N = 75).Individuals who report sexual identity-uncertainty are at-risk for heavyalcohol consumption and alcohol use disorder symptomology. The current studyexamined the impact of states of aversive self-focus on subsequentconsumption of ostensibly alcohol-containing beverages among a sample ofwomen in early adulthood with varying levels of sexual identity-uncertainty × 2 between-subjects design with 3 within-person assessments ofsalivary cortisol, both a moderation model and mixed-effects general linearmodel were tested.Utilizing a 2 (States of aversive self-focus caused increases in overall consumption amongwomen higher in sexual identity-uncertainty. Findings suggested consumptionof ostensibly alcohol-containing beverages was more likely among womenhigher in sexual identity-uncertainty who also reported consuming beveragesto cope with distress. Among women who reported higher levels of sexualidentity-uncertainty and drinking-to-cope motives, salivary cortisolconcentrations dampened more quickly over time, as they supposedly consumedalcohol.Findings demonstrate that, among women reporting sexual identity-uncertaintywho are motivated to consume alcohol to forget about troubles or worries,situations which evoke states of aversive self-focus may contribute todifferences in alcohol consumption in early adulthood. Sexual identity-uncertainty is positively related to alcohol misuse, even amongindividuals who may not self-identify as non-heterosexual. Experimental findingsshow that, among individuals with sexual identity-uncertainty, situations thatexplicitly focus on their sexuality contribute to bias in approachingalcohol-related cues , as well as greaterconsumption in an ad libitum drinking task. The current study extended prior work by testing whether: (a) general statesof aversive self-focus also lead to greater consumption of an ostensiblyalcohol-containing beverage among women in early adulthood who report sexualidentity-uncertainty, (b) the aforementioned effect would be more robust among thosemotivated to drink-to-cope with psychological distress, and (c) a biophysiologicalstress response, namely reductions in salivary cortisol, would support thereinforcing effects of such motivated consumption.Alcohol misuse is often observed among youth and adults who report being uncertain orunsure about their sexual orientation.) and the timing of developmental milestones is typically later for women,compared to men . Individuals who report being “unsure” of their sexual identity areoften engaged in exploring various aspects of their sexuality and may be more likely to identify as nonmonosexual . Individuals expressing sexual identity-uncertainty often reportperceived pressure to adhere to the presumed sexual orientation binary . As a result of external and internalized pressures,sexual identity development and periods of relative sexual identity-uncertaintyare theorized to be important identity-related contributors to alcohol misuse.Individuals with diverse sexual identities report various trajectories withregard to sexual identity development  source of minority stress that may contribute to alcohol misuse. Proximal minority stressors are a specific, self-directedform of sexual stigma, promulgated by heterosexist norms and assumptionsinherent in the larger culture, which can tax coping resources. Sexualidentity-uncertainty is expected to contribute to psychological distress, atheorized mechanism  that accounts, in part, for differences in alcohol use and misuse insexual minority populations. Uncertainty-identity theory14 alsoprovides a general motivational account of how social-identity processes canlead to risk-taking behaviors, including alcohol misuse. Specifically, whenindividuals feel uncertain about core aspects of their identity, such as theirsexual orientation, they are believed to enter an aversive psychological state,akin to cognitive dissonance , and are motivated to reduce psychological and physiological stressresponses stemming from their identity-uncertainty (see).Within the minority stress framework,) through negative reinforcement, especially as drinking behavior becomesmore compulsive . Specifically, when self-relevant aspects of the environment aresalient, a self-evaluative process is initiated, which potentiates states ofaversive self-focus when individuals are confronted with aspects of the selfthat are deemed unfavorable . Alcohol consumption istheorized to inhibit states of aversive self-focus by indirectly reducing one'sawareness and encoding of self-relevant “sources of tension,” . Sourcesof proximal stress, including sexual identity-uncertainty, catalyzed byanti-bisexual experiences and heterosexist norms that oblige binary sexualorientations , may contribute to psychological tension for women withsexual identity-uncertainty.Within drinking contexts, alcohol is expected to alleviate aversive psychologicalstates . Individual differences in the tendency to drink to cope with distresscan exacerbate risk of alcohol misuse among adults expressing sexualidentity-uncertainty , whereby heavy drinkingis used as a means to cope with feelings of psychological distress, in part, bydampening one's physiological stress response. Such accounts of motivateddrinking are consistent with general models of addiction  as well as those that offer intraindividual, situational accounts ofrisk for drinking behavior among at-risk individuals .Alcohol consumption may alleviate self-relevant sources of tension brought on byincreased self-focus among individuals with concealable minoritized identities , in predicting consumption of andphysiological responses to alcohol has received attention.25 The HPAaxis regulates release of cortisol, and baseline axis activity is critical forhomeostasis. This axis also aids response to and recovery from stressors and impacts health. Cortisol output, over time, has been used to examine stress responsivityin sexual minority samples in response to acuteidentity-specific and socio-evaluative stressors . Exposure tominority-identity stressors relates to increased cortisol and other stressresponses, which are linked to health disparities in at-risk sexual minoritywomen . Theoretical perspectives argue that individuals reportingidentity-uncertainty who encounter states of aversive self-focus may show agreater cortisol stress response , which is expected to bedampened, or reduced, following the ostensible consumption of alcohol, throughnegative reinforcement processes .Addiction science has sought to identify neurobiological mechanisms potentiatinga person's vulnerability to alcohol misuse.). As argued previously, sexual identity-uncertainty functions as apotential source of proximal stress . This source ofinternalized stress may contribute to a greater sensitivity to self-relevantsources of psychological distress for some sexual minority individuals,especially nonmonosexual women, and relate to unique physiological stressresponses .Individuals expressing sexual identity-uncertainty are expected to encounter bothgeneral states of aversive self-focus as well as those brought on byidentity-specific stressors, which tax psychological resources ,particularly among women in early adulthood. Understanding how aversiveself-focus predicts drinking behavior is important because sexualidentity-uncertainty is associated with alcohol-related health disparities,especially among women in early adulthood. Using a sample composed entirely ofself-identified women drinkers between the ages of 21 and 35, we examinedconsumption behavior in a double-blind alcohol administration experiment. At thebetween-person level, we predicted: 1) women with higher levels of sexualidentity-uncertainty would show greater consumption of an ostensiblyalcohol-containing beverage after exposure to states of aversive self-focuscompared to their counterparts in neutral settings, and 2) women experiencingsexual identity-uncertainty who report a higher likelihood of drinking to copewith stress would consume greater amounts in response to general states ofaversive self-focus. Within-person, we expected that greater consumption amongwomen with heightened sexual identity-uncertainty who reportedly drank to copewith stress would relate to a more robust dampening of salivary cortisol levelsover time.Evidence is accumulating to support that sexual identity-uncertainty isassociated with alcohol-related health disparities . Women were told they would be engaging in a verbalfluency task and then rating the appeal and taste of “alcoholic beverages.”Women who reported drinking at least one alcohol-containing beverage in the pastthree months and who were between the ages of 21 and 35 were recruited. SeeParticipants were self-identified women who agreed to be part of an experimentalstudy ostensibly examining “Verbal Fluency and Taste Preferences.” Participantsagreed to provide saliva throughout the experimental study. The first between-subjects factor manipulated aversive self-focus. The presence of amirror or audience is a well-established means of evoking heightened states ofself-focus in experimental studies.Supplemental Materials). To prime states of aversiveself-focus during this verbal task, participants in the experimentalcondition (n = 45) were seated in front of the reflectivesurface of a large (3′ × 5′) mirror. Participants in the control conditionwere asked to complete the same solvable vocabulary and anagrams questions;however, they were seated in front of the non-reflective surface of thelarge mirror (n = 30). We did not anticipate the completionof these solvable anagrams to be particularly aversive on their own, asquestions were not graded and all were solvable, corresponding to ahigh-school level of difficulty. In a subset of participants, a briefcomment followed the verbal task and conveyed that the room often madepeople feel uncomfortable. We included an indicator of whether or not thiscomment was made as a covariate in the primary models.All participants were asked to complete a paper-and-pencil task consisting ofvocabulary and anagram questions ( The amount consumed by each participant (ml) was determined bymeasuring the amount of beer left in the bottles, subtracted from theinitial amount given.During the taste-test portion of the study, each participant was providedwith three de-identified 12 oz. bottles of ostensibly alcohol-containingbeer, labeled ‘A’, ‘B’, and ‘C’ respectively. All participants were toldthey were receiving alcoholic beverages and given 20 min to taste and ratethe beer, with three additional beers provided upon requested. Participantsanswered questions regarding the taste and appeal of the drinks. - higher scores indicate greater acknowledgement that aspects ofone's sexual orientation are uncertain. Ten items  were rated on a Likert-type scale that ranged from 1(‘Strongly Disagree’) to 4 (‘Strongly Agree’). Internal consistency was goodin the current sample (α = .87).Sexual identity-uncertainty was measured with the Sexual OrientationSelf-Concept Ambiguity (SSA) scaleFor each of three beers that were part of the taste test, one item asked towhat extent the person could anticipate drinking the beer “to forgettroubles or worries.” The three items  weremeasured with a six-point Likert-type scale .To improve reliability, the rating for Beer “B” was dropped, and the othertwo items were averaged to create a score reflecting the extent to whichparticipants reported being motivated to drink to cope with distress(α = .74). Participants provided saliva samples throughout the lab session usingthe passive drooling method (0.5 ml per collection). The first salivacollection occurred at the start of the session, prior to any experimentalmanipulation and following a 10-min resting period . Subsequently, a saliva collection occurred after the verbal-fluency task,reflecting stress responses occurring during the first five minutes of thetask. Finally, the last saliva collection occurred at the conclusion of thetaste test , reflecting stress responses during thefirst five minutes of tasting of ostensibly alcohol-containingbeverages.Blood levels of cortisol rise within 3–10 min of stressor onset, andcorresponding salivary cortisol increases shortly afterward, peaking around20–22 min after exposure to psychological stress. The standard curve ranged from 0.012 (∼92% bound) to 3.00 µg/dl (∼7%bound). Sample duplicate coefficient of variation (CV) values were all under10%. High and low kit controls were run in each assay plate, average intra-and inter-assay CVs were 10.4% and 15.2%, respectively. Cortisolconcentration was recorded as micrograms of hormone per deciliter of saliva(µg/dl); data were log transformed prior to analysis to correct positiveskew.Salivary samples were assayed in duplicate using a commercially availableenzyme immunoassay kit according to the manufacturer's instructions, as we have done previously.M = 5.33,SD = 1.61; year of sample recruitment, 0 = prior toCOVID-19 pandemic [2018], 1 = COVID-19 onset [2020]).Covariates were selected because they were relevant for alternativeexplanations of levels of salivary cortisol  or consumption behavior  whoacknowledged same-sex sexual attractions, or (c) who reported any history ofsame-sex sexual behavior, to acquire greater variation in SSA scores. Allpersons who completed the pre-screening survey were compensated with a $5 giftcard to Amazon.com. Women who expressed any degree of SSA, as indicated bynon-zero SSA scores , or any women who reported a non-exclusively heterosexual identity (54%)or any degree of same-sex attraction (64%) or behavior (28%) in their lifetimewere invited to participate.The pre-screening survey assessed SSA scores and sought to oversample women (a)who did not identify as ). Different experimenters were used for each task in the study such thatthe experimenter leading the taste-test portion of the study was blind to theparticipants’ assigned experimental condition.At the start of the session, participants verified eligibility and adherence topre-experimental protocols . All relevant federal and institutionalresearch ethical standards were met with regard to the treatment ofparticipants.Because we were interested in whether aversive states of self-focus precipitatedthe Prior to their inclusion in models, variable scores that were non-normal weretransformed to improve normality . Bivariateassociations, as well as observed means and standard deviations, are