Clinical Utility of an Artificial Intelligence Radiomics-based Tool for Risk Stratification of Pulmonary Nodules.

BACKGROUND: Clinical utility data on pulmonary nodule (PN) risk stratification biomarkers are lacking. We aimed to determine the incremental predictive value and clinical utility of using an artificial intelligence (AI) radiomics-based computer-aided diagnosis (CAD) tool in addition to routine clinical information to risk stratify PNs among real-world patients. METHODS: We performed a retrospective cohort study of patients with PNs who underwent lung biopsy. We collected clinical data and used a commercially available AI radiomics-based CAD tool to calculate a Lung Cancer Prediction (LCP) score. We developed logistic regression models to evaluate a well-validated clinical risk prediction model (the Mayo Clinic model) with and without the LCP score (Mayo vs Mayo + LCP) using area under the curve (AUC), risk stratification table, and standardized net benefit analyses. RESULTS: Among the 134 patients undergoing PN biopsy, cancer prevalence was 61%. Addition of the radiomics-based LCP score to the Mayo model was associated with increased predictive accuracy (likelihood ratio test, P = .012). The AUCs for the Mayo and Mayo + LCP models were 0.58 (95% CI, 0.48-0.69) and 0.65 (95% CI, 0.56-0.75), respectively. At the 65% risk threshold, the Mayo + LCP model was associated with increased sensitivity (56% vs 38%; P = .019), similar false positive rate (33% vs 35%; P = .8), and increased standardized net benefit (18% vs -3.3%) compared to the Mayo model. CONCLUSIONS: Use of a commercially available AI radiomics-based CAD tool as a supplement to clinical information improved PN cancer risk prediction and may result in clinically meaningful changes in risk stratification.

Lung cancer mortality trends and disparities: A cross-sectional analysis 1999-2020.

BACKGROUND: Lung cancer remains a leading cause of morbidity and mortality in the United States. Given the importance of epidemiological insight on lung cancer outcomes as the foundation for targeted interventions, we aimed to examine lung cancer death trends in the United States in the recent 22-year period, exploring demographic disparities and yearly mortality shifts. METHODS: Mortality information was obtained from the CDC Wide-ranging Online Data for Epidemiologic Research database from the years 1999-2020. Demographic information included age, sex, race or ethnicity, and area of residence. We performed log-linear regression models to assess temporal mortality shifts and calculated average annual percentage change (AAPC) and compared age-adjusted mortality rates (AAMR) across demographic subpopulations. RESULTS: A total of 3,380,830 lung cancer deaths were identified. The AAMR decreased from 55.4 in 1999-31.8 in 2020 (p<0.001). Males (AAMR 57.6) and non-Hispanic (NH) (AAMR 47.5) populations were disproportionately impacted compared to females (AAMR 36.0) and Hispanic (AAMR 19.1) populations, respectively. NH Black populations had the highest AAMR (48.5) despite an overall reduction in lung cancer deaths (AAPC -3.3 %) over the study period. Although non-metropolitan regions were affected by higher mortality rates, the annual decrease in mortality among metropolitan regions (AAPC -2.8 %, p<0.001) was greater compared to non-metropolitan regions (AAPC -1.7 %, p<0.001). Individuals living in the Western US (AAPC -3.4 %, p<0.001) experienced the greatest decline in lung cancer mortality compared to other US census regions. CONCLUSIONS: Our findings revealed lung cancer mortality inequalities in the US. By contextualizing these mortality shifts, we provide a larger framework of data-driven initiatives for societal and health policy changes for improving access to care, minimizing healthcare inequalities, and improving outcomes.

Perceived discrimination and mental health among female migrant domestic workers in Hong Kong: A sequential explanatory mixed-methods study.

