally, the recommendations included in this paper provide some references, quality parameters, and genomic tools aimed to standardize and facilitate the clinical genomic diagnosis of OC.Germline/somatic  TP53 mutations and about 50% have homologous recombination deficiency (HRD) due to alterations in genes involved in the homologous recombination DNA repair pathway [Ovarian cancer (OC), the most aggressive gynecologic malignancy, produced 42,704 deaths in Europe in 2012 (1). This high lethality can be attributed, among other factors, to its frequent late stage at presentation and limited treatment options. OC is a heterogeneous disease that includes five main histological types : high-gr pathway .BRCA genes: 9% germline mutations in BRCA1, 8% germline mutations in BRCA2, and 3% somatic mutations affecting one of the two genes [BRCA1/2 mutations (both somatic and germline) have been reported in 15% and 10% of EOC and CCC, respectively [In the TCGA cohort, 20% of HGSC carried mutations in wo genes . In addiectively .BRCA-mutated OC patients is important for the following reasons:BRCA germline mutation carriers (40% of patients with OC and pathogenic germline BRCA1/2 variants have no family history of breast or ovarian cancer).To identify BRCA-mutated OCs are associated with better response to platinum-based chemotherapy (the standard of care in patients with late-stage OC) and long-term prognosis than non-BRCA-associated OCs.Germline/somatic BRCA mutation is a predictive factor to response to PARPi.Germline/somatic Identification of BRCA1/2 mutations for all patients with non-mucinous ovarian cancer [Current recommendations for BRCA testing vary among European countries. Thus, Vergote et al.  observedn cancer .BRCA1/2 mutation testing should be offered to all patients with high-grade non-mucinous ovarian carcinomas. In addition, somatic testing should be considered in cases negative for germline mutations. At present, although the consensus recognizes the potential role of testing other HRD genes, its clinical implementation is still low [In Spain, a national consensus issued by the Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Medical Oncology (SEOM) recommended that germline till low .BRCA1/2 mutations in the molecular diagnostic testing strategy for OC. Under the coordination of Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH), these recommendations have been developed by pathologists and geneticists taking into account previously published recommendations [Provided that the clinical recommendations have been addressed by different positioning papers , 49, 66,ndations  and theiBRCA testing as a predictive factor is whether to initiate the workflow by testing germline mutations in the blood or by testing both germline and somatic mutations in tumor tissue. Distinctive features of both strategies are summarized in Table An important question regarding the implementation of Any pre-analytical factor that modifies the quality of the sample could potentially impact in the results , 33. A rBriefly, in tissue samples, the cold ischemia time (Cit), the period of time since tissue is obtained and its fixation, impacts on DNA, RNA, and proteins in large surgery specimens, like ovary tumors . To limiBlood samples should be collected in tubes with either EDTA or nucleic acids preserving agents, and processed immediately after its extraction, ideally in less than 30 min when tubes contain no preservatives  and follDNA extraction based on precipitation, magnetic beads, or silica-based membrane columns shows similar efficiency upon quality and concentration of the DNA obtained . HoweverMeasures for assuring full traceability of the sample and its derived products to avoid cross-contamination are critical. For tissue samples, precautions such as the preparation of free nucleic acid plastic containers, the use of PCR quality water for floating baths, and the systematic cleaning of devices with DNA and RNA removers  should bThe use of spectrophotometry (Nanodrop), fluorometry (Qubit), and/or PCR fragment  systems is recommended for the quantification and qualification of the DNA obtained, especially in tissue sample where formaldehyde and paraffin treatments produce the fragmentation of the DNA . IsolateLibrary preparation adapts the nucleic acids to be sequenced, including the fragmentation of the DNA by either physical (sonication) or enzymatic (endonuclease cocktail or transposase-mediated fragmentation) methods, adapter linkage, and the enrichment of the regions of interest (ROI). There are several strategies for ROI enrichment, and differences should be taken into account prior to the sequencing process. Amplicon-based methods are characterized by shortened and simplified preparation protocols also requiring smaller DNA inputs and no fragmentation step. This is especially relevant when working with small FFPE clinical specimens. This technology provides higher on-target values, while hybridization capture-based methods demonstrated higher uniformity rate. However both approaches have demonstrated technical and clinical utility in diagnostic procedures .Molecular barcodes are introduced to allow sample identification and combination in a unique sequencing reaction . After lBRCA1/2 testing is the most common praxis in clinical routine. Since they have been validated by the manufacturers, their implementation has turned out to be easier. BRCA1/2 genes are included in several NGS panels, such as BRCA panels in