in  for SPSS, which allows for the calculation of regression effectsusing 10,000 bootstrapped samples to derive coefficient estimates andconfidence intervals. PROCESS estimates simple slopes, for continuousscores, at the 16th, 50th, and 84th percentiles of each respectivemoderator, to ensure estimates are derived from observable data. Covariatesincluded session time-of-day, presence of room comment, and liking of thetaste-test beverages.A linear regression model tested moderation hypotheses to predict overallbeverage consumption. Moderation hypotheses were tested using the HayesPROCESS MacroGiven our expectation that women who reported higher levels of sexualidentity-uncertainty and were assigned to the aversive self-focus conditionwould consume more beverages than in the control condition, we first testeda two-way interaction between condition and sexual identity-uncertainty(SIU) scores see . There wIn a test of the three-way interaction hypothesis, we expected that women whoreported higher levels of SIU and drinking-to-cope motives would consume thegreatest amount of alcohol in the experimental condition, compared to thecontrol condition. At average and even higher levels of drinking-to-cope, the respective interactions between experimentalcondition and SIU scores in predicting consumption were significant .Decompoβ = −.02, SE = .01,p = .009, 95% CI , see A mixed-effects general linear model (with a random intercept) was used totest hypotheses regarding within-person changes in salivary cortisolconcentrations across the laboratory session among those with varying levelsof sexual identity-uncertainty, see decreases insalivary cortisol concentrations, from the beginning of the experiment tothe taste-testing session, b = −.083,SE = .014, p < .001, 95% CI , suggesting the ostensible consumption of alcohol-containingbeverages dampened salivary cortisol output over time. Among those withlower levels of sexual identity-uncertainty, there were also reliabledecreases in cortisol response over time for thoselower, b = −.034, SE = .015,p = .023, 95% CI  and higher,b = −.033, SE = .016,p = .044, 95% CI , in drinking-to-copemotives, but these effects were smaller in magnitude.Changes in salivary cortisol throughout the session, in each of the randomlyassigned conditions, depended on participants’ SIU scores anddrinking-to-cope motives. Women who were higher in sexualidentity-uncertainty  and reported higher levels ofdrinking-to-cope with distress showed robust 42) and compeldrinking behavior motivated by efforts to alleviate psychological discomfort. Thepattern of effects was supportive of hypotheses and consistent with theorizing thatsexual identity-uncertainty may serve as an internal source of minority stress, particularly for individuals with nonmonosexual sexual identities. Women withheightened sexual identity-uncertainty are expected to regularly encountersituations characterized by states of aversive self-focus, given ubiquitous sexualstigma and discrimination from the larger society . When encountering situations that evoke states of aversive self-focus andthe opportunity to drink presents itself, women with heightened sexualidentity-uncertainty who typically use alcohol to cope with distress may be at-riskto consume greater amounts of alcohol.Situations which evoke aversive states of self-focus among adults with higher levelsof sexual identity-uncertainty are expected to catalyze cognitive dissonanceprocesses  and other research linking sexual identity-uncertainty to risky alcoholuse,4 the currentwork extends prior work on approach motivations toward cues related to alcoholic,versus neutral, beverages in response to sexual identity-specific stressors by examining biopsychosocial stress responses to general self-relevantstressors. Prior work also suggests that elevations in distress, resulting fromstates of aversive self-focus among those with sexual identity-uncertainty, may bemoderated by multiple factors, including identity-related factors that wentunmeasured in the current report . Other psychosocial variables, such as alcohol expectancies , may also alter theactual or perceived effects of stress on alcohol and relate to drinking as a meansof negative reinforcement.Consistent with theory . This isone of a few experimental studies to show that general states of aversiveself-focus can also cause increased consumption among women with sexualidentity-uncertainty and result in biophysiological changes in one's stressresponse. Results are also consistent with findings showing that distal  LGBT-specific stressors positively relate to acutesalivary cortisol response. Third, the current sample was composed entirely of self-identified women.Theoretical relations may be distinct based on gender or sex assigned at birthin that individual differences in stress-based hormonal systems may relate todistinct changes in salivary cortisol concentrations depending on consumption. Future research should seek to replicate these effects amongindividuals of different genders who are experiencing sexualidentity-uncertainty. Finally, among gender minority adults, mirrors can be “triggering” and evoke distress due to gender dysphoria, or negative feelings spurnedby a perceived mismatch between a person's gender identity and how others mayview them. In addition, transgender and gender non-conforming adults may experienceheightened body shame or otherwise respond uniquely. Although women in the current work were not asked about their genderidentity, future work examining this process within samples of transgender andgender non-conforming adults may use other means of evoking self-focus, such asthe presence of an audience.Findings have notable limitations. First, despite enhanced statistical powerprovided by within-person repeated-assessments, the current sample was somewhatsmall. Nevertheless, effect sizes are small-to-moderate, suggesting effects arereliable. Second, the experimental manipulation was subtle and intended to evokegeneral states of aversive self-focus. Prior work has supported the theorizedrelation between exposure to identity-) and beverages contain actual ethanol . Studies thathave examined the impact of ethanol consumption on the HPA response, show thatethanol decreases the HPA axis response to stress. More data onthe role of acute, voluntary alcohol consumption on the HPA axis response toproximal and distal sources of minority stress in various social settings amongsexual minorities or those experiencing sexual identity-uncertainty are needed.The current work has relevance for clinical settings, as internalized sources ofminority stress may be alleviated by greater societal support for exploration ofnonmonosexual sexual identities. Policymakers and community stakeholders could assist in improving alcoholmisuse in this population by encouraging bipositivity, refuting and dismantlingstereotypes pertaining to sexual identity-uncertainty, as well as nonmonosexualidentities, and enacting policies that disallow discrimination on the basis ofidentifying with diverse sexual identities.Findings contribute to a growing literature that examines biopsychosocialpredictors of alcohol consumption and misuse among at-risk populations,including persons expressing sexual identity-uncertainty. Associations shown inthe current report are expected to be even more robust when identity-specificstressors are encountered (eg,Click here for additional data file.Supplemental material, sj-docx-1-css-10.1177_24705470221118308 for AversiveSelf-Focus and Alcohol Consumption Behavior in Women with SexualIdentity-Uncertainty: Changes in Salivary Cortisol Stress Response Among Thosewho Drink-to-Cope by Amelia E. Talley, Breanna N. Harris, Tran H. Le and ZacharyP. Hohman in Chronic Stress


The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology.We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs).Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/μl HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients  by the above 5 methods.We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance.The online version contains supplementary material available at 10.1007/s12072-022-10311-0. Hepatitis B virus (HBV) infection continues to be a major health burden worldwide. The global HBV chronic infection is approximately 257 million people . EliminaIn clinical treatment, functional cure of HBV infection refers to the disappearance of sustained HBsAg with or without the presence of anti-HBs, while complete cure is defined as eradication of HBV covalently closed circular DNA (HBV cccDNA) , 4. ViraDespite the critical role of cccDNA in the chronicity and duration of HBV infection, there is a lack of effective and accurate detection methods to monitor cccDNA. There are two characteristics that limit the detection of HBV cccDNA . The firThe clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system is an acquired immune system for the cleavage of foreign genetic elements from invading viruses and phages and was first identified in bacteria and archaea . ImportaHere, we established a high-sensitivity and high-specificity HBV cccDNA detection method using the CRISPR-Cas system combined with RCA and PCR methods. For a more comprehensive evaluation, we collected clinical samples that were detected by quantitative real-time PCR (qPCR), droplet digital PCR (ddPCR) and our CRISPR-based cccDNA assay.Liver samples were collected from 24 patients with HBV-related Hepatocellular carcinoma (HCC), 6 patients with HBV-related Liver cancer (LC), 10 patients with CHB and 3 individuals with normal livers. Normal liver specimens were collected from hepatic resection for liver transplantation. CHB samples were obtained from patients undergoing liver puncture biopsy. LC and HCC liver samples were obtained from the livers of patients with HBV infection undergoing liver transplantation. Whole blood, plasma, and peripheral blood mononuclear cells (PBMCs) were collected from 24 patients with HBV affected by different HBV viral loads and 3 healthy subjects. The study was approved by the medical ethics committee of Beijing Youan Hospital, Capital Medical University, and written informed consent was obtained from each patient. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki declaration of 1975, as revised in 1983.t-test and Fisher’s exact test. All statistical tests were two-sided, and p < 0.05 was considered statistically significant.The HBV cccDNA results are expressed as copies/µL (template concentration). Means and standard deviations were calculated by GraphPad Prism software version 8.0 . Mean differences in quantification were determined by paired Other methods in supplementary materials.In this study, we established a novel strategy named the CRISPR-based cccDNA assay for the highly sensitive and specific detection of HBV cccDNA. As illustrated in Fig. HindIII and PSAD   that were specific to different sites in the same HBV cccDNA amplification region . HBV cccDNA was not detected by any method in the samples of 3 healthy people, while in 40 HBV-associated patients, 18 HBV cccDNA-positive samples were detected by ddPCR , 4 samples were detected by qPCR , 14 samples were detected by PCR-CRISPR , 18 samples were detected by RCA-qPCR , and 29 samples were detected by RCA-PCR-CRISPR  (Supplementary Table 1). The Venn diagram compares several detection methods and the Cas13a-based detection assay further enhanced the positive rate of HBV cccDNA in 40 liver tissue samples of clinical patients Fig. a–c.Fig. Then, we tested HBV cccDNA in plasma, whole blood and PBMC samples of 24 HBV-associated patients and 3 healthy individuals with qPCR, PCR-CRISPR, RCA-qPCR, and RCA-PCR-CRISPR methods. HBV cccDNA was detected by ddPCR in one plasma, three whole blood and one PBMC sample, by RCA-qPCR in two whole blood and one PBMC sample, and by RCA-PCR-CRISPR in two whole blood and one PBMC sample (Supplementary Table 2). The results suggested that the RCA-PCR-CRISPR has a high positive detection rate for HBV cccDNA; moreover, HBV cccDNA was rarely detected by these methods in the plasma, whole blood and PBMC samples of HBV-associated patients.In this study, we developed a CRISPR-based detection method for HBV cccDNA by a method combining RCA and PCR called the CRISPR-based cccDNA assay. Compared with the existing HBV cccDNA detection methods, CRISPR-based cccDNA detection has theoretically significant sensitivity and specificity because it has the following characteristics: (1) the “collateral effect” of Cas13a further increases the amplified signals of RCA and PCR to improve the detection sensitivity, and (2) the crRNA design combine with Hind III digestion and PSAD-treated sample processing and cross-gap region primer design of PCR to further improve the specificity of detection. Therefore, the CRISPR-based cccDNA assay has enhanced sensitivity and specificity for detecting HBV cccDNA.To improve the treatment of patients with HBV infection, we need better methods for detecting HBV cccDNA. In 2011, a study reported the use of nested qPCR to quantify cccDNA in PBMCs and bone marrow mononuclear cells (MMNCs); 42% of MMNC samples and 36% of PBMC samples tested positive for HBV cccDNA . In thisHBV cccDNA is well known to exist in HBV-infected liver cells. However, whether HBV cccDNA exists outside the liver has caused great controversy. Several reports have shown that HBV cccDNA was detected in plasma samples of patients and had a significant correlation with HBsAg titer . Neverth6–101 HBV DNA copies). We need to continue to explore the linearity of Cas13 detection. The approach involves two-step RCA amplification and PCR transcription, which are slightly complicated and need to be further optimized. Additionally, the liver tissues, plasma, whole blood and PBMCs of HBV-associated patients were detected by this novel method, which possesses high sensitivity for liver tissue detection. However, the plasma, whole blood and PBMC samples and liver tissue samples were not from the same patients, preventing the observation of a clear relationship between liver tissue and extrahepatic HBV cccDNA. Further experiments are required to investigate this issue.This study had several limitations. Although our CRISPR-cccDNA assay detects HBV cccDNA with high specificity and sensitivity, cccDNA quantification is not linear compared with qPCR and ddPCR Below is the link to the electronic supplementary material.


