Polycystic ovarian syndrome (PCOS) is the most common endocrinological disorder in women, in which, besides chronic anovulation/oligomenorrhea and ovarian cysts, hyperandrogenism plays a critical role in a large fraction of subjects. Inositol isomers—myo-Inositol and D-Chiro-Inositol—have recently been pharmacologically effective in managing many PCOS symptoms while rescuing ovarian fertility. However, some disappointing clinical results prompted the reconsideration of their specific biological functions. Surprisingly, D-Chiro-Ins stimulates androgen synthesis and decreases the ovarian estrogen pathway; on the contrary, myo-Ins activates FSH response and aromatase activity, finally mitigating ovarian hyperandrogenism. However, when the two isomers are given in association—according to the physiological ratio of 40:1—patients could benefit from myo-Ins enhanced FSH and estrogen responsiveness, while taking advantage of the insulin-sensitizing effects displayed mostly by D-Chiro-Ins. We need not postulate insulin resistance to explain PCOS pathogenesis, given that insulin hypersensitivity is likely a shared feature of PCOS ovaries. Indeed, even in the presence of physiological insulin stimulation, the PCOS ovary synthesizes D-Chiro-Ins four times more than that measured in control theca cells. The increased D-Chiro-Ins within the ovary is detrimental in preserving steroidogenic control, and this failure can easily explain why treatment strategies based upon high D-Chiro-Ins have been recognized as poorly effective. Within this perspective, two factors emerge as major determinants in PCOS: hyperandrogenism and reduced aromatase expression. Therefore, PCOS could no longer be considered a disease only due to increased androgen synthesis without considering the contemporary downregulation of aromatase and FSH receptors. Furthermore, these findings suggest that inositols can be specifically effective only for those PCOS phenotypes featured by hyperandrogenism. Polycystic ovarian syndrome (PCOS) is the most common cause of anovulation , initialWomen suffering from PCOS—excluding the phenotype D—share several clinical features of hyperandrogenism, including hirsutism, acne, and alopecia . ExcessiA thought-provoking paper published almost twenty years ago reported very intriguing results . In PCOSHow could we explain this abnormal sensitivity? A significant contribution in explaining this puzzle has been provided by Larner’s seminal paper demonstrating that ovarian abnormal response to insulin alters the specific myo-Inositol/D-Chiro-Inositol (myo-Ins/D-Chiro-Ins) ratio within the ovary . PreviouNevertheless, it is well established that ovarian cells from PCOS subjects retain insulin sensitivity, even in those patients showing insulin resistance, as both insulin and hyperinsulinemia still stimulate ovarian androgen production in PCOS . TherefoMoreover, the increase in D-Chiro-Ins availability within the ovary following insulin stimulation would provide increased chiro-inositol to be incorporated into precursor GPI-phospholipid and or precursor GPI-protein. Insulin activates phospholipase anchored to the external layer of cell membranes , thus fuThese results have outstanding consequences, given that IPGs have been shown to be required for androgen synthesis downstream of insulin stimulation . NoticeaA study performed on granulosa cells obtained from PCOS women has shown that D-Chiro-Ins reduces the mRNA expression of both aromatase and cytochrome P450 side-chain cleavage genes in a dose–response fashion . PrelimiThese recent data can help explain some puzzling clinical results provided by treating PCOS patients with high doses of D-Chiro-Ins. In a study authored by Nestler’s group, a positive correlation between changes in IPG-P release and in insulin sensitivity in PCOS patients treated with very high levels of D-Chiro-Ins (1500 mg twice daily for 6 weeks) was observed . UnfortuNonetheless, these assumptions have been challenged for a while by controversial clinical results obtained in PCOS patients treated with inositol formulations, in which both myo-Ins and D-Chiro-Ins have been associated according to different ratios . Again, this is a paradox that prompted us to reconsider the basic physiology of inositol to assess if inositol isomers exert either complementary or opposite actions upon ovarian steroidogenesis.In the middle of the 19th century, Johann Joseph Scherer extracted a polyol-a hexa-hydroxy-cyclohexane compound—hence named “inositol”—from muscle cells . The che−/− animals showed a general modification in the global gene expression profile and a significant increase in embryonic lethality [In