OBJECTIVES: Female migrant domestic workers (MDW), often unemployed in their home country, are household workers that migrate abroad for better wages. Although poor employment conditions have shown detrimental effects on MDWs health, the mental health effect of perceived discrimination remains understudied among MDWs. This mixed-methods study seeks to (a) assess the association between perceived discrimination and mental health among female MDWs and (b) explore in-depth the common ways MDWs experience discrimination. METHOD: A cross-sectional self-administered survey (n = 1965) was conducted among Filipino and Indonesian MDWs from August 2020 to August 2021 in Hong Kong. A multivariable logistic regression model, controlling for background characteristics, assessed associations between perceived discrimination with anxiety and depression. Qualitative semistructured interviews were then conducted (n = 20) to provide in-depth information about perceived discrimination. Thematic analysis was used to identify the contexts and types of discrimination experienced. RESULTS: Among survey respondents, 60.4% reported ever experiencing discrimination, and 10.5% reported often/always feeling discriminated against. Of MDWs, 18.1% and 31.5% were classified with anxiety and depression, respectively. MDWs reporting higher frequency of discrimination were at increased risk of anxiety (ORadj: 2.30-6.60) and depression (ORadj: 2.06-5.91). In-depth interviews revealed that perceived discrimination inside the workplace (from overwork, lack of autonomy, and employer-imposed restrictions) and outside the workplace (from MDW policies) had strong effects on MDWs' mental health. CONCLUSIONS: Increased availability to mental health services should be considered. To improve MDW mental health, policymakers may also regulate maximum weekly working hours and ensure minimum standards for living environments. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Serologic Testing of US Blood Donations to Identify Severe Acute Respiratory Syndrome Coronavirus 2 and Other Coronaviruses, December 2019 to July 2020.

Background: The first coronavirus disease 2019 (COVID-19) case in the United States was recognized on 19 January 2020, but the time of introduction of the virus into the United States is unknown. An existing sample cohort was examined for serologic evidence of early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Methods: A repository of 46 120 samples from healthy routine blood donors, representing 46 states and the District of Columbia, was tested for total antibodies to SARS-CoV-2 nucleocapsid (anti-N) using a commercial test. All reactive samples were further tested using an experimental receptor-binding domain (RBD)-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay. Further testing was also conducted for anti-spike (anti-S) antibodies by commercial tests, experimental anti-S immunologic blocking, and for antibodies to the 4 human cold coronaviruses. Results: Anti-N reactivity was observed in 92 tested samples (0.2%), 91 of which had adequate volume for further testing; of these, 55 were confirmed positive by anti-RBD. None of these reactive findings were attributable to the other human coronaviruses tested. The confirmed-positive frequency increased over time paralleling patterns observed for COVID-19 cases reported in the United States (in contrast to stable patterns over time for the cold coronaviruses). Nine confirmed positive samples (0.07%) were identified among the 13 364 donations collected between 13 December 2019 and 22 January 2020. None of these early confirmed-positive samples were reactive by commercial anti-S tests suggesting very recent infection. Conclusions: The samples tested in this study were broadly representative of the United States, and all were from individuals who had successfully donated blood. The antibody-reactive results of this study suggest that SARS-CoV-2 was likely present in the United States before 19 January 2020.

Dissociative photoionization of acetaldehyde in the 10.2-19.5 eV VUV range.

The valence-shell dissociative photoionization of acetaldehyde has been investigated by means of the photoion photoelectron coincidence technique in conjunction with tuneable synchrotron radiation. The experimental results consist of threshold photoelectron spectra for the parent ion and for each fragment ion in the 10.2-19.5 eV photon energy range, along with (ion, e) kinetic energy coincidence diagrams obtained from measurements at fixed photon energies. The results are complemented by high-level ab initio calculations of potential energy curves as a function of the C-H bond distance. The nudged elastic band (NEB) method has been employed to connect the parent ion Franck-Condon region to the formation of the HCO+, CH3+ and CH4+ ion fragments. Appearance energies have been determined for six fragment ions with an improved accuracy, including two fragmentation channels, which to the best of our knowledge have not been reported previously, i.e. the formation of CH2CO+, lying at 13.10 ± 0.05 eV, and the formation of CH2+ at 15.1 ± 0.1 eV. Based on both experimental and theoretical results, the dissociation dynamics following ionization of acetaldehyde into the different fragmentation channels are discussed.