Patients with ventricular tachyarrhythmias (VT) are at high risk of sudden cardiac death. When appropriate, catheter ablation is modestly effective, with relatively high VT recurrence and complication rates. Personalized models that incorporate imaging and computational approaches have advanced VT management. However, 3D patient-specific functional electrical information is typically not considered. We hypothesize that incorporating non-invasive 3D electrical and structural characterization in a patient-specific model improves VT-substrate recognition and ablation targeting.In a 53-year-old male with ischemic cardiomyopathy and recurrent monomorphic VT, we built a structural-functional model based on high-resolution 3D late-gadolinium enhancement (LGE) cardiac magnetic resonance imaging (3D-LGE CMR), multi-detector computed tomography (CT), and electrocardiographic imaging (ECGI). Invasive data from high-density contact and pace mapping obtained during endocardial VT-substrate modification were also incorporated. The integrated 3D electro-anatomic model was analyzed off-line.Merging the invasive voltage maps and 3D-LGE CMR endocardial geometry led to a mean Euclidean node-to-node distance of 5 ± 2 mm. Inferolateral and apical areas of low bipolar voltage (<1.5 mV) were associated with high 3D-LGE CMR signal intensity (>0.4) and with higher transmurality of fibrosis. Areas of functional conduction delay or block  were in close proximity to 3D-LGE CMR-derived heterogeneous tissue corridors. ECGI pinpointed the epicardial VT exit at ∼10 mm from the endocardial site of origin, both juxtaposed to the distal ends of two heterogeneous tissue corridors in the inferobasal left ventricle. Radiofrequency ablation at the entrances of these corridors, eliminating all EDPs, and at the VT site of origin rendered the patient non-inducible and arrhythmia-free until the present day (20 months follow-up). Off-line analysis in our model uncovered dynamic electrical instability of the LV inferolateral heterogeneous scar region which set the stage for an evolving VT circuit.We developed a personalized 3D model that integrates high-resolution structural and electrical information and allows the investigation of their dynamic interaction during arrhythmia formation. This model enhances our mechanistic understanding of scar-related VT and provides an advanced, non-invasive roadmap for catheter ablation. Catheter ablation of arrhythmogenic substrate and triggers of ventricular tachycardia (VT) reduces arrhythmia burden and implantable cardioverter-defibrillator (ICD) shocks , 2 but rRecent advances in the field of catheter ablation for scar-related VT have primarily focused on defining target locations for ablation through pre-procedural characterization of the ventricular scar architecture by combining imaging techniques [such as computed tomography (CT) or cardiac magnetic resonance imaging (CMR)] with electrocardiogram (ECG)-VT algorithms. Image-guided or -aided substrate ablation may increase long-term success, decrease recurrence rates and reduce procedural duration , 8. ImagIn this study, we combined ECGI with high-resolution 3D dark-blood late gadolinium enhancement (3D-LGE) CMR and cardiac CT in a case with ischemic cardiomyopathy and recurrent monomorphic VT, providing a patient-specific structural-functional model to improve mechanistic understanding and to provide a road map for future personalized catheter ablation for VT. Additionally, we examined the accuracy of non-invasively determined parameters compared to standard invasive VT-substrate characteristics.We constructed a personalized 3D cardiac model that integrates the individualized structural-functional characteristics of this case to identify mechanisms of arrhythmia formation and to develop an individualized framework for future VT ablation guidance.The model incorporates (1) 3D dark-blood LGE CMR and cardiac CT delineating the myocardial anatomy and scar ; and (2) functional-electrical aspects by non-invasive ECGI. Model outcomes were compared to data from invasive electro-anatomical mapping (EAM), and arrhythmia mechanisms were investigated.A 53-year-old male was admitted to our hospital for electrical storm due to recurrent hemodynamically tolerated, sustained monomorphic VTs with an inferobasal to mid, septal left-ventricular (LV) origin  11). He. He11). The patient underwent a free-breathing 3D-LGE CMR with high isotropic resolution  and dark-blood nulling  on a 1.5Cardiac magnetic resonance-image processing was performed using a research software tool  . Pixel-sPrior to the VT ablation procedure, an ECG-gated helical dual-source CT scan  using intravenous iodine contrast medium was performed during breath-hold, to visualize cardiac geometry. Additionally, the patient underwent a low-dose thoracic CT scan to locate the body-surface electrodes, required for ECGI.The ECGI was performed as previously described and validated , 18. BriEpicardial ventricular unipolar electrograms (UEGs) were reconstructed for selected beats through previously detailed methods . For eacThe patient underwent continued body-surface potential mapping during the VT ablation procedure . The proThe geometries from 3D-LGE CMR, CT, ECGI and invasive EAM were manually aligned and registered digitally in 3D Slicer . The mosInformation from 3D-LGE CMR and ECGI was combined to examine triggers, substrate, and their interactions. 3D-LGE CMR scar delineation was compared to areas of low invasive bipolar voltage, by finding the corresponding nearest neighbors of the CMR anatomy and the invasive electroanatomic anatomy. Any neighbors with a corresponding distance of >10 mm from each other were disregarded. The colocalization of endocardial EDPs with MRI-derived heterogeneous tissue corridors was also evaluated, disregarding EDPs that were tagged >10 mm from the endocardial 3D-LGE CMR shell.A sustained monomorphic VT arising from the inferobasal LV, closely resembling the documented arrhythmia, was repeatedly induced by programed electrical stimulation  and led to hemodynamic compromise. The high-density LV-endocardial bipolar voltage map  revealed two remote areas of scar in the lateral and apical regions of the LV, colocalizing with areas of increased endocardial PSI based on 3D-LGE CMR. 21 EDPs were tagged. Pace mapping at the inferobasal LV resulted in a 92% match with the induced VT. Radiofrequency delivery targeted at the inferolateral EDPs and the site of VT origin, eliminated the EDPs and rendered the patient non-inducible at the end of the procedure. Total procedural time was 330 min; fluoroscopy time 10 min. The procedure was without any complications. A single-chamber ICD was implanted afterward. No VT had recurred at 20 months follow-up.Contrast-enhanced multidetector CT showed wall thinning in the inferolateral LV and apex . The CMRManual image integration of invasive EAM and 3D-LGE CMR endocardial geometry led to a mean Euclidean node-to-node distance 5 ± 2 mm. Increased PSI (>0.4) of the innermost endocardial layer of 3D dark-blood LGE CMR closely resembled reduced endocardial bipolar voltage (<1.5 mV) using previously defined cutoffs , while aA total of 17/21 EDPs were within 10 mm of the endocardial CMR-wall. 15/17 (88%) of these EDPs were located in an area of endocardial scar detected by 3D dark-blood LGE CMR . EDPs weStructural-functional overlay of the 3D-LGE CMR and ECGI data  allowed the investigation of the VT trajectory and mechanism. In Although the monomorphic VT (with exception of its first beat) appeared stable on the ECG , ECGI rePrior to the electrophysiological study, a premature ventricular complex (PVC) was recorded with ECGI . The PVCIn this proof-of-concept study, we developed a 3D structural-functional model for scar-related VT by combining 3D dark-blood LGE CMR and ECGI (of PVC and VT) with high spatial and temporal resolution. This model was developed in an exemplary case with recurrent monomorphic VT in the setting of ischemic cardiomyopathy. Patient-specific VT substrates, triggers, and their interactions were detailed, in a non-invasive manner, serving as a roadmap for future individualized ablation strategies. Integrating high-density invasive EAM advanced the understanding of possible VT mechanisms.In our integrated model, the (invasive and non-invasive) VT exit colocalized at the end of CMR-detected heterogeneous tissue corridors. Furthermore, the patient’s spontaneous PVC originated in close proximity to the VT exit and CMR-detected corridors. This suggests that the PVC may be involved in patient’s VT formation. Moreover, EDPs were found close to heterogeneous tissue corridors, suggesting a strong link between functional endocardial conduction slowing/block and structural border zone channels.Acquiring 3D-LGE CMR with high isotropic resolution visualizes myocardial fibrosis from every desired direction compared to a fixed viewing angle when using (non-isotropic) 3D or standard 2D imaging . Subtle Additionally, we have shown a good correlation between the 3D dark-blood LGE CMR-derived myocardial heterogeneous tissue corridors and the invasively measured areas of functional electrical conduction slowing or block . These cECGI has been validated in multiple studies. For a recent overview we refer to Stoks and Cluitmans . Its strMoreover, ECGI can generate a complete 3D-epicardial activation map in a single beat, while several VT cycle lengths are needed during invasive contact mapping EAM to construct an activation or propagation map . This may be relevant for investigating the dynamic behavior of VT, especially in polymorphic VT but also during the onset of monomorphic VTs. In our case, ECGI uncovered marked initial activation dynamicity, only stabilizing after several beats. An area of electrical instability (shifting early activation) during VT was present close to the VT exit and structural-functional corridors , 5B.Non-invasive modalities identifying critical components of the VT circuit to guide catheter ablation have evolved considerably over the last decades. Historical milestones in the human heart are shown in In 1972, Wellens et al. found that, in (mostly) post-infarction patients, VTs were based on reentrant excitation, that could be terminated through