humans, myo-Ins (~1 g/day) primarily comes from dietary intake, albeit a relevant fraction is synthesized endogenously, in a variable range (from 1 to 4 g/day), depending on several factors, yet requiring a full assessment . Within ethality . It is wethality , in mammethality . Glucoseethality . This efethality . ReducedThe cell’s machinery uses myo-Ins to build up several derived inositol-phosphates (InsPs) as well as complex macromolecules, including phosphatidyl-inositol (PI), its seven phosphoinositides , and glycosylphosphatidylinositol-anchored proteins (GPI), localized mostly at the surface of cell membranes . Myo-InsAside from the functions exerted by inositol-derived metabolites, myo-Ins, as such, has been described as a modifier of a number of processes involving endocrine signaling, morphogenesis, and reproduction, just to mention a few ,70,71. TAs previously recalled, under insulin stimulation, tissue-specific epimerases convert myo-Ins into its stereoisomer D-Chiro-ins. Unknown factors finely tune this irreversible reaction to deliver inositol isomers according to variable tissue-dependent needs . The twoThe insulin-sensitizing capabilities of both myo-Ins and D-Chiro-Ins have been confirmed by several clinical studies ,83. HoweIndeed, as previously recalled, D-Chiro-Ins given in isolation enhances androgen synthesis. Yet, as important as it could be, this effect does not explain in full the androgenic phenotype of PCOS patients, given that increased androgen production would, in turn, increase aromatase activity, leading to a proportional increase in estrogen release . Theca cIntriguingly, clinical investigations in which myo-Ins is associated with very low doses of D-Chiro-Ins provided unexpected results, indicating that, while retaining the beneficial, insulin-sensitizing effects exerted by D-Chiro-Ins, the addition of myo-Ins allowed rescuing almost completely the ovarian function ,91. ThesTreatment with myo-Ins (2 g twice a day) significantly decreases the LH/FSH ratio in the plasma of PCOS women ,94, whilThese data indicate that myo-Ins and D-Chiro-Ins play an opposite role in ovarian steroidogenesis, namely acting upon aromatase, androgen release, and FSHr expression. Again, let us outline that the downregulation of FSH receptors and aromatase in granulosa cells represents a hallmark of PCOS . In thisSome observational studies have confirmed these findings, indicating that treatment based upon a proper myo-Ins/D-Chiro-Ins ratio (40:1) can efficiently counteract the foremost PCOS signs and symptoms . ConclusRemarkably, under insulin stimulation, the highest conversion rate of myo-Ins into D-Chiro-Ins is close to 9%, and was detected in the liver and muscle, two important insulin-sensitive areas, whereas in the heart and brain, this is less than 2%. These differences highlight that tissues display differential needs of both isomers to fulfill their specific metabolic demands. Insulin resistance can dramatically impair D-Chiro-Ins levels in many tissues, resulting in low intracellular levels of D-Chiro-Ins . HoweverInsulin resistance cannot explain the complexity of the polycystic ovary syndrome, given that this hypothesis is biased by assuming as a premise that PCOS pathogenesis essentially relies upon defective insulin transduction due to impaired availability of inositolphosphoglycans. This model leaves aside the opposite effects directly triggered by myo-Ins and D-Chiro-Ins upon ovarian steroidogenesis, and underestimates the specific insulin hypersensitivity displayed by the ovary in PCOS subjects. Obviously, the increased insulin secretion, as that observed in insulin-resistant subjects, can amplify the steroidogenic response of the ovary by raising D-Chiro-Ins levels in the gonads. Nevertheless, it cannot explain the abnormal insulin sensitivity displayed by the ovaries of PCOS patients, which show an increased presence of androgens and their related enzymes when stimulated by LH and insulin with respect to normal controls . Thus, iConclusively, insulin resistance is not a general feature of PCOS, since the prevalence of insulin alterations involves barely around 60% of patients . FurtherThe conclusive pathogenic hypothesis based on the preliminary hints provided by Larner  and UnfeWhen the two isomers are given in association—according to the physiological ratio of 40:1—patients could benefit from the insulin-sensitizing effects displayed mostly by D-Chiro-Ins. Yet, Larner’s study indicates clearly that we need not postulate insulin resistance to explain PCOS pathogenesis, given that insulin hypersensitivity is likely a common feature of PCOS ovaries. Indeed, even in the