which only these genes are tested, hereditary cancer panels including other cancer-predisposing genes, and somatic cancer panels designed to detect oncogenic mechanisms in FFPE samples.The combination of sequencing platforms and commercially available sequencing kits offers a variety of options for next-generation sequencing (NGS) studies , 30, 37.BRCA1/2 genes should allow the identification of single nucleotide variants (SNVs) and small insertions-deletions (indels) in all coding exons and exon-intron boundaries, as well as CNVs, although the latest can be determined by other techniques such as multiplex ligation-dependent probe amplification (MLPA).In conclusion, the choice of the NGS panel for library preparation will depend on the type of study  and the sequencing strategy available in the institution . A good primary analysis consists of base calling and demultiplexing processes and is performed automatically by specific software on the sequencer. Base calling is the conversion of raw data to nucleotide reads, and the demultiplexing process separates data from each sample according to specific barcodes. A quality control of the run is also performed at this stage by the technical staff to verify the amount of the library pool loaded as well as the general sequencing process. Although there are different file formats to store primary analysis results, FASTQ and BAM are the most commonly used.Data analysis is performed after the sequencing process and can be divided into three stages: primary, secondary, and tertiary analysis. The secondary analysis can be done on the same sequencer or employing external pipelines or workflows [paired-end reads [Variant calling is the step where the variants (bases that differ from the reference genome) are identified for each sample. Variant calling format (VCF) is the standard file to store the information of variants (one line per variant). The variant information from different individuals can be stored in a single VCF file. Variant filtering criteria vary depending on the NGS platform and the enrichment system used, like the minimum number of reads covering the base, as well as the minimum average coverage and depth accepted for a variant. In the case of tumor samples, the variant allele frequency (VAF) varies in function of the tumoral cellularity present in the sample and the tumor proportion with the alteration [BRCA1/2 the most frequent variants observed are single nucleotide polymorphisms (SNPs), indels, single nucleotide variants (SNVs), and copy number variations (CNVs) [The orkflows . Qualitynd reads . The mosnd reads . Dependind reads . After and reads . Finallynd reads . Variantteration  (the minteration  with a cs (CNVs) . One lims (CNVs) .tertiary analysis consists of the functional annotation of the variants identified in the individuals and the selection of pathogenic variants . In this stage, the technical and biological information is integrated to interpret the variants detected through the sequencing process [The  process , 55.BRCA1/2 genes analysis, which is even more crucial when tumor samples are studied.Data analysis is the most complex and time-consuming stage of the entire NGS process; therefore, qualified personnel are required, either a geneticist or a molecular biologist appropriately trained to handle the complex classification of genetic variants across BRCA genes. In case of somatic mutation analysis, the importance of the pathologist expertise is stressed, since the impact of pre-analytical conditions, selection of the appropriate tissue fragment, and interpretation of the results in the light of microscopical appearance are crucial, providing an integrated molecular pathology report. BRCA1/2 genes have a large size, and many clinically relevant variants have been located throughout the whole coding region of the genes that have to be entirely analyzed. The quality of the DNA from FFPE samples is typically low, and the tumor percentage is variable; thus the variants can be identified at allelic frequencies less than 50%, leading to a complex identification of the variants, compared to peripheral blood samples. For all these reasons, the accurate pipeline validation and the staff experience play an important role in the evidence-based network for the interpretation of germline mutant alleles) ENIGMA consortium [BRCA1/2). The ACMG/AMP guidelines propose 28 classification criteria , categorize them according to their strength for a benign or pathogenic assertion, and propose rules to combine different criteria into a final five-tier classification of variants that reflects their probabilities of being clinically relevant  , 27, 54 nsortium  .Most genetic variants will be readily classified as  variant , 25. The variant , 47, 48  variant , which cvariants of unknown significance (VUS), which are typically missense or intronic variants. A high proportion of the former VUS in BRCA1/2 has been classified, thanks to international efforts that have allowed compilation of evidences and standardization of classification rules [BRCA1/2 variants classification based on multiple evidences from different sources combined into a Bayesian model to generate a posterior probability and validated by an expert panel [BRCA1/2 testing in France. It is important to highlight that other databases or levels of curation  might not be reliable. For that reason, variant interpretation requires expertise and should be performed by a specialist, avoiding automatic