electrical stimulation . In 1981In 1999, Wittkampf et al. first validated an impedance-based 3D catheter navigation system in humans for EAM . In 2000In 2005, pioneering work on image integration was done by Dickfeld et al. who coregistered MRI acquisitions with the electromagnetic catheter positioning system to guide catheter navigation in the human right atrium and ventricle . In the In 2008, Codreanu et al. performed image integration of EAM and LGE CMR to conclude that transmural scar (by CMR) could be detected by spiky electrograms, reduced voltage amplitudes or prolonged bipolar electrogram durations . A year The aforementioned advances in the field of catheter-based ablation of scar-related VT have primarily focused on defining target locations for ablation through analysis of structural imaging modalities, typically CMR , 44 and/Combined, we argue that the integration  of ECGI with high resolution 3D-LGE dark-blood CMR may be synergistic for the development of (non-invasive) individualized VT models to improve our understanding of VT mechanisms and treatment.Prospective studies are needed to investigate the procedural efficacy and cost-effectiveness of such personalized structural-functional models. This would require optimization of the modeling pipeline, including automated segmentation and merging of different modalities and automation of analyses, which is currently being developed.Ideally, in future cases, ECGI recordings should be performed pre-procedurally during spontaneous VT or non-invasive programed stimulation  to allow preprocedural structural-functional analysis of the VT trajectory and its mechanism. This would guide targeted RF delivery to critical components of the VT circuit without the need for extensive intraprocedural voltage and pace mapping. For instance, CMR-derived heterogeneous tissue corridors  connecting to ECGI-derived VT exit sites could be ablated. ECGI activation maps should be compared to invasively acquired  VT activation/propagation maps to investigate excitation accuracy, VT dynamicity and trajectories.In absence of an ICD, CMR may replace CT for localization of ECGI electrodes and the heart geometry, thereby omitting radiation. Beyond ischemic cardiomyopathies, this model could be extended other cardiac diseases with increased susceptibility for VT/VF, including non-ischemic and idiopathic etiologies. In the latter, ECGI uncovered previously undetected functional repolarization abnormalities, crucial for arrhythmia formation . Next, ttransmural fibrosis, although this method is most widely applied and the most extensively validated (This was a proof-of-concept study based on one exemplary case. We aimed to first develop the model before recruiting more patients, which is currently ongoing. Firstly, although the ECGI VT activation map and exit site were acquired during the procedure, the study data were merged and analyzed retrospectively. The VT ablation procedure was not guided by our model. Secondly, anatomies were merged based on visual inspection. Advanced merging methods may further improve results of invasive EAM vs. ECGI/3D-LGE CMR comparisons . Thirdlyalidated . Lastly,In this proof-of-concept study, we combined 3D-LGE CMR, CT and ECGI imaging modalities to develop a patient-tailored high-resolution non-invasive structural-functional model to investigate VT substrate and trigger in a case of recurrent VT in the setting of ischemic cardiomyopathy. The model accurately pinpointed the VT exit site, collocated areas of structural and functional corridors and provided insight into scar transmurality and endocardial voltage characteristics. Beyond existing methods, our model encompasses beat-by-beat functional electrical information, such as dynamic 3D activation patterns of spontaneous ventricular ectopics and the VT circuit.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the METC azM/UM. The patients/participants provided their written informed consent to participate in this study.JS, MC, PV, and RTB: study setup. JS: ECGI data acquisition and processing, data integration and analysis, and manuscript draft. YK: CT wall thickness analysis. RH and SG: CMR data acquisition. RTB: EAM and clinical data acquisition. JS, MC, BH, BB, PV, and RTB: conceptualization. MC, BH, RH, SG, YK, KV, BB, PV, and RTB: critical review of manuscript. All authors have read and approved the final manuscript and data interpretation.


Results: The meta-analysis of six selected studies (126 patients) provided a DR of 85.9% for PET imaging with PSMA-targeting radiopharmaceuticals in the diagnosis of HCC. Moderate statistical heterogeneity among the included studies was found (I2 = 56%). Conclusions: The quantitative data provided demonstrate the high DR of PET/CT or PET/MRI with PSMA-targeting radiopharmaceuticals for HCC lesion detection. However, more studies are needed to confirm the promising role of PSMA-targeted PET in HCC.Background: Several studies proposed the use of positron emission tomography (PET) with Prostate-Specific Membrane Antigen (PSMA)-targeting radiopharmaceuticals in hepatocellular carcinoma (HCC). Our aim is to calculate the detection rate (DR) of this examination in HCC with a meta-analysis. Methods: A comprehensive literature search of studies on the DR of PET/CT or PET/MRI with PSMA-targeting radiopharmaceuticals in HCC was performed. Original articles evaluating these imaging examinations both in newly diagnosed HCC patients and HCC patients with disease relapse were included. Pooled DR including 95% confidence intervals (95% CI) was calculated. Statistical heterogeneity was also assessed using the I Liver cancer is the sixth most common cancer worldwide and the fourth most prevalent cause of cancer-related death globally, and hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for about 90% of cases ,2. The hImaging is essential to HCC diagnosis and management, contributing to primary liver tumor typing and HCC staging . To date18F]FDG) and radiolabeled choline (with 18F or 11C) positron emission tomography/computed tomography (PET/CT) [18F]FDG PET/CT showed no valuable additional information for the early diagnosis of HCC because of its low diagnostic accuracy (lower than 40%), especially in well-differentiated HCCs [18F]FDG PET might have an added value in the detection of extrahepatic metastatic disease in advanced tumors but, considering the high diagnostic accuracy of CT and MRI imaging, its employment in clinical practice is poor in this setting [18F]FDG PET, its use is limited by the overwhelming CT and MRI performances FDG uptake, evaluating other molecular pathways than the glucose metabolism. Moreover, since first-line therapy in locally advanced and metastatic HCC consists of a combination of immunotherapy and anti-neoangiogenic treatment FDG PET/CT in one study [18F]FDG PET/CT with additional MRI in one study [18F]FDG PET/CT with additional ceCT and MRI in one study FDne study , [18F]FDne study , and ceCne study ,33.68Ga]Ga-PSMA-11, and the injected activity ranged from 148 to 185 MBq  and between 2 and 2.5 MBq/Kg . In contrast, the time interval between PSMA-targeting radiopharmaceutical injection and PET imaging acquisition ranged from 60 to 90 min.Several differences among the included studies were found in the index test key characteristics . In all All the included studies co-registered PET imaging with CT, although in one study, five of the included patients underwent PET/MRI instead of PET/CT .max, SUVmin, and SUVmean). Target-to-background uptake ratio (TBR) was calculated in all the included studies and was obtained by dividing lesions’ SUVmax for the healthy liver SUVmean.PET imaging interpretation was performed both with qualitative and semi-quantitative analyses in all the included studies. Semi-quantitative analyses included the calculation of the maximal, minimal, and mean standardized uptake values  with laboratory exam results. No statistically significant relationships were found with the serum concentrations of alpha-fetoprotein, CA 19-9, and CEA FDG PET/CT has limitations for staging and restaging of HCC [18F]FDG uptake is usually observed in metabolically active cells; thus, cancer cells should show higher avidity for this radiopharmaceutical than normal cells. In most normal and tumoral cells, [18F]-FDG is phosphorylated by hexokinase to [18F]FDG-6-phosphate, which cannot be metabolized and remains within the cell as a polar metabolite that can be visualized by PET imaging; nevertheless, in liver cells, [18F]FDG-6-phosphate can undergo dephosphorylation and can exit the cells. Since the enzymes of well-differentiated HCC are similar to the ones observed in normal liver, this could explain the mild [18F]FDG uptake expressed by well-differentiated HCC and the subsequent low sensitivity of [18F]FDG PET imaging [18F]FDG uptake compared to well-differentiated HCC [18F]FDG PET/CT in HCC is a strong predictor of prognosis and has a role in the assessment of tumor size or number of tumor nodules (as defined by the Milan criteria) [18F]FDG PET/CT. This finding may be explained by the different mechanisms underlying the uptake of the compared tracers as neo-angiogenesis is a rate-limiting factor in tumor growth [18F]FDG metabolism may be highly variable in HCC depending on its differentiation. In this context, it is fundamental to specify that not all kinds of intra-tumor neo-vascular endothelium has significant PSMA expression and that some kinds of tumors show weak neo-angiogenesis; this implies that PSMA-targeting radiopharmaceuticals might not be employed in every type of cancer [[g of HCC . [18F]FD imaging . Moderatated HCC ,42. Consriteria) . Based oriteria) ,29,31, Pr growth , whereasf cancer .11C]acetate and choline-labeled (with [18F] or [11C]) radiopharmaceuticals were found in the literature search.No articles comparing PET with PSMA-targeting radiopharmaceuticals to [11C]acetate PET is a valuable instrument for studying tumors that rely more on fatty acid metabolism than glycolysis, such as low-grade HCC [11C]acetate has the potential to play a complementary role to [18F]FDG PET/CT in the diagnostics of HCC [18F]FDG and [11C]acetate in PET imaging, it is possible that PSMA-targeting radiopharmaceuticals might demonstrate