presence of a physiological insulin stimulus, the PCOS ovary synthesizes D-Chiro-Ins four times more than that measured in control theca cells. The increased D-Chiro-Ins within the ovary is detrimental to ensuring proper steroidogenic control, and this failure can easily explain why treatment strategies based upon high D-Chiro-Ins have been poorly effective. Within this perspective, two factors emerge as major determinants in PCOS: hyperandrogenism and reduced aromatase expression. Therefore, PCOS could no longer be considered a disease due to only increased androgen synthesis, without considering the contemporary downregulation of aromatase and FSH receptors.However, that model raises several unanswered issues, which should be addressed by future studies.First, why do different tissues show different myo-Ins/D-Chiro-Ins ratios, and how could differences in epimerase activity explain these data? Is there any difference in the structure of the D-Chiro-Ins epimerase among different tissues? In other words, what factors actually shape the different epimerase sensitivity to insulin in different histological contexts? Second, are there any other factors that prevent theca androgens from undergoing the physiological conversion into estrogens when ovaries are treated with D-Chiro-Ins? Third, how do inositols modify other endocrine receptors, LHr, AR, and ER, when both theca and granulosa cells are treated with both the two inositol isomers? Some preliminary reports show indeed that alternative splicing dramatically alters AR recruitment and androgen-induced expression of genes related to folliculogenesis in human granulosa cells, namely by impairing aromatase expression . It is tMoreover, how does myo-Ins supplementation modify the complex inositol-related network, and how could inositol-related metabolites influence the overall picture? To obtain a reliable picture, we should be able to capture the whole metabolic fate of myo-Ins when added to a specific tissue/cellular context and correlate changes in inositol metabolism with the associated biochemical pathways .Finally, maybe we should start thinking of tailored therapies for the different PCOS phenotypes, particularly for the non-hyperandrogenic D phenotype, which could be properly managed by natural, active compounds other than inositols.Overall, these tasks are challenging. Integrated, multidisciplinary approaches—performed in in vitro and in animal studies—are warranted to address those issues, hence providing a compelling confirmation of the proposed pathogenetic hypothesis.


Dexmedetomidine has been subjected to an extensive evaluation for its’ role inthe prevention of postoperative delirium following cardiac surgery. In strikingcontrast to the preexisting meta-analysis supporting postoperativedelirium-reduction with dexmedetomidine, few recently concluded multicentriclarge scale randomized controlled trials suggest otherwise. This article aims topresent a nuanced perspective of the evolving controversy by attempting todecode the apparent incongruences in the literature accumulating off-late, whichis particularly pertinent amidst an ever-escalating heterogeneity in the currentresearch ecosystem. Turanet al.8]. Li11,12][. An eleven-RCT meta-analysis by Abowali et al.11][ highlighted that Abowali et al.11][ meta-analysis excluded theRCTs which did not rule out a preoperative cognitive impairment.Looking beyond the categorical details of the meta-analysis13][.Even from a holistic research standpoint, the traditionally cited evidence hierarchy ofmeta-analysis over and above multiple and single RCTs is an oversimplistic rendition,mandating a cautious interpretation


The overall patterns are similar to what has been observed in the close relative P. berghei, but here we show that gametocyte-associated pir genes are distinct from those involved in chronic blood-stage infection and highlight a male-associated pir gene which should be the focus of future studies.Infected red blood cells containing female and male The online version contains supplementary material available at 10.1186/s13104-023-06322-1. Plasmodium species contain several multigene families, the largest of which is the pir multigene family shared by Plasmodium vivax which infects humans, Plasmodium species infecting monkey and apes and members of the Vinckei subgenus infecting rodents . The rofe cycle . We haveirulence , 4. We h RNA-seq , 6, theschabaudi . Here wepir genes in Plasmodium chabaudi chabaudi (AS) gametocytes during a blood-stage infection in C57BL/6 mice. Using transgenic parasites, in which male and female gametocytes are specifically tagged with a fluorochrome via Dynein  .