interpretations.We will focus here on the on rules , 52, 54.on rules , 46 thatrt panel : ClinVarrt panel  expert prt panel , includirt panel  compilesBRCA1/2 testing, Align GVGD has been calibrated to predict the probability for any missense change in these two genes to be pathogenic [Most variants remaining of uncertain significance after filtering are rare missense, synonymous, or intronic variants (MAF < 0.01). If pathogenic, missense variants are often assumed to impact the protein structure/function, but the possibility of the underlying nucleotide change leading to aberrant splicing should not be disregarded . Over 40thogenic .prior probability of any possible substitution in BRCA1/2 to be pathogenic due to an impact on splicing [BRCA1 exons 8, 9, 10, 13, and 14 and BRCA2 exon 12 and variants located in the donor site of BRCA1 exons 9 and 10 and BRCA2 exon 12 should be considered VUS. Align GVGD- and MaxEntScan-calibrated predictions are updated periodically [In silico tools predicting splicing alterations are far more accurate. MaxEntScan has been calibrated to predict the splicing . Due to odically  and haveonly PTC-NMD transcripts and/or transcripts lacking the coding sequence of critical functional domain, it can be classified as pathogenic. If RNA from carriers is not available, or if the splicing outcome observed does not permit a definitive classification, complementary studies performed by validated reporter minigenes might be useful.The most likely mechanism underlying the pathogenic nature (if any) of synonymous and intronic variants is aberrant splicing. A recent report by the ENIGMA consortium describes some aspects relevant to design, perform, and interpret adequately studies performed by RT-PCR in blood derived RNA from carriers. . If the BRCA1/2 genes, there is a broad international consensus to use the five-classes classification [The laboratory must determine which variants, according to its clinical significance, are informed and how the results will be communicated. For fication . All patThe report should contain the identification data of the patient and his/her diagnosis, the name of the physician requesting the test, and the reason for referral. Methodology of the diagnostic test performed and its scope should be accurately described  and include procedures and a quality manual, document control, define non-conformities (NCs) and corrective and preventive actions (CAPA), perform internal audits, and enhance customer satisfaction, but it does not necessarily include requirements of technical or analytical competence.Accreditation is defined by ISO as the “Procedure by which an authoritative body gives formal recognition that a body or person is competent to carry out specific tasks.” The standard developed by ISO most frequently used in medical laboratories is the ISO15189 and ensures technical competence of a laboratory to perform specific types of testing by complying with specific management and technical requirements. The accreditation process also considers the QMS, like certification, but it has additional formal requirements of technical competence, including initial and continuous training of personnel, validation of methods and instruments, as well as internal and external quality control [Entidad Nacional de Acreditación (ENAC).There is also a difference in the body that carries out the assessment and delivers the certification or accreditation certificate. Laboratories applying for ISO 9001 certification will be audited by a certification body, a third party that is accredited by an accreditation body. Each country has multiple certification bodies, but there is only one recognized national accreditation body (NAB) in each country that assesses laboratories against internationally agreed standards (Regulation (EC) No 765/2008). In Spain this is BRCA1/2 assessment, this is covered by the quality programs of the EMQN and GENQA.As part of the accreditation process, it is mandatory to provide internal quality controls (IQC) and to participate in external quality assessment (EQA). IQC is an internal verification that the test yields consistent results day after day and is defined as “the set of procedures undertaken by the staff of a laboratory for continuously assessing laboratory work and the emergent results, in order to decide whether they are reliable enough to be released” . EQA impBRCA assessment, this is of utmost importance as mutational assessment may not only include the detection of pathogenic variants but also VUS or benign variants, which should be correctly identified. For this reason, in recent years, some quality control assessments have been only based in the interpretation of the BRCA1/2 variants, without performing wet-lab testing.These programs evaluate not only the technical performance of producing analytical results from samples shared to all participating laboratories but also the accuracy of the report produced , as well as the interpretation of the genetic results. In the case of BRCA1/2 have been reported in approximately 10% of ovarian carcinomas, including both serous and non-serous histologies [BRCA1/2 mutations [RAD51C/D, BRIP1, and PALB2, should be considered in patient with ovarian cancer. Current assays of HR function, although promising, cannot be used to exclude patients from PARP inhibitor therapy [Germline or somatic mutations in HR genes other than tologies , 63. In utations . Finallyutations . In addiutations , 40. In  therapy .