superior diagnostic performance than [11C]acetate PET imaging; nevertheless, since no studies compared the two examinations, it is not possible to express a definitive comparison between the examinations.Acetate can be used by cells to synthesize cholesterol and fatty acids or oxidized via the tricarboxylic acid cycle to produce energy . Thanks rade HCC . Therefos of HCC . Based oCholine-labeled tracers allow the study of lipid metabolism. Their use is based on the presence of large amounts of choline in HCC, as reported by proton magnetic resonance (MR) spectroscopy studies . Choline18F]-labelled compounds as [18F]F-DCFPyL and [18F]F-PSMA-1007 [68Ga]Ga-PSMA-11 [18F]-labeled PSMA-targeting radiopharmaceuticals seems to rely on several advantages since it can count on large-scale production, reduced costs, and high-quality images through lower positron energy and a longer half-life [18F]F-PSMA-1007 shows a different biodistribution from the other PSMA-targeting radiopharmaceuticals, demonstrating higher lipophilicity and, consequently, may not be found in the ureters and bladder since its clearance is liver-dominant [18F]F-PSMA-1007 less suitable for HCC imaging than the others PSMA-targeting radiopharmaceuticals. In this context, more studies are needed to assess the actual feasibility of PSMA-radiopharmaceuticals other than [68Ga]Ga-PSMA-11 to be employed in the diagnostics of HCC.To date, an increasing amount of PSMA-targeting radiopharmaceuticals is available for nuclear medicine physicians, including novel Lu-PSMA-targeting radiopharmaceuticals provided excellent results in metastatic castration-resistant prostate cancer patients [177Lu]Lu-PSMA-617 RLT in two patients who showed liver lesions with high uptake of [68Ga]Ga-PSMA-11 in PET images and had no other local or systemic treatment options [One of the most exciting aspects of PSMA expression in HCC lesions relies on the concept of PSMA-targeted theragnostic, hence the use of PSMA both as a diagnostic and a therapeutic target. As reported by the recent VISION trial, PSMA-targeted therapy with [patients . Based opatients . One of  options . In both options , furtherA previous systematic review published by Dondi et al. investigated the role of PSMA-targeting radiopharmaceuticals in detecting HCC lesions  and did In a recent review published by our group , we partWith regard to the limitations and biases of this meta-analysis, a restricted number of studies was available. Furthermore, a verification bias cannot be excluded since the authors utilized a composite reference standard in some studies. Finally, a lesion-based meta-analysis was not performed due to missing data. Overall, the authors suggest updating this evidence-based analysis when more original articles in this field of interest are published to accomplish a more accurate estimation of the diagnostic performance of the index test.2 was 56%. A subsequent subgroup analysis omitting the only study that employed PET/MRI as hybrid imaging compound in some of the included patients [Heterogeneity among the included papers  may be a source of bias in a meta-analysis . We founpatients  was perfBased on the current literature data, more prospective multicenter studies with larger sample sizes on the diagnostic accuracy of PSMA-targeted PET imaging in HCC patients are required as well as studies exploring its role in prediction and assessment of treatment response. Moreover, studies evaluating the impact of PSMA-targeted PET imaging on the management of HCC and cost-effectiveness analyses (comparing a diagnostic approach with or without the index test) are needed to assess its role in the management of HCC patients.The qualitative and quantitative data provided by this systematic review and meta-analysis highlighted the emerging role of PET/CT or PET/MRI with PSMA-targeting radiopharmaceuticals for the detection of HCC lesions. Nevertheless, more studies are required to corroborate these findings and to better define the role of PET imaging with PSMA-targeting radiopharmaceuticals in this setting , establishing specific clinical recommendations on this imaging method.


Astrocytes produce extracellular matrix (ECM) glycoproteins contributing to the blood-brain barrier and regulating the immune response in the central nervous system (CNS). The aim of this study was to investigate the impact of astrocyte depletion upon the clinical outcome and the composition of ECM glycoproteins in a virus-induced animal model of demyelination. Glial fibrillary acidic protein (GFAP)-thymidine-kinase transgenic SJL (GFAP-knockout) and wildtype mice were infected with Theiler’s murine encephalomyelitis virus (TMEV). Astrocyte depletion was induced during the progressive, demyelinating disease phase by ganciclovir administration once daily between 56 and 77 days post infection (dpi). At 77 dpi GFAP-knockout mice showed a significant deterioration of clinical signs associated with a reduction of azan and picrosirius red stained ECM-molecules in the thoracic spinal cord. Basement-membrane-associated ECM-molecules including laminin, entactin/nidogen-1 and Kir4.1 as well as non-basement membrane-associated ECM-molecules like collagen I, decorin, tenascin-R and CD44 were significantly reduced in the spinal cord of GFAP-knockout mice. The reduction of the investigated ECM-molecules demonstrates that astrocytes play a key role in the production of ECM-molecules. The present findings indicate that the detected loss of Kir4.1 and CD44 as well as the disruption of the integrity of perineuronal nets led to the deterioration of clinical signs in GFAP-knockout mice. Astrocytes play an important role in diseases of man such as multiple sclerosis (MS) as well as in animals like canine distemper encephalitis and Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) –5. TMEV-Astrocytes express multiple pattern recognition receptors (PRRs) and can mediate the innate immune response by directly regulating the entry of inflammatory cells through the blood-brain barrier (BBB) / blood-spinal cord barrier (BSCB) as well as by secretion of various cytokines including MCP-1, RANTES, CXCL10, CXCL12, and IL-8, all attracting peripheral immune cells and microglia , 12. FolAstrocyte derived ECM glycoproteins belong to the group of basement and non-basement molecules  13]. Th. Th13]. Non-basement membrane ECM molecules include, among others, collagen I, decorin, tenascin-R and brevican  13]. Te. Te13]. SJL.Cg-Tg(Gfap-TK)7.1Mvs (GFAP knockout) mice to investigate the effects of astrocyte depletion upon inflammation, demyelination, and axonal damage during TMEV-IDD .–50.48–50Immunohistochemistry targeting Kir 4.1 was applied to further evaluate the effects of the observed ECM molecule loss upon Kir 4.1 potassium channels . StatistAs astrocytes are involved in remodeling and maintenance of neuronal function and plasticity, immunohistochemistry targeting astrocyte associated, non-basement membrane ECM molecules such as collagen I, decorin, tenascin-R, and brevican was applied , 35. PreDuring CNS injury the, non-basement membrane ECM molecule decorin is upregulated by astrocytes and endothelial cells promoting axonal growth, reducing inflammatory reactions and stimulating angiogenesis –54. In tFurthermore the amount of the non-basement membrane ECM molecule tenascin-R was investigated using immunohistochemistry . GSTG miPrevious publications reported an interaction between tenascin-R and brevican . To inveUsing a hyaluronan backbone, non-basement membrane ECM molecules like tenascin-R and brevican are anchored to astrocyte foot processes by the cell-surface glycoprotein CD44 contributing to the integrity of perineuronal nets , 37. Theet al. (2012) demonstrated an association between clinical deterioration and deposition of ECM components during TMEV-IDD. The deposition of astrocyte derived ECM glycoproteins in the CNS plays a crucial role at later stages of demyelinating and inflammatory diseases of the CNS such as multiple sclerosis [Progression of demyelinating diseases is frequently accompanied by deterioration of clinical signs in human diseases like multiple sclerosis and its animal models , 55. Haiclerosis . During clerosis . Furtherclerosis . TherefoIn a recent study, we evaluated the impact of astrocyte depletion upon clinical manifestation, inflammation and demyelination using TMEV-IDD as a virus-induced animal model for multiple sclerosis. However, the mechanisms leading to clinical deterioration remained elusive and an altered composition of the ECM was postulated to contribute to the clinical deterioration . TherefoIn the present study, a significant reduction of ECM glycoproteins in the thoracic spinal cords of GFAP knockout compared to control mice was identified using azan and picrosirius red stainings . These dAssembly and organization of the basement membrane is not restricted to the presence of laminin, but also requires direct linkage to entactin/nidogen-1 for assurance of basement membrane integrity . TherefoDifferent studies already postulated a co-expression of Kir 4.1 and AQP4 in a laminin dependent manner , 61–63. During CNS injury, activation of astrocytes leads to an upregulation of ECM glycoprotein secretion and intralesional ECM accumulation . UltimatAstrocytes are not only participating in formation of the BSCB, they also play a crucial role in formation of PNNs . TherebyCD44 represents another indispensable glycoprotein sustaining the integrity of tripartide synapses and PNNs . The conAccording to previous studies, CD44 is critically involved in synaptic excitatory transmission as well as functional and structural plasticity of dendritic spines . TherefoS1 FigIdentification and quantification of collagen I accumulation in the thoracic spinal cord white matter was performed using immunohistochemistry. Statistical analysis revealed a significant reduction of collagen I in the lesioned thoracic spinal cord white matter in TMEV infected, ganciclovir treated, GFAP-transgenic (GSTG) mice (A) compared to TMEV infected, NaCl treated, GFAP-transgenic (GSTP) controls (B). Inserts visualize in more detail the intralesional accumulation of collagen I. Bars represent 100 μm in overviews and 50 μm in the inserts.(TIF)Click here for additional data file.