P < .001) in states that prohibited short-term limited duration insurance plans vs did not regulate short-term limited duration insurance plans. State policies resulting in unavailability of short-term limited duration insurance plans were associated with an increased percentage of early-stage diagnoses.Short-term limited duration insurance plans, which proliferated following 2018 federal regulations, may not provide adequate protections for patients with suspected or newly diagnosed cancer and can destabilize insurance markets for comprehensive insurance plan enrollees. Individuals aged 18-64 years with newly diagnosed cancer from 11 states during 2016-2017 and 2019 were identified from the Surveillance, Epidemiology, and End Results program. Difference-in-differences analyses were used to compare changes in early-stage cancer diagnoses from 2016-2017 to 2019 in states that prohibited vs did not regulate short-term limited duration insurance plans. In adjusted difference-in-differences analyses, early-stage diagnoses increased 0.95 percentage points (95% confidence interval = 0.53 to 1.38,  In 2018, federal regulations expanded access to short-term limited duration insurance plans, which can exclude people with preexisting conditions like cancer, limit coverage of medical services including cancer screening, and have no cost-sharing caps .Uptake of short-term limited duration insurance plans by healthy individuals can increase premium costs for comprehensive insurance plan enrollees by changing risk pools and destabilizing markets for individual insurance . SeveralIndividuals aged 18-64 years with staged first primary malignancies diagnosed 2016-2019 were identified from Surveillance, Epidemiology, and End Results (SEER) registry data from 11 states covering 26.5% of the US population  in states that prohibited short-term limited duration insurance plans vs did not regulate short-term limited duration insurance plans, translating to approximately 650 more early-stage diagnoses in 2019 in short-term limited duration insurance–prohibiting states, with less change in states with other short-term limited duration insurance policies  (P = .05) percentage point increase in early-stage diagnoses in states where legislation stopped the sale of short-term limited duration insurance plans   . Relativ < .001) , translaRelative to states without policies enhancing access to care, states with increases in early-stage diagnosis percentages associated with 2018-2019 health policy legislation included California, Hawaii, New Jersey, and New Mexico (Using SEER data spanning 11 states, state legislation prohibiting short-term limited duration insurance plans was associated with a differentially increased percentage of early-stage cancer diagnoses, translating to approximately 650-800 more individuals diagnosed with early rather than late-stage cancers. In other words, states allowing the sale of short-term limited duration insurance plans experienced a relative decrease in percentages of early-stage cancer diagnoses. However, limited legislation regulating but still enabling sale of short-term limited duration insurance plans was not associated with changes in the percentage of early-stage diagnoses.State-level short-term limited duration insurance policies may influence stage at diagnosis in at least 2 ways. First, short-term limited duration insurance plan enrollees may experience coverage gaps and delays for cancer screening and/or diagnostic workup because of inadequate health-care access . Second,This is the first study to our knowledge to assess potential impacts of short-term limited duration insurance plans and associated policies on cancer diagnosis. However, the study is limited by its retrospective nature. Many states enacted other policies in the time period that could affect the health insurance market and care delivery and ultimately confound our findings. However, in a sensitivity analysis, all states with statistically significant increases in early-stage diagnoses severely restricted short-term limited duration insurance plans, and those states shared no other contemporary health insurance policies. Additionally, our study was limited to 11 states available within the SEER program, which may not be representative of other states. Our study period is limited, with only 1-year follow-up after short-term limited duration insurance plan expansion, so long-term impacts are unclear. Furthermore, it is unclear whether individuals who otherwise would be uninsured might benefit from short-term limited duration insurance plans, though the difference-in-differences results suggest a net harm. Finally, individual insurance plan data are lacking; however, to the best of our knowledge, there are no existing databases that adequately capture short-term limited duration insurance plan information, so a quasi-experimental analysis such as this may provide the best evidence regarding the clinical impacts of short-term limited duration insurance plans.In conclusion, state policies prohibiting short-term limited duration insurance plans were associated with increased percentages of early-stage diagnoses. Our findings can inform state and federal policy makers about the adverse and unintended consequences of short-term limited duration insurance plans as part of efforts to improve cancer outcomes, particularly in the context of a new proposed federal rule limiting short-term limited duration insurance plans .pkad060_Supplementary_DataClick here for additional data file.