Mesenchymal stem cells (MSCs) give rise to adipocytes, osteocytes, and chondrocytes and reside in various tissues, including bone marrow and adipose tissue. The differentiation choices of MSCs are controlled by several signaling pathways, including the Wnt/β-catenin signaling. When MSCs undergo adipogenesis, they first differentiate into preadipocytes, a proliferative adipocyte precursor cell, after which they undergo terminal differentiation into mature adipocytes. These two steps are controlled by the Wnt/β-catenin pathway, in such a way that when signaling is abrogated, the next step in adipocyte differentiation can start. This sequence suggests that the main role of Wnt/β-catenin signaling is to suppress differentiation while increasing MSC and preadipocytes cell mass. During later steps of MSC differentiation, however, active Wnt signaling can promote osteogenesis instead of keeping the MSCs undifferentiated and proliferative. The exact mechanisms behind the various functions of Wnt signaling remain elusive, although recent research has revealed that during lineage commitment of MSCs into preadipocytes, Wnt signaling is inactivated by endogenous Wnt inhibitors. In part, this process is regulated by histone-modifying enzymes, which can lead to increased or decreased Wnt gene expression. The role of Wnt in adipogenesis, as well as in osteogenesis, has implications for metabolic diseases since Wnt signaling may serve as a therapeutic target. Decades of research has established a main role of Wnt signaling in stem cell fate and control Nusse, . For exaAdipose tissue is a potential source of easily accessible MSCs  and Wnt/Calcium pathways. Wnt signaling plays important roles during embryonic development as well as in adults Nusse, . When WnSecreted Wnt inhibitors factors include Wnt inhibitor protein (WIF) and secreted Frizzled-related proteins (sFRPs). Dickkopf (DKK) is a secreted factor that binds and inhibits the LRP5/6 co-receptors  and brown adipose tissue (BAT). These are both derived from adipose-residing MSCs and are generated in a stepwise differentiation program. MSCs first differentiate into an intermediate cell type called preadipocytes, during which the cells undergo growth arrest and mitotic clonal expansion , resulting in the upregulation of the adipogenic transcription factors PPARγ and C/EBPα  marks the promoters of Additionally, the activity of epigenetic enzymes also affects how MSCs and preadipocytes respond to Wnt signaling. Histone Acetyltransferase GCN5 (GCN5), an enzyme that acetylates histone 3 on lysine 9 (H3K9ac), is known to prime the activation of Wnt genes, resulting in reduced adipogenesis , happens at the expense of adipocyte differentiation, and this bifurcation is regulated by Wnt signaling. Two distinct mechanisms are involved in skeletal bone development, intramembranous and endochondral ossification. The first is characterized by direct formation of osteoblasts while the latter proceeds through an intermediate step of chondrocytes transplantation. Although isolated human MSCs are self-renewing stem cells, they have limited doubling potential when cultured TCF7L2) (Chen et al., In vivo experiments with inactive mutations of TCF7L2 results in impaired adipogenesis characterized by increased subcutaneous adipose tissue mass, adipocyte hypertrophy, and inflammation. All mechanisms that are manifested when there is an excess of lipids. Moreover, this was accompanied by whole-body glucose intolerance and hepatic insulin resistance, a T2D hallmark (Kahn et al., Impaired Wnt signaling can lead to disease, and this is also true for mesenchymal tissues. Since mesenchymal tissue types are constantly renewed (Alfaro et al., Adipogenesis, the process of the formation of adipocytes from MSCs, is tightly regulated by signaling pathways. The Wnt signaling pathways are among these and have been proven to be crucial for MSC lineage specification. It is widely documented that canonical Wnt signaling regulates several steps in this differentiation process, but the precise underlying mechanisms remain poorly understood. In recent years, MSCs have been shown to be promising for use in regenerative medicine (Hoeppner et al., In adipose tissue, Wnt signaling promotes stem cell maintenance of preadipocytes. The