Absence of evidence of transfusion transmission risk of Creutzfeldt-Jakob disease in the United States: Results froma 28-year lookback study.

BACKGROUND: For many years, there has been concern about the risk of transmission of classic forms of Creutzfeldt-Jakob disease (CJD) by blood transfusion, particularly after the recognition of such transmission of variant CJD (vCJD). We report on a 28-year lookback study of recipients of blood from donors who subsequently developed CJD. METHODS: Patients with diagnosed CJD and a history of blood donation were identified. Blood centers were asked to provide information about the distribution of the donations and consignees were requested to provide information about the recipients of the donations. Vital status of each available recipient was determined and, if deceased, the reported cause(s) of death were obtained primarily from the National Death Index. All recipients included in the study database contributed person-time up to the last recorded review of vital status. RESULTS: There were 84 eligible donors who gave 3284 transfusable components, and it was possible to evaluate 1245 recipients, totaling 6495 person-years of observation. The mean observation period per recipient was 5.5 years with a maximum of 51 years. No case of CJD or prion disease was reported among the recipient population. DISCUSSION: The study suggests that CJD may not be transfusion-transmissible, a position in agreement with similar findings from two similar European reports amounting to an overall observation period of 15,500 person-years. These studies have supported the conclusion that the risk, if any, of transmission of CJD by blood products is extremely small and remains theoretical.

Changes in transfusion-transmissible infection prevalence and demographics among US blood donors during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic impacted the US blood supply. We compared blood donor demography and infectious disease prevalence before and during the pandemic using a large multicenter database. METHODS: Data were categorized as "Before COVID-19" (March 2018-February 2020) or "During COVID-19" (March 2020-February 2022). Donor demographics, donation frequency, and infectious marker prevalence of HIV, HBV, and HCV were compared for the two time periods. The odds of a donor testing positive for these infections among the two time periods were calculated using multivariable logistic regression. RESULTS: Our study assessed a total of 26,672,213 donations including 13,430,380 before and 13,241,833 during COVID-19. There were significantly more donations from donors who were female, aged 40 and older, white, and repeat, during COVID-19. Donation frequency comparison quantified the increase in donations from donors who were white, female, older, and repeat during the pandemic. The prevalence of HIV and HCV decreased significantly during COVID-19 compared to before, but not for HBV. For HIV, the adjusted odds of infection during the pandemic did not differ but for HBV, the odds were significantly more likely during the pandemic and were significantly lower for HCV. DISCUSSION: Demographics and infectious disease marker prevalence changed during the COVID-19 pandemic in the United States. Prevalence of each infection in the donor population will continue to be monitored to determine if changes were specific to the pandemic period.

Radiomics and artificial intelligence for risk stratification of pulmonary nodules: Ready for primetime?

Pulmonary nodules are ubiquitously found on computed tomography (CT) imaging either incidentally or via lung cancer screening and require careful diagnostic evaluation and management to both diagnose malignancy when present and avoid unnecessary biopsy of benign lesions. To engage in this complex decision-making, clinicians must first risk stratify pulmonary nodules to determine what the best course of action should be. Recent developments in imaging technology, computer processing power, and artificial intelligence algorithms have yielded radiomics-based computer-aided diagnosis tools that use CT imaging data including features invisible to the naked human eye to predict pulmonary nodule malignancy risk and are designed to be used as a supplement to routine clinical risk assessment. These tools vary widely in their algorithm construction, internal and external validation populations, intended-use populations, and commercial availability. While several clinical validation studies have been published, robust clinical utility and clinical effectiveness data are not yet currently available. However, there is reason for optimism as ongoing and future studies aim to target this knowledge gap, in the hopes of improving the diagnostic process for patients with pulmonary nodules.

Rates of Downstream Procedures and Complications Associated With Lung Cancer Screening in Routine Clinical Practice : A Retrospective Cohort Study.