Numerous post-vaccine complications have been reported secondary to the COVID-19 vaccine. Many of these complications are believed to be due to a hyperactive immune system. A 59-year-old woman developed diffuse abdominal pain two days after receiving the mRNA-1273 COVID-19 vaccine (Moderna). A computerized tomography (CT) angiogram of the abdomen and pelvis revealed the presence of numerous vascular irregularities in the celiac axis, bilateral renal arteries, and inferior mesenteric artery consistent with polyarteritis nodosa (PAN), a medium-vessel vasculitis. The patient was managed with intravenous methylprednisolone 500 mg daily for three days and was then placed on oral methotrexate (MTX) 12.5 mg daily for immunosuppressive maintenance treatment. Until now, a limited number of cases of polyarteritis nodosa secondary to the COVID-19 vaccine have been reported. Major mechanisms of post-vaccine autoimmunity are molecular mimicry and autoantibody production. Although rare adverse events from COVID-19 vaccination are possible, there remains an immense benefit to vaccination in preventing COVID-19-related morbidity and mortality. Coronavirus disease 2019 (COVID-19) is a pandemic that has had a significant burden on healthcare systems and patient outcomes around the world. The introduction of vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has allowed for improved outcomes in morbidity and mortality . The casA healthy 59-year-old woman presented to the emergency department with a two-day history of progressively worsening abdominal pain, melena, and postprandial nausea. The patient had no significant chronic medical history and no recent infections. She was recently vaccinated with the second dose of the mRNA-1273 vaccine against SARS-CoV-2 approximately two days before her presentation. Physical examination was significant for diffuse tenderness to palpation of the abdomen with rebound tenderness of the right upper quadrant. Laboratory values were significant for a mild leukocytosis with a WBC count of 12,550 cells/mL , and an elevated erythrocyte sedimentation rate (ESR) of 91 mm/h (0-21). Serology for both hepatitis B and C was negative. Serum antibody testing was significant for a positive antinuclear antibody (ANA) with a level of titer of 1:160. Both myeloperoxidase-anti neutrophil cytoplasmic antibody (ANCA), and proteinase-3 antibody screening were negative. Urine analysis showed no gross blood and 3-5 red blood cells/high power field with no WBCs. The patient tested negative for SARS-CoV-2 via polymerase chain reaction (PCR). A computerized tomography (CT) scan of the abdomen showed numerous vascular irregularities in the celiac axis, bilateral renal arteries, and inferior mesenteric artery consistent with vasculitis. There was significant stenosis of the distal branch of the right renal artery resulting in renal parenchyma ischemia with heterogenous enhancement  12.5 mg daily for remission maintenance immunosuppression. At a three-month follow-up, the patient had a subsequent CT angiogram of the abdomen which showed resolution of the previous vascular irregularities and renal ischemia . The clinical presentation of PAN often involves fever, weight loss, malaise, and joint pain. In patients with gastrointestinal involvement; abdominal pain, melena, and vomiting are often reported. Other organ systems involved include the coronary arteries ~35%), kidneys (~60%), and skin ~40%) [0% [4]. M%, kidneyPAN involving the abdomen includes inflammation manifested as stenosis and aneurysmal dilatation of various medium-sized vessels. Renal artery involvement is the most prevalent. A retrospective review of seven patients with PAN involving the abdomen reported five cases of renal artery aneurysms and six cases with involvement of the mesenteric artery . SystemiFor non-HBV-related PAN, the management is similar to other forms of vasculitis. First-line management for moderate and severe PAN includes high-dose glucocorticoids and/or cyclophosphamide. In cases of HBV-associated PAN antiviral therapy or plasmapheresis can be administered. Maintenance immunosuppression therapy can include azathioprine and MTX. Immunosuppressive therapy is often used for a total duration of 18 months . Ohkubo The COVID-19 mRNA vaccine may lead to an acute increase in inflammatory markers after vaccination, particularly after the second dose. The mRNA has been shown to bind pattern recognition receptors prior to translation and lead to the activation of multiple pro-inflammatory cascades. Up-regulation of these immunological mechanisms may lead to the observed autoimmune conditions . MoleculA descriptive cohort analysis of 3.9 million individuals vaccinated with an mRNA-based (BNT162b2) and a non-mRNA-based (CoronaVac) vaccine revealed a similar incidence of autoimmune conditions compared to nonvaccinated individuals . Larger The case of a 59-year-old woman who was diagnosed with polyarteritis nodosa two days after mRNA-1273 COVID-19 vaccination is reported. CT angiography of the abdomen and pelvis revealed multifocal aneurysmal dilatation of the renal artery, stenosis of the splenic artery, and dilatation of the proximal inferior mesenteric artery. Management included high-dose intravenous methylprednisolone for three days and methotrexate for maintenance immunosuppression. Few cases of PAN secondary to COVID-19 vaccination have been reported thus far. Immunologic mechanisms associated with post-vaccine autoimmunity include cytokine storm, autoantibody production, and molecular mimicry. Although rare AEs secondary to the COVID-19 vaccine are possible, there remains an immense benefit to COVID-19-related morbidity and mortality.


Nature has evolved numerous supramolecular machineries for modulating various cellular functions. Inspired by the assembly of these sophisticated structures in nature, the controlled assembly of synthetic peptides emerges as a promising approach to therapeutically relevant applications. The self-assembling biomimetic peptides could form well-ordered architectures through non-covalent interactions such as π-π stacking, van der Waals, electrostatic, and hydrogen bonding. In addition, the peptidic building blocks are highly biocompatible and allow facile chemical manipulation with diverse functionalities. For decades, a serious of engineered self-assembling peptides have been extensively studied as functional hydrogels for various applications. Meanwhile, the surface modification strategies based on self-assembling peptide matrices have also raised the attention of biomaterials researchers due to their programmability and 3D porous morphologies. This concise review will cover recent advances in self-assembling peptide matrices as functional coatings for implantable devices. The opportunities and challenges in this field will also be discussed. Peptide self-assembly has emerged as an attractive strategy for achieving diverse supramolecular structures for therapeutically relevant applications . These nin vivo and clinical performance, particularly the inflammatory responses and potential foreign body reactions induced by the implantable devices. As a result, functional coatings of implantable devices have been vigorously studied to conquer the potential risks to patients  constitute an important category of self-assembling peptides with enormous biological functionalities, which are featuring with a hydrophobic segment such as lipids at the termini . In 20014 or RADA16 (RADARADARADARADA) peptide, which forms a regular nanofiber structure. Their physicochemical properties and biomedical applications have been extensively investigated so far  that could form β-sheets with ionic-complementarity properties . After td so far . These iin vitro or in vivo. The formation of the hydrogels generally includes the bond formation of the hydrogelator or the removal of a masking group from the hydrogelator precursor. A rich number of enzymes have been reported so far, such as kinase, β-lactamase, or phosphatase , consisting of two phenylalanine residues, represents a minimalistic self-assembling building block derived from the core motif of pathogenic amyloid-β peptide . Previousphatase .Amphipathic peptides have been reported to form supramolecular structures in water. One of the representative categories is the surfactant-like peptide which is composed of consecutive hydrophobic residues  and one or two hydrophilic residues  in the peptide sequence . AnotherDespite the applications of linear peptides as supramolecular building blocks, cyclic peptides have also attracted considerable attention as a more stable peptidic self-assembly building block. One of the cyclic peptides was reported to form a β-sheet-like tubular structure through intermolecular hydrogen bonding. The backbone of the cyclic peptide is positioned to the inner side of the cylinder with the side chain residues positioned outside the cylinder . Li et aFunctional peptides have been widely used in the surface modification of various biomaterials to enhance their biocompatibility. Conventional surface modification of medical implants by functional peptides is based on covalent interactions between peptide and surface. Compared to peptide grafting, the surface coating strategies based on self-assembling peptide matrices have apparent advantages in peptide loading capacity, 3D porous structure, multifunction, and relatively simple fabrication due to their materials’ nature. For decades, several self-assembling peptide-based surface functionalization techniques have been investigated for improving the biocompatibility of implantable materials, including cardiovascular and blood-contacting devices, orthopedic and dental implants, and other implants with antimicrobial and antifouling coatings .4 hydrogel coating with the nitric oxide (NO)-generating activity for blood-contacting devices (316L stainless steel (316L SS) as an example)  of glutathione peroxidase functionalized (RADA)example) . Moreoveexample) . These nDopa  molecule is a biocompatible mussel-inspired adhesive structure for biomolecule immobilization on metal surfaces. The REDV motif selectively promotes the growth of endothelial cells. Nanofibers formed by Dopa- and REDV-conjugated PAs were used as a surface coating on 316L SS, which is widely used as the backbone of vascular stents .Currently, the most used stent is a drug-eluting stent that contains a drug-eluting polymer coating on its surface. It has been reported that the diphenylalanine (FF) nanotubes can be used as an alternate drug-eluting system for polymer coating in stents .6V4 alloy, which creates an osteoconductive interface and inhibits the growth of fibroblasts  and its alloys-based orthopedic and dental implants are increasingly used in the treatment of bone defects and tooth loss due to their excellent corrosion resistance and good hard-tissue compatibility, which are commonly limited due to insufficient bone integration, and potential infections. PAs functionalized with adhesive Dopa and osteogenic peptide motif KRSR can be self-assembled as nanofiber coating on the surface of TiAlroblasts . Antimicfections . It has fections .Hydroxyapatite (HA) coatings are one of the conventional surface modification techniques for Ti alloys to improve biocompatibility and bioactivity. It has been reported that HA coatings can be further functionalized by EAbuK 16-II self-assembling peptide overlayer on their surface .A self-assembling peptide-mediated biomimetic strategy can also be adapted to the functionalization of 3D graphene foam with a motif-specific peptide (LLVFGAKMLPHHGA), which exhibits biomimetic mineralization ability. Such peptide sequence consists of two functional motifs: the β-sheet self-assembling LLVFGAK sequence and the biomimetic mineralization motif MLPHHGA .4 self-assembling peptide can build a cellular microenvironment that promotes bone reconstruction. The bone reconstruction of demineralized bone matrix (DBM) was enhanced by (RADA)4 nanofiber matrix which induced smaller pore size and stronger charge interaction eraction .Other than orthopedic and dental implants, surface modification strategies based on antimicrobial peptides (AMPs) have been extensively developed for preventing bacterial infections in other biomedical implants.As previously described by Dopa-mediated surface adhesion strategies, mussel-inspired adhesive molecular or materials design plays an important role in the surface modification of implantable biomaterials. Recently, it