stem cell niche of preadipocytes, however, does not solely consist of preadipocytes and adipocytes. Other cell types that reside in the cell type niche are fibroblasts, immune cells, endothelial cells, and a small fraction of MSCs. Preadipocytes can be found in the stromal vascular fraction of adipose tissue and this is believed to be in close proximity of the blood vessels (Tsuji et al., A limitation of many studies related to adipogenesis and MSCs biology is the use of non-human permanent cell lines 3T3-L1 and C3H10T1/2 (Ross et al., In conclusion, Wnt signaling mainly plays a role in suppressing adipogenesis during several stages of differentiation. It seems that the main role of suppression is to increase the MSC and preadipocytes cell mass. Epigenetic and transcriptional regulators have a significant impact on this process, but it is still poorly understood how transcriptional regulation and Wnt signaling interact during adipogenesis.TW performed the literature review, analyzed and interpreted the findings, and wrote the manuscript. RN supervised the writing process and edited the manuscript. All authors contributed to the article and approved the submitted version.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.


Influenza persists as a common communicable disease and remains a significant cause of disease burden across the world. Despite preventative therapies, such as influenza vaccination to reduce its spread and transmission, influenza continues to be a source of morbidity and mortality, even in developed countries. For the population over the age of 65, the effects of influenza virus may be more severe when they are compounded by pre-existing conditions and reduced natural immune function. In light of plateauing vaccination rates, a scoping review was conducted to map the literature and determine why seniors aged 65 and above refuse or fail to receive seasonal influenza vaccination. Nine peer-reviewed academic databases covering both social sciences and medical research were searched, along with the grey literature. A total of 6,562 references were identified; after the screening process, 118 references were included in the final review. Thematic analysis focused on the broad areas that positively or negatively influence older adults’ decision-making regarding influenza vaccination, and this resulted in five main themes: (1) barriers to obtaining vaccination; (2) social factors; (3) personal characteristics; (4) individual subjectivity; and (5) direct clinical interventions. This review aims to identify gaps in knowledge and synthesize currently available information to make recommendations for future research, policy development and clinical practice. Increasing the vaccination rate among Canadian older adults will contribute to ongoing efforts to reduce the spread of the influenza virus among the population, reducing influenza-associated hospital admissions and deaths.


Ozone mixing ratios in suburbs are usually higher than in the center of cities and may reduce photosynthetic productivity at a relatively higher degree. Consequently, carbon-based defense capacities of plants may be suppressed by elevated O3 more in the suburbs. However, contrary to this expectation, grazing damages by leaf beetles have been severe in some urban centers in comparison with the suburbs. To explain differences in grazing damages between urban areas and suburbs, the disruption of atmospheric communication signals by elevated O3 via changes in plant-regulated biogenic volatile organic compounds and long-chain fatty acids are considered. The ecological roles of plant volatiles and the effects of O3 from both a chemical and a biological perspective are presented. Ozone-disrupted plant volatiles should be considered to explain herbivory phenomena in urban and suburban systems.Plant–insect interactions are basic components of biodiversity conservation. To attain the international Sustainable Development Goals (SDGs), the interactions in urban and in suburban systems should be better understood to maintain the health of green infrastructure. The role of ground-level ozone  to authorized users. In addition to their ecological importance, pollinators provide great benefits to horticultural as well as to agricultural activities. Approximately 