BACKGROUND: Lung cancer screening (LCS) using low-dose computed tomography (LDCT) reduces lung cancer mortality but can lead to downstream procedures, complications, and other potential harms. Estimates of these events outside NLST (National Lung Screening Trial) have been variable and lacked evaluation by screening result, which allows more direct comparison with trials. OBJECTIVE: To identify rates of downstream procedures and complications associated with LCS. DESIGN: Retrospective cohort study. SETTING: 5 U.S. health care systems. PATIENTS: Individuals who completed a baseline LDCT scan for LCS between 2014 and 2018. MEASUREMENTS: Outcomes included downstream imaging, invasive diagnostic procedures, and procedural complications. For each, absolute rates were calculated overall and stratified by screening result and by lung cancer detection, and positive and negative predictive values were calculated. RESULTS: Among the 9266 screened patients, 1472 (15.9%) had a baseline LDCT scan showing abnormalities, of whom 140 (9.5%) were diagnosed with lung cancer within 12 months (positive predictive value, 9.5% [95% CI, 8.0% to 11.0%]; negative predictive value, 99.8% [CI, 99.7% to 99.9%]; sensitivity, 92.7% [CI, 88.6% to 96.9%]; specificity, 84.4% [CI, 83.7% to 85.2%]). Absolute rates of downstream imaging and invasive procedures in screened patients were 31.9% and 2.8%, respectively. In patients undergoing invasive procedures after abnormal findings, complication rates were substantially higher than those in NLST (30.6% vs. 17.7% for any complication; 20.6% vs. 9.4% for major complications). LIMITATION: Assessment of outcomes was retrospective and was based on procedural coding. CONCLUSION: The results indicate substantially higher rates of downstream procedures and complications associated with LCS in practice than observed in NLST. Diagnostic management likely needs to be assessed and improved to ensure that screening benefits outweigh potential harms. PRIMARY FUNDING SOURCE: National Cancer Institute and Gordon and Betty Moore Foundation.

Employment conditions and mental health of overseas female migrant domestic workers in Hong Kong: a parallel mediation analysis.

BACKGROUND: Female migrant domestic workers (MDW), approximately 8.5 million globally, often live in their employer's home under vulnerable conditions. In Hong Kong, MDWs currently comprise 5% of the population. This study was conducted to assess the association between employment conditions and mental health, and the mediating roles stress and job satisfaction have, among female MDWs in Hong Kong. METHODS: Participants completed an online cross-sectional survey. A total of 1,965 survey were collected between August 2020 and August 2021. Questions in the survey were related to MDWs background information, employment conditions, stress, job satisfaction, and two mental health outcomes: anxiety and depression. An employment conditions score was created to assess the cumulative effect poor employment conditions had on mental health. A multicategorical parallel mediation analysis was used to assess the direct effect employment conditions have on mental health and the indirect effects through stress and job satisfaction. RESULTS: Overall, 17.7% of MDWs were reported to be suffering from anxiety and 30.8% from depression. An increase in poor employment conditions was statistically associated with an increase in both outcomes, while stress levels and job satisfaction mediated this association. CONCLUSIONS: The findings call for increased scrutiny of employment conditions and mental well-being of MDWs.

Syphilis seroprevalence and incidence in US blood donors from 2020 to 2022.

BACKGROUND: HIV, HBV, and HCV infections for ~60% of the US blood supply are monitored by TTIMS with syphilis added in 2020. STUDY DESIGN AND METHODS: Data were compiled from October 2020 to September 2022. Syphilis prevalence was estimated for allogeneic and directed donors who were consensus positive (CP) and the subset of those with confirmed-active infections (AI). Prevalence and incidence were stratified by demographics for two consecutive 1-year periods, starting October 1, 2020 and for both years combined. Incidence was estimated for repeat donors. Associations between syphilis positivity and other infections were evaluated. RESULTS: Among 14.75 million donations, syphilis prevalence was 28.4/100,000 donations and significantly higher during the second year compared to the first year. Overall, syphilis incidence for the two-year period was 10.8/100,000 person-years. The adjusted odds of a CP infection were 1.18 (95% CI: 1.11, 1.26) times higher in the second year compared to the first, and for AI, 1.22 (95% CI: 1.10, 1.35) times higher in year 2. Highest rates occurred among males, first-time, Black, and younger (ages 18-39) donors, and those in the South US Census region. Syphilis CP donors were 64 (95% CI: 46, 89) times more likely to be HIV CP, and AI donors 77 (95% CI: 52, 114) times more likely to be HIV CP than non-CP donors, when controlling for confounders. SUMMARY/CONCLUSIONS: Syphilis prevalence increased over the study period mirroring national trends reported by CDC and is significantly associated with HIV CP.