was demonstrated that a peptide derived from mussel foot protein, displays antibacterial properties, which inspired the design of self-assembling peptide (MIKA2) based adhesive hydrogels with antimicrobial activity against drug-resistant Gram-positive bacteria on Ti .Polyurethane (PU) based biomaterials have favorable mechanical properties and biocompatibility that have great potential in implant devices. It has been demonstrated that polyethylene glycol (PEG) motif functionalized Fmoc-diphenylalanine (Fmoc-FF) self-assembling peptide endows waterborne PU surfaces with enhanced biocompatibility, antifouling ability, and water resistance, simultaneously .42–), can be dephosphorylated into Fmoc-FFY and form an antibacterial hydrogel coating by previously deposited alkaline phosphatase functionalized NPs@AP nanoparticles on the surface. The resulting Fmoc-FFY peptides then self-assemble into β-sheets nanofiber hydrogel coating, which inhibits the growth of Gram-positive Staphylococcus aureus (S. aureus) (Enzyme-triggered self-assembly of Fmoc-FF-based short peptides was developed. Fmoc-FFpY peptide (p: PO aureus) .For antifouling coatings, two simple PAs functionalized with PFB , namely, PFB-VVD and PFB-LLE was designed and can self-assemble into functional coatings with anti-parallel β-sheet secondary conformation on the surface. As described, PFB-VVD exhibits better antifouling activity compared to PFB-LLE .2 surface, which has a protentional to support cell attachment and growth while against infections by the antimicrobial nature of chitosan. The deposited EAK16-II possesses layer-by-layer and ordered structures depending on the preparative stoichiometry and path (Chitosan grafted EAK16-II self-assembling peptides-based hydrogel coating on TiOand path .in vivo applications. Moreover, introducing surface adhesive elements or using intermediate coating for effective immobilization of nanostructures on the surface is essential for ensuring their long-term stability. Although challenges remain, the peptide self-assembly holds great promise to further broaden its applications in biology and nanotechnology.The utilization of peptide self-assembly to form well-ordered structures offers great potential in biomedical engineering. This concise review summarizes the most used peptide building blocks and their applications as self-assembled functional coatings for implantable devices. Despite the significant advances in peptide-based self-assembly nanomaterials. How to precisely control the assembly of the peptide and understand the mechanistic insight of the assembly process will be highly desired to drive the discovery of new peptidic building blocks’ superior properties in the future. Another concern regarding the self-assembling peptide is their stability issues for


Breast cancer therapeutic intervention continues to be ambiguous owing to the lack of strategies for targeted transport and receptor-mediated uptake of drugs by cancer cells. In addition to this, sporadic tumor microenvironment, prominent restrictions with conventional chemotherapy, and multidrug-resistant mechanisms of breast cancer cells possess a big challenge to even otherwise optimal and efficacious breast cancer treatment strategies. Surface-modified nanomedicines can expedite the cellular uptake and delivery of drug-loaded nanoparticulate constructs through binding with specific receptors overexpressed aberrantly on the tumor cell. The present review elucidates the interesting yet challenging concept of targeted delivery approaches by exploiting different types of nanoparticulate systems with multiple targeting ligands to target overexpressed receptors of breast cancer cells. The therapeutic efficacy of these novel approaches in preclinical models is also comprehensively discussed in this review. It is concluded from critical analysis of related literature that insight into the translational gap between laboratories and clinical settings would provide the possible future directions to plug the loopholes in the process of development of these receptor-targeted nanomedicines for the treatment of breast cancer. Breast cancer has an extensive history, dating back more than 3500 years to around 1500 B.C. when it was first mentioned by the ancient Egyptians ,3. BreasBreast cancer is a multifaceted disease characterized by a wide range of molecular profiles with characteristics of biological and clinical features . DependiIn this review, notable challenges in breast cancer therapeutic intervention are emphasized. A comprehensive account of surface-modified nanomedicines is discussed that improve the efficacy of loaded therapeutics for breast cancer treatment. The key challenges that need to be addressed to improve the utility of receptor-targeted nanomedicines in clinical settings are also discussed.Despite considerable advancements in breast cancer treatment, there are still numerous hurdles in accomplishing optimal therapeutic response. Chemotherapy is the primary therapeutic option, either alone or combined with other therapeutic approaches such as radiation or surgery ,11,12,13MDR is a complicated mechanism that includes aberrant vasculature, localized hypoxia, a low pH environment, aerobic glycolysis, enzymatic degradation, up-regulated ABC-transporters, an enhanced apoptotic threshold, and increased interstitial fluid pressure, among other conditions that limit the therapeutic action of drug ,20. The Among various ABC transporters, the main ABC transporters primarily linked to MDR in breast cancer are P-glycoprotein (P-gp/MDR1/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) ,22,24. BBCRP was first isolated from MDR human breast cancer cells . It is mAs discussed earlier, the clinical subtypes of breast cancer have different therapeutic approaches. However, the molecular pathway revealed that this disease shares several common characteristics, specifically, disruption to the phosphatidylinositide 3-kinase (PI3K)/Akt pathway, miRNA levels, and epigenetic gene transcription modulation . The PI3Most of the cancer chemotherapeutics are typically hydrophobic, nontargeted, and toxic in nature that can have serious adverse effects . Over thThe lipid-based nanocarriers have been proved to be superior over polymeric and inorganic nanocarriers in terms of biocompatibility, safety, and biodegradability. Lipid nanocarrier drug delivery system can be sub-categorized into nanoemulsion, self-nano emulsifying drug delivery system (SNEDDS), liposomes, solid lipid nanoparticles (SLN), and nanostructured lipid carrier (NLC). In SNEDDS and nanoemulsion (oil-in-water), the modifying agent such as probes or imaging agents and lipophilic anticancer drugs are enclosed in oil droplets for specific tumor site delivery. In liposomes, the imaging/diagnostic agent can be enclosed with anticancer therapeutics either in an aqueous core or lipid bilayer according to their physicochemical characteristics (hydrophilicity/lipophilicity). Solid matrix lipid nanoparticles such as SLN (composed of solid lipid only) and NLC (composed of both solid and liquid lipid) entrapped the lipophilic therapeutics and imaging agents into the lipid phase of this delivery system. The various attributes of liposomes  make it promising therapeutic/imaging compared to other lipidic nanocarrier systems. However, optimizing lipid nanomedicines with desirable size distribution, shape, surface charge, and stability is quite challenging in itself. Recently, exploring cancer cell-specific drug delivery to improve the antitumor efficacy of lipid-based nanocarriers by surface functionalization with various ligands and peptides is contemplated in extensive research work. These lipid nanocarriers are now being designed to encapsulate or conjugate multifunctional components such as anticancer agents, targeting moieties, antibodies, etc. in one single formulation, therefore can be called complex nanoproducts ,15,16,18Lipid-polymer hybrid is a promising new generation nanocarrier system and concept has originated from the fusion of liposomes and polymeric nanoparticles. It has polymeric core enclosed by a similar kind of lipid bilayer shell. Many researchers are exploring its potential as drug delivery nanocarrier system particularly for anticancer therapeutics in current scenario ,45.®. It has been used against Kaposi’s sarcoma, refractory breast and ovarian cancer with a circulating half-life of 74 h in breast cancer patients, compared to approximately 5 min t1/2 for free drug ..93].In another research, tamoxifen, an ER antagonist, surface-grafted liposomes loaded with DOX showed enhanced cellular and nuclear uptake of DOX in comparison to DOX-loaded liposomes and free DOX solution. In vitro investigations demonstrated that tamoxifen and DOX co-loaded liposomes were more cytotoxic to ER overexpressing MCF-7 cells as compared to DOX liposomes, DOX solution, and tamoxifen-DOX solution. Tamoxifen-DOX liposomes exhibited remarkably increased inhibition of tumor growth in animal model compared to DOX solution and DOX liposomes .Estrone decorated DOX-loaded stealth liposomes exhibited significant cytotoxicity, accumulation, and antitumor efficacy compared to untargeted formulations in ER-positive MCF-7 tumor-bearing female Balb/c-nude mice . In a prThese studies demonstrated that estrone-targeted nanomedicine may advance the treatment of various estrogen receptor overexpressing breast cancers by utilizing receptor-mediated targeting of anticancer agents.The glycosaminoglycan hyaluronan is a naturally occurring component of the extracellular matrix. Hyaluronic acid (HA) plays a crucial role in cell proliferation, migration, and invasion, and as these processes are involved in inflammation and cancer progression, therefore HA is significant in cancer pathophysiology. The HA receptor CD44 is found sparsely on the surface of epithelial, hematopoietic, and neuronal cells, and is abundantly overexpressed in various cancer cells ,98,99. C50 value with HA decorated NLCs in comparison to baicalein and DOX loaded NLCs, and baicalein and DOX solution, respectively. In vivo antitumor efficacy study using Kunming mice bearing MCF-7/ADR breast cancer cells xenograft showed higher inhibition in tumor growth with targeted NLCs as compared to untargeted baicalein and DOX loaded NLCs, baicalein NLCs, DOX NLCs, and baicalein and free DOX solution [A novel redox-responsive and HA-functionalized chitosan lipoic acid NPs (HA-CSLA-NPs) loaded with cytoplasmic 17α-methyltestosterone showed significantly higher cellular internalization through CD44 receptors with rapid drug release, and enhanced cytotoxicity against CD44 overexpressing BT-20 breast cancer cell line as opposed to CD44 negative MCF-7 cell line . Previousolution .Zhong et al. developed HA L-lysine methyl ester-lipoic acid (HA Lys-LA) NPs. These HA Lys-LA NPs exhibited 20-folds higher tumor uptake of DOX compared to free DOX. DOX-loaded crosslinked HA-Lys-LA NPs possessed significantly higher targetability and excellent antitumor activity towards CD44 positive MCF-7/ADR cells. Moreover, the developed NPs exhibited significantly greater survival of mice throughout the experimental period of 44 days and showed lesser side effects compared to untargeted NPs and free drugs . In anotDocetaxel-loaded self-assembled NPs of PLGA/HA block copolymers exhibited significantly higher uptake, enhanced cytotoxicity in MDA-MB-231 cells by CD44-mediated endocytosis compared to untargeted NPs and free drug. In vivo studies with Balb/c nude mice showed that HA decorated NPs exhibited significantly improved cancer targeting and anticancer activity compared to untargeted NPs and free drug . Yang etThese studies suggest that HA could be employed as a promising targeting ligand to target various nanomaterials carrying the anticancer payload to the CD44 receptor for the treatment of breast cancer.Luteinizing hormone-releasing hormone (LHRH) is a hormonal decapeptide produced by the hypothalamus. It is also known as Gonadotropin-releasing hormone (GnRH). It plays an important role in the regulation of the pituitary-gonadal axis and reproduction. LHRH receptors are overexpressed in 80% of endometrial and ovarian, 86% of prostate, 50% of breast, and 80% of renal cancers . These LRecently scientists conjugated LHRH with anticancer drugs, prodigiosin or paclitaxel, separately and evaluated these LHRH drug conjugates for treatment and specific targeting in MDA-MB-231 TNBC cells and tumor-bearing female nude mice. The