75% of crops humans depend on the activities of pollinators to maintain their productivity  is an indicator of physical defense to insect herbivory  and biotic  . They are classified into glandular and non-glandular trichomes, and provide a means for plants to defend against stresses, both abiotic  can induce positive effects, including enhanced photosynthesis, and improved photosystem functioning, resulting in higher leaf areas and/or enhancement of defensive capacities by trees and other plant species. These suggest that O3 effects on leaf quality is not a one-way but a two-way direction.An important development in understanding the mechanism is the recognition that plant responses to O3 leaves (over ambient O3 leaves) when birch leaves had low contents of condensed tannin and phenolics under elevated O3  of plants and insect grazing  alteration of BVOCs emissions (an effect on plant metabolism), and (2) post-emission disruption of BVOCs in the atmosphere. In the former, BVOCs emissions can be negatively or positively affected by elevated O3, potentially altering the behavior of insects that detect the BVOCs and orient to space. There are some tree species that significantly change their emissions due to O3, while others show little or no effect, depending on the volatile compounds , the emission of some volatile compounds were reduced at lower O3 levels but increased at higher levels , insects can be easily disoriented and wander away from their host plants in elevated O3 , sesquiterpenes (SQT), and others , show different physiological traits between early and late leaves  and alder (Alnus japonica (Thunb.) Steud) are positive controls; Japanese rowan (Sorbus commixta Hedl) and Korean mulberry  are negative controls that grow in the same area and time with positive controls in Hokkaido, Japan .By comparing BVOCs among tree species that pests commonly graze (positive controls), compounds of high importance for the attractant property can be found. There are numerous studies that have clarified the composition of BVOCs of targeted trees but only showed the BVOCs profile, being often difficult to refer to the relationship between BVOCs and insect behavior in detail  were attracted to LCFAs lauric, palmitic and oleic fatty acids, and red pumpkin beetle (Aulacophora foveicollis Lucas) to myristic, palmitoleic, α-linolenic, and nonadecanoic acids  and their composition can also regulate the behavior of insects via oxidation by elevated O3 based on a GC/MS analysis, shows the dynamics of LCFA composition in elevated O3. Previously, the relationship was studied by evaluating malondialdehyde (MDA), an indicator of lipid peroxidation , linolenic acid (C18:3), linoleic acid (C18:2), oleic acid (C18:1) and stearic acid (C18:0) can be relatively easily analyzed with standard samples.Comparisons between pre- and post-exposure to O3 exposure can disrupt plant–insect communication , which enables the detection of whether each compound is active on the insect’s antennae  from the other arm at the same flow rate, an insect has the task to move from the mouth of Y-shaped glass tube and select one of the two arms (Air A or Air B) to visit   in terms of time, effort and resources needed. New studies of plant volatiles with forest and avenue trees should be conducted to conserve healthy forest ecosystems in different environments facing the threat of elevated O3.Recently considerable research of O3 in the atmosphere, thereby altering the activity of insects before they arrive at the leaves and are affected by the chemical and physical foliage quality. This review serves as the basis to encourage further studies based on this insight centered on plant–insect communication via plant volatiles.In conclusion, biological communication via plant volatiles  between plants and insects in urban and suburban areas should be of concern. Traditional discussions were based on foliage quality , which was rightly assumed to directly affect pests, pollinators, and natural enemies. However, a new insight based on communication between plants and insects should be also considered. Plant volatiles as olfactory cues can be altered by air pollutants such as OSupplementary material 1 (DOCX 18 KB)Below is the link to the electronic supplementary material.