Resonant Photoionization of CO2 up to the Fourth Ionization Threshold.

We present a comprehensive theoretical study of valence-shell photoionization of the CO2 molecule by using the XCHEM methodology. This method makes use of a fully correlated molecular electronic continuum at a level comparable to that provided by state-of-the-art quantum chemistry packages in bound-state calculations. The calculated total and angularly resolved photoionization cross sections are presented and discussed, with particular emphasis on the series of autoionizing resonances that appear between the first and the fourth ionization thresholds. Ten series of Rydberg autoionizing states are identified, including some not previously reported in the literature, and their energy positions and widths are provided. This is relevant in the context of ongoing experimental and theoretical efforts aimed at observing in real-time (attosecond time scale) the autoionization dynamics in molecules.

Perineuronal nets and the neuronal extracellular matrix can be imaged by genetically encoded labeling of HAPLN1 in vitro and in vivo.

Neuronal extracellular matrix (ECM) and a specific form of ECM called the perineuronal net (PNN) are important structures for central nervous system (CNS) integrity and synaptic plasticity. PNNs are distinctive, dense extracellular structures that surround parvalbumin (PV)-positive inhibitory interneurons with openings at mature synapses. Enzyme-mediated PNN disruption can erase established memories and re-open critical periods in animals, suggesting that PNNs are important for memory stabilization and conservation. Here, we characterized the structure and distribution of several ECM/PNN molecules around neurons in culture, brain slice, and whole mouse brain. While specific lectins are well-established as PNN markers and label a distinct, fenestrated structure around PV neurons, we show that other CNS neurons possess similar extracellular structures assembled around hyaluronic acid, suggesting a PNN-like structure of different composition that is more widespread. We additionally report that genetically encoded labeling of hyaluronan and proteoglycan link protein 1 (HAPLN1) reveals a PNN-like structure around many neurons in vitro and in vivo. Our findings add to our understanding of neuronal extracellular structures and describe a new mouse model for monitoring live ECM dynamics.

Social vulnerability and lung malignancy mortality.

INTRODUCTION: Lung cancer is a major cause of death in the United States. Social determinants of health (SDOH) are important factors that impact the treatment and prognosis of lung cancer. The social vulnerability index (SVI) is a validated measure of SDOH. This cross-sectional study aimed to investigate the impact of the SVI on lung cancer mortality using descriptive epidemiology. METHODS: Mortality data for lung malignancies from 2014 to 2018 was obtained from the CDC database and was age-adjusted and standardized to the population in the year 2000. The SVI for the same years was obtained from the CDC Agency for Toxic Substances and Disease Registry database. Age-adjusted mortality rates (AAMR) were estimated for each SVI quartile (SVI-Q) and demographic subgroup. RESULTS: We found that counties in SVI-Q4 (most vulnerable) had a higher cumulative AAMR compared to counties in SVI-Q1 (least vulnerable), accounting for a 4.48 excess death rate per 100,000 person-years. AAMR among males in SVI-Q4 was higher compared to SVI-Q1, accounting for a 9.96 excess death rate per 100,000 person-years, whereas no mortality differences were observed for female populations between SVI-Q4 and SVI-Q1. AAMR in SVI-Q4 was higher for both Hispanic and non-Hispanic populations, except for American Indian/Alaska Native populations. Similar trends were observed in both metropolitan and non-metropolitan counties. CONCLUSION: Our study suggests that the SVI may play a significant role in lung cancer mortality and highlights the need for interventions targeting vulnerable populations to improve outcomes.