designed conjugates showed promising efficiency to target TNBC cells specifically with the ability to cause significant regression in tumor growth without any significant toxicity . Previou50 value against LHRH overexpressing MCF-7 cells compared to nontargeted NPs [Varshosaz et al. developed magnetic LHRH chitosan bioconjugated NPs which demonstrated significantly enhanced cellular uptake and a 2-fold reduction in ICeted NPs . In anoteted NPs . Furthereted NPs .The studies discussed above supports further exploration of LHRH conjugated nanomedicine for treatment and diagnosis of breast cancer including its most complicated version, TNBC. Thus, LHRH can be used as a potential targeting ligand for the targeted treatment of LHRH overexpressed breast cancers.3+) through TfRs on the plasma membrane. The delivery of Fe3+ is mediated via clathrin-mediated endocytosis and the TfRs recycle back to the cell surface. TfRs are overexpressed in cancer cells owing to their higher necessity of iron and there exists a correlation between the number of TfRs expressed and the proliferative ability of the tumors [Transferrin (Tf) is a serum nonheme iron-binding blood plasma glycoprotein responsible for the transport of ferric ions  to increase internalization in MCF-7 breast cancer cells via Tf/TfR mediated endocytosis. In vivo studies showed that there was a significant inhibition in tumor growth and reduced side effects through cell apoptosis as determined by TUNEL assay . Cui et 50 value compared to untargeted NPs [Tf has also been conjugated with lipid-coated PLGA NPs carrying the aromatase inhibitor, 7α-(4′-amino) phenylthio-1,4-androstadiene-3,17-dione (7α-APTADD) which exhibited significantly higher drug uptake in SK-BR-3 breast cancer cells compared to untargeted NPs. Moreover, Tf-decorated NPs exhibited a 2.8-fold reduction in ICeted NPs . In anoteted NPs .Recently, Li et al. developed Tf-decorated piperine-loaded polymeric nanoparticles for targeted treatment of breast tumors . In vitrThus, a nanoparticulate system can be successfully employed for TfR targeted treatment of breast cancers using Tf as a targeting ligand.Integrins represent the family of heterodimeric integral membrane proteins encompassing not less than 24 combinations of 18α and 8β subunits that mediate cell adhesion. Integrin receptors are intricately involved in cell–cell and cell–extracellular matrix interactions, cell adhesion, migration, survival, growth, cytoskeleton organization, and cell signaling. In cancer, integrins stimulate the proliferation of tumor cells and tumor vascular endothelial cells . Integri5β1, thereby providing a tool to target α5β1-expressing cancer cells in vitro as well as in vivo. In vitro binding efficacy study showed that functionalized PEGylated liposomes exhibited enhanced binding as compared to the untargeted PEGylated liposomes, and their binding efficiency increased with increasing fibronectin-mimetic peptide concentration. Furthermore, functionalized PEGylated liposomes exhibited higher cytotoxicity to MDA-MB-231 cells compared to untargeted PEGylated liposomes [vβ3 integrins on cancer cells. The conjugated NPs exhibited significantly higher uptake in comparison to untargeted NPs in MCF-7, MCF7-MFP1, DU145, DU145LN2, PC3, PC3MLN4 cell lines. In vivo cytotoxicity study in female nude mice xenograft bearing MCF-7-MFP1 breast cancer cells showed that RGD-targeted cisplatin encapsulated NPs exhibited significantly higher cytotoxicity compared to nontargeted NPs [Researchers have designed leukocyte-mimicking nanovesicles, i.e., leukosomes, for effective delivery of DOX for the treatment of breast cancer and melanoma. They observed significantly higher tumor accumulation of leukosomes with reduced tumor volume and prolonged survival span and thus, more potent anticancer activity in comparison to free DOX solution. There was a significant inhibition in tumor growth with DOX-loaded leukosomes as compared with free DOX in both breast and melanoma tumors . Shroff iposomes . Graf eteted NPs .From the studies discussed here, we could conclude that integrin receptor-targeted nanomedicine could be explored as a promising therapeutic intervention for breast cancer treatment.Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide of the glucagon-secretin class which is pervasive in both the central and peripheral nervous systems. VIP receptors are five times more profuse in breast cancer cells than in normal breast cells . The VIP50 up to 14.2 ± 1.2 μM in comparison that forms free curcumin which was 26.1 ± 3.0 μM. Furthermore, this nanomedicine also showed inhibition in the formation of tumorsphere up to 20% at a dose of 5 μM [Pirarubicin, an anticancer drug, which is an analog of the anthracycline antineoplastic antibiotic DOX and causes serious side effects, was loaded into sterically stabilized micelles of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- methoxy-- polyethylene glycol 2000 (DSPE-PEG2000). Furthermore, these micelles were surface-functionalized with VIP to target breast cancer cells. In the in vitro studies with MCF-7 breast cancer cells as well as in nude mice xenograft model of 4T1 breast cancer, these VIP functionalized pirarubicin-loaded DSPE-PEG2000 micelles showed higher anticancer activity without significant toxicity . Cancer  of 5 μM .50 value compared to free paclitaxel. Furthermore, the in vivo studies in female Sprague Dawley rats demonstrated that there was a significantly higher accumulation of paclitaxel delivered in the targeted carrier in the breast tumors due to its size, long circulation, and interaction with target receptors. A minimal accumulation of paclitaxel in healthy tissues particularly those associated with systemic toxicities (such as bone marrow) owing to target-specific delivery was observed [Paclitaxel-loaded VIP surface grafted sterically stabilized phospholipid nanomicelles were designed by Dagar and coworkers to target VIP receptors. This VIP receptor-targeted nanomedicine exhibited a 2-fold improvement in EDobserved . Onyukseobserved . In anotobserved .From the above studies, it could be concluded that VIP can be utilized promisingly for receptor-specific targeted treatment of breast cancers including CSCs enriched breast cancers.58-b-polystyrene13058-b-PSt130) NPs, which showed that NAG conjugation enhanced the internalization and targeting ability of NAG-P58-b-PSt130 NPs. In vitro anticancer activity in MCF-7 cells showed that DOX loaded NAG-P58-b-PSt130 NPs exhibited an enhanced anticancer activity compared to NPs without NAG, which could be attributed to NAG receptor-mediated endocytosis [Scientists have also explored some other receptors targeted nanomedicine for improving the treatment protocol of the world’s most reported cancer, breast cancer, in women. Tian et al. developed N-acetyl-D-glucosamine (NAG) decorated DOX loaded poly ocytosis . Guo et ocytosis .2000-DSPE800) micelles which showed much higher cellular uptake, cytotoxicity, and apoptosis than that of unmodified micelles for either A549 or MCF-7 cells. In vivo antitumor efficacy study in MCF-7 tumor-bearing female nude mice demonstrated that targeted micelles showed significantly enhanced suppression of tumor growth during the experiment up to 18 days compared to untargeted micelles and Taxol [A ligand-gated ion channel, Alpha7 nicotinic acetylcholine receptor (α7 nAChR), is being investigated by scientists for targeted delivery of anticancer drugs due to its specific expression in cancer. In an attempt to target α7 nAChR, Mei et al. developed paclitaxel loaded and α-conotoxin ImI (it is a disulfide-rich toxin which has shown a strong affinity for α7 nAChR) conjugated poly (ethylene glycol) -(distearoyl-sn-glycero-3-phosphoethanolamine)  peptide conjugated paclitaxel loaded aldehyde PEG–PLA NPs exhibited significantly higher uptake compared to untargeted NPs and free Taxol. The pharmacokinetic study showed that K237 conjugated paclitaxel-loaded NPs and unconjugated paclitaxel NPs exhibited an extended half-life of paclitaxel from 0.9 h to 8.77 h and 8.94 h, respectively compared to Taxol. Meanwhile, the AUC improved about 30-fold for K237 conjugated paclitaxel-loaded NPs and 27-fold for unconjugated NPs compared to Taxol. K237 conjugated paclitaxel-loaded NPs also exhibited significantly stronger antitumor efficacy compared to unconjugated NPs in female Balb/c nude mice bearing MDA-MB-231 breast tumors .The prognosis of receptor-mediated targeted therapy in breast cancer is mainly dependent upon the extent of overexpression of the breast cancer-specific cellular receptors particularly human epidermal growth factor receptor-2 (HER2/Neu/ErbB2), estrogen and progesterone receptors. Triple-negative breast cancers (TNBC), which do not express any of these targets, i.e., estrogen receptor, progesterone receptor, and HER2 receptor, are the most challenging to treat as these show a poor response to both HER2 targeted and hormonal receptor  targeted therapies. Theranostic nanomedicines are emerging as a novel paradigm with the ability for concurrent delivery of imaging (with contrasting agents), targeting (with biomarkers), and anticancer therapeutics through the single delivery vehicle as cancer theranostics. It can transpire as a promising strategy to overcome various limitations for effective management of breast cancer including its most aggressive form, TNBC . RecentlThe potential of different receptor-targeted nanomedicines explored for the treatments of breast cancer is concisely summarized in ™, a liposomal formulation of daunorubicin and cytarabine in several countries, including the USA and EU against acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes demonstrated remarkably improved survival and response rates, with tolerable toxicity in older patients [Owing to the significant advances in the field of nanobiotechnology, multiple nanomedicines-based anticancer therapeutic agents are now available in the market and accessible for clinical use, and some of the products are in different stages of clinical trials. Most of the anticancer nanomedicine available in the market or under clinical trials utilized previously approved active therapeutic moieties encapsulated into different nanocarriers including liposome, polymeric micelles, dendrimers, nanoemulsion, and inorganic nanoparticles . Despitepatients ,199.Even after obtaining promising results in preclinical investigations, there are still numerous obstacles in development of targeted cancer nanomedicines that restrict its success in clinical settings. The various biological and translational barriers are being identified. These barriers need to be overcome in order to improve the clinical outcomes of targeted therapies. Among various challenges, the greatest is the production of targeted nanoparticles at a large scale since it faces continuous issues such as multistep synthetic procedure, higher manufacturing cost, and reproducibility of biopharmaceutical attributes ,201,202.In a nutshell, receptor-targeted modified nanomedicines foster a great prospect of optimal therapeutic and drug delivery approaches. Modified nanomedicine possesses the remarkable potential to address the shortcomings of the conventional chemotherapy approach. Strenuous research on tumor milieu, pathophysiology, and progression stemmed exploration of novel propitious targets for breast cancer alleviation. In the interim, substantial improvement in the fabrication of various nanoparticulate systems and insights of their physicochemical and pathophysiological obstacles made them a promising targeting system cognizant for fixing the clinical effect of nanomedicine for breast cancer therapeutic intervention. Various advancements in nanomedicines such as stimuli-responsive nanoparticulate systems were developed until now to deliver anticancer agents for treating breast cancer. Several ligands were utilized for specific targeting of receptors that are overexpressed in breast cancer cells. The findings of the preclinical investigation established improved cellular internalization, mitigated toxicity issues, and multidrug resistance in breast cancer cells through a targeted nanoparticle–drug conjugation/encapsulation approach. Such encouraging outcomes propel the clinical translation of receptor-targeted surface-modified nanomedicines. Nonetheless, still, many more arduous investigations are highly needed for selecting the ideal target receptor and for exploring the ligand–receptor interaction.