Breast cancer is the most common malignant tumor in women globally. Currently, due to limited data, there are no international guidelines for addressing the management of a large group of patients during infectious disease pandemics. Coronavirus disease 2019 (COVID-19), declared as a pandemic by the World Health Organization (WHO), has rapidly spread globally. The COVID-19 pandemic changed our daily routines and forced us to rethink the management of breast cancer patients. Clinicians need to take into account multiple factors such as the timing and delivery of cancer care, epidemic prevention and control, and the allocation of medical resources. Determining ways to reasonably adjust the treatment strategy is a real challenge. In this review, we aim to discuss particular challenges associated with managing breast cancer patients during the COVID-19 pandemic, share experience from Chinese oncologists and surgeons and propose some practical approaches to the management of early-stage breast cancer patients from a surgical standpoint. The emergence of COVID-19 has caused a global public health emergency. In December 2019, an outbreak of respiratory disease caused by a novel coronavirus first emerged and has now spread to more than 150 counties Breast cancer ranks first in the incidence of female malignant tumors in the world, and new patients with cumulative follow-up and adjuvant and salvage treatment continue to accumulate Accumulating evidence has suggested that cancer patients are at higher risk of COVID-19 infection and are more likely to have higher morbidity and mortality than the general population During the pandemic period of COVID-19, the principles of breast cancer treatment should be made scientifically and adjusted reasonably. If breast cancer patients present with a COVID-19 coinfection, the treatment should have an anti-pneumonia focus, and the patients should be admitted to a COVID-19-designated hospital The treatment of breast cancer should be based on the principle of nearby treatment For patients with difficulties in obtaining systemic treatment, we may make corresponding adjustments in the stages of adjuvant therapy, neoadjuvant therapy and even rescue therapy. For patients with difficulties in coming to the hospital, oral drugs are preferred Under the COVID-19 pandemic, patients should make an appointment to see a doctor in the hospital to ensure safety and protection from the disease and undergo specialized breast examination after COVID-19 screening. First, patients should undergo imaging examination, mainly mammography and ultrasonography; some patients may also need MRI examination. If the imaging classification is less than BI-RADS 4, we recommend regular follow-up and re-examination for 3 to 6 months According to the guidelines for breast cancer diagnosis and treatment, the indications for neoadjuvant therapy for breast cancer include stage II or stage III TNBC (triple-negative breast cancer) or HER2 -positive breast cancer, stage II or stage III HR (hormone receptor)-positive breast cancer that is expected to undergo adjuvant chemotherapy, and certain breast cancer patients who need better surgical treatment, i.e., patients who require shrinking of the tumor to be able to undergo breast-conserving surgery, those who need to achieve pathological complete response of the axillary metastatic lymph nodes to achieve axilla preservation and those who need to reduce tumor burden to achieve R0 resection During the COVID-19 pandemic, neoadjuvant therapy is recommended primarily for patients whose tumor diameters are larger than 2 cm or who are clinical node-positive. For breast cancer patients with small tumors who cannot receive surgical treatment within a limited time, the indications of neoadjuvant therapy can be appropriately broadened according to IHC (immunohistochemistry) results For HR-positive and HER2-negative breast cancer, neoadjuvant endocrine therapy should be considered first and aromatase inhibitors should be given priority, in addition, the use of ovarian function suppressants should be included in premenopausal women Patients who have completed the assigned neoadjuvant regimens should undergo the designated operation within 4 weeks, but in this special situation, patients for whom the therapy was effective can delay the operation by 2 to 4 weeks Currently, patients in China need to receive body temperature measurements, routine blood tests, lung CT, and IgG, IgM and nucleic acid testing for COVID-19 before admission for surgical treatment Breast cancer patients who have been diagnosed with or are suspected of novel coronavirus disease might postpone the administration of cancer treatment for safety reasons; especially patients who need to receive anti-coronavirus disease treatment, which is preferred. Patients who have contacted COVID-19 patients or suspected COVID-19 patients should receive lung CT, routine blood tests, IgG, IgM and nucleic acid testing, and isolate for 14 to 21 days In this special period, surgery for benign breast tumors is postponed. For malignant breast tumors, we should attempt to perform a relatively minimal operation with quick recovery. Due to the limited medical resources, we should adjust the treatment strategy for early breast cancer accordingly. For patients with primary tumors less than 2 cm and who are node-negative, surgical treatment should be actively performed when local medical conditions permit, preferably breast-conserving surgery and SLNB (sentinel lymph node biopsy), and if the patients refuse breast-conserving surgery, mastectomy and SLNB can be performed For postoperative follow-up, most patients recover well after breast surgery with few major side effects. Wound care is simple and patients will be taught and given specific instructions in the management of various types of wound coverage