Bivalent non-human gal-α1-3-gal glycan epitopes in the Fc region of a monoclonal antibody model can be recognized by anti-Gal-α1-3-Gal IgE antibodies.

Monoclonal antibody (mAb) production using non-human cells can introduce non-human glycan epitopes including terminal galactosyl-α1-3-galactose (α1-3-Gal) moieties. Cetuximab is a commercial mAb associated with causing anaphylaxis in some patients due to the binding of endogenous anti-α1-3-Gal IgE to the Fab (containing bi-α1-3-galactosylated glycans) but not to the Fc region (containing mono-α1-3-galactosylated glycans). Despite being low in abundance in typical commercial mAbs, the inherent sensitivity of cell culture conditions on glycosylation profiles, and the development of novel glycoengineering strategies, novel antibody-based modalities, and biosimilars by various manufacturers with varying procedures, necessitates a better understanding of the structural requirements for anti-α1-3-Gal IgE binding to the Fc region. Herein, we synthesized mAb glycoforms with varying degrees and regioisomers of α1-3-galactosylation and tested their binding to two commercial anti-α1-3-Gal human IgE antibodies derived from a human patient with allergies to red meat (comprising α1-3-Gal epitopes), as well as to the FcγRIIIA receptor. Our results demonstrate that unexpectedly, anti-α1-3-Gal human IgE antibodies can bind to Fc glycans, with bi-α1-3-galactosylation being the most important factor, highlighting that their presence in the Fc region may be considered as a potential critical quality attribute, particularly when using novel platforms in mAb-based biotherapeutics.

Tissue-Engineered Disease Modeling of Lymphangioleiomyomatosis Exposes a Therapeutic Vulnerability to HDAC Inhibition.

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2-/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.

Structural and photophysical characterization of the small ultra-red fluorescent protein.

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

Trajectory and Demographic Correlates of Antibodies to SARS-CoV-2 Nucleocapsid in Recently Infected Blood Donors, United States.

We evaluated antibodies to the nucleocapsid protein of SARS-CoV-2 in a large cohort of blood donors in the United States who were recently infected with the virus. Antibodies to the nucleocapsid protein of SARS-CoV-2 indicate previous infection but are subject to waning, potentially affecting epidemiologic studies. We longitudinally evaluated a cohort of 19,323 blood donors who had evidence of recent infection by using a widely available serologic test to determine the dynamics of such waning. We analyzed overall signal-to-cutoff values for 48,330 donations (average 2.5 donations/person) that had an average observation period of 102 days. The observed peak signal-to-cutoff value varied widely, but the waning rate was consistent across the range, with a half-life of 122 days. Within the cohort, only 0.75% of persons became seronegative. Factors predictive of higher peak values and longer time to seroreversion included increasing age, male sex, higher body mass index, and non-Caucasian race.

Do perineuronal nets stabilize the engram of a synaptic circuit?

Perineuronal nets (PNN), a specialized form of ECM (?), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesis that PNNs serve as gatekeepers that guard and protect synaptic territory, and thus may stabilize an engram circuit. We present high-resolution, and 3D EM images of PNN- engulfed neurons showing that synapses occupy the PNN holes, and that invasion of other cellular components are rare. PNN constituents are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or MMP9 knockout mice allowed normal fear memory acquisition but diminished remote-memory stabilization, supporting the above hypothesis. Significance: In this multidisciplinary work, we challenge the hypothesis that the pattern of holes in the perineuronal nets (PNN) hold the code for very-long-term memories. The scope of this work might lead us closer to the understanding of how we can vividly remember events from childhood to death bed. We postulate that the PNN holes hold the code for the engram. To test this hypothesis, we used three independent experimental strategies; high-resolution 3D electron microscopy, Stable Isotop Labeling in Mammals (SILAM) for proteins longevity, and pharmacologically and genetically interruption of memory consolidation in fear conditioning experiments. All of these experimental results did not dispute the PNN hypothesis.