and the drain care. However, there are still some patients may have postoperative complications, based on this, most hospitals in China have set up specific wound care clinics to deal with postoperative complications during the pandemic. Postoperative return appointments usually take place 7-10 days after the surgery date and should be scheduled with surgical team after surgery. During the return appointment, the surgical team will check the wound recovery, review the pathology report and discuss the next treatment steps with breast cancer patients.2 IV day 1, doxorubicin 50 mg/m2 IV day 1, cyclophosphamide 500 mg/m2 IV day 1, cycled every 21 days for 6 cycles) chemotherapy regimens and dose-dense AC  followed by paclitaxel or weekly paclitaxel regimens should be avoided During the COVID-19 pandemic period, it is necessary to restrict the indications of adjuvant chemotherapy and avoid unnecessary chemotherapy. Oncologists should carefully weigh the advantages and disadvantages of chemotherapy, try to choose adjuvant regimens with a low risk of neutropenia, strictly calculate the dosage, and not exceed the standard recommended dose. Furthermore, TAC  and DFS  in breast cancer patients, and an interval between 60 to 90 days had no significant effect on OS; however, a delay of more than 12 weeks might significantly affect the prognosis of breast cancer patients Another important issue is the management of patients who have currently completed neoadjuvant therapy and are waiting for surgery. Although many studies have suggested that delayed surgical treatment could lead to poor prognosis in breast cancer patients, the delay between neoadjuvant therapy and surgery has been ignored by many prospective clinical trials of neoadjuvant chemotherapy The timing of adjuvant chemotherapy after breast cancer surgery is still controversial. The guidelines of the European Society of Oncology (ESMO) recommend that the best time for adjuvant chemotherapy is 2 to 6 weeks after surgery. The American Society of Clinical Oncology (ASCO) recommended that patients receive adjuvant chemotherapy no more than 120 days from the diagnosis of breast cancer. Although some studies suggest that the short-term delay of adjuvant chemotherapy will not affect survival, many studies have indicated as delay time increases, the benefit of adjuvant chemotherapy is reduced, affecting the prognosis of breast cancer patients Under the COVID-19 pandemic, patients with HR-positive breast cancer in some areas may face interruption or delay of adjuvant endocrine therapy. Retrospective analyses of compliance with endocrine therapy for breast cancer patients have indicated that early withdrawal or poor compliance of postoperative adjuvant endocrine therapy can increase the risk of recurrence and death, while insisting on endocrine therapy would achieve better long-term survival HER2-positive patients in some areas may face an interruption or delay of adjuvant targeted therapy. Although the best time for starting adjuvant trastuzumab treatment is still unclear, the HERA trial showed that the therapeutic effect of 2-year trastuzumab is not better than that of 1-year treatment, indicating that the best treatment period may be within 12 months in situ) who underwent breast-conserving surgery, and an interval of more than 12 weeks led to poor DFS and OS in patients with early-stage breast cancer who underwent breast-conserving surgery The impact of radiotherapy delay is different for patients with different stage or various surgeries. Retrospective studies showed that an interval of more than 12 weeks between surgery and radiotherapy resulted in a significantly higher risk of IBCR  in patients with DCIS (ductal carcinoma DCIS: In patients requiring radiotherapy for DCIS, radiation can be safely delayed up to 12 weeks after breast-conserving surgery.Invasive breast cancer: Patients with early-stage, node-negative, HR-positive breast cancer can safely begin radiotherapy 8 to 12 weeks after breast-conserving surgery without compromising disease control or survival, with several large studies showing that a delay of up to 20 weeks may be safe in an appropriate subset. There is limited evidence to guide the interval from chemotherapy to radiotherapy and extrapolation from the aforementioned literature suggests that an interval of up to 12 weeks from chemotherapy to radiotherapy may be reasonable Under extreme circumstances, it may be necessary to prioritize which patients with breast cancer can receive radiotherapy services. Prioritization of patients for whom radiotherapy is anticipated to provide a survival benefit is paramount. Based on available evidence and nascent clinical judgment, we have defined tiers of elevated priority, with prioritization made based on patient age, comorbidity, risk of exposure, and predicted benefit of radiotherapy.In conclusion, under the COVID-19 pandemic, breast oncologists should make scientific decisions and reasonable adjustments to minimize its impact on breast cancer treatment. Moreover, breast oncologists should protect patients from COVID-19 infection during breast cancer treatment and ensure the continuity of breast cancer treatment to the greatest extent. All treatment decisions should be made based on a multidisciplinary tumor board, and all treatment decision-making should balance the risks and benefits of treatment in the context of a specific pandemic level on a case-by-case discussion, always including patient preferences. Here we summarize the effective managements of breast cancer patients accumulated by Chinese breast oncologists during the COVID-19 pandemic and to provide guidelines according to the pandemic scenario in each country on how to triage, prioritize and develop diagnostic procedures, surgical, radiation and medical treatments for early-stage breast cancer patients.