Antibody-drug conjugates are attractive targeted agents in anticancer treatment because of their unique mechanism of action and reduced toxicity. Little is known about the spectrum of adverse events associated with antibody-drug conjugates, despite tens of clinical trials.A systematic review of randomized controlled trials evaluating antibody-drug conjugate efficacy in anticancer treatment was conducted. PubMed, EMBASE, and ClinicalTrial.gov were searched for relevant studies. Meta-analyses assessed the odds ratios (ORs) of 12 treatment-related symptoms and toxicities in patients treated with antibody-drug conjugates compared with those receiving other anticancer agents without antibody-drug conjugates. All-grade and high-grade (grade ≥3) toxicities were examined.Twenty studies involving 10 075 patients were included. Compared with control groups, antibody-drug conjugates were associated with a higher risk of all-grade fatigue , anorexia , nausea , and sensory neuropathy  as treatment-related symptoms. Patients treated with antibody-drug conjugates had a statistically significantly lower risk of all-grade febrile neutropenia . Conversely, they had a higher risk of thrombocytopenia , increased alanine aminotransferase , and increased aspartate aminotransferase . Subgroup analysis showed a similar toxicity profile when comparing the solid tumors with hematologic malignancy groups and the antibody-drug conjugate vs antibody-drug conjugate plus chemotherapy groups, except for some neurologic and hematologic adverse events.This comprehensive profile of adverse events associated with antibody-drug conjugate–based treatment shows an increase in various types of all-grade treatment-related symptoms and adverse events, although no increase in high-grade adverse events was seen. The use of antibody-drug conjugates in cancer treatment has seen a substantial rise, yet the toxicity profiles of these agents remain largely unknown. Antibody-drug conjugates constitute a class of anticancer agents that consist of cytotoxic payloads, monoclonal antibodies, and chemical linkers joining them. Antibody-drug conjugates are directed toward a target antigen overexpressed on the cancer cell surface ,2. When Despite the growing interest in and enthusiasm for antibody-drug conjugates, challenges remain to find strategies for further optimization of molecular targeted therapies with greater efficacy and less toxicity. Although antibody-drug conjugates may reduce systemic toxicities compared with conventional cytotoxic drugs by minimizing off-target effects, previous studies have shown that antibody-drug conjugates are associated with unexpected dose-limiting toxicities . These tWe conducted a systematic review and meta-analysis of treatment-related adverse events in patients receiving antibody-drug conjugate–based therapy to provide oncologists with a better understanding of how to monitor and manage these antibody-drug conjugate–related toxicities.This meta-analysis was conducted under using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses  reportinWe used a 2-level strategy to search for all randomized controlled trials investigating the effectiveness of antibody-drug conjugates. As a first step, we used PubMed, EMBASE, and ClinicalTrial.gov for a comprehensive search on January 10, 2023. We used (“antibody-drug conjugate” or “ADC”) and  as key search terms. We then performed an additional manual search of secondary sources, including studies referenced in articles retrieved during the first step, to accomplish a comprehensive review.Studies meeting the following criteria were eligible for inclusion in our review:  the studData were independently extracted from eligible studies by 2 investigators (S.Z. and Y.S.). Any discrepancies between reviewers were resolved by consensus. The following information was recorded for each study: first author’s name, publication year, trial phase, blinding, malignancy type, number of patients available for analysis, type of the antibody-drug conjugate in the treatment arm, placebo or treatment drug in control arm, and the grade of toxicity. The Cochrane Risk of Bias Tool was used to evaluate the risk of bias for each randomized controlled trial . The quaP < .05 was considered statistically significant. Cochran Q test and I2 statistics were used to assess the heterogeneity in each adverse event analysis. I2 values of greater than 50% were considered evidence of substantial heterogeneity in the study. Egger linear regression tests and funnel plots were applied in ProMeta, version 3.0, to adverse events to show statistically significant differences in risk and evaluate publication bias. Subgroup analyses were also conducted based on cancer type  and antibody-drug conjugate regimen (antibody-drug conjugate vs antibody-drug conjugate plus chemotherapy). The P value for interaction was calculated to determine whether the risk of each adverse event differed between the subgroups. P < .1 for interaction was considered statistically significant in our study  of each all-grade adverse event associated with the use of antibody-drug conjugates. Additionally, we examined the pooled odds ratios of high-grade adverse events (grade ≥3). The number of treated patients and the number of patients who developed adverse events in each treatment arm were recorded from each trial. Adverse events reported in at least 3 randomized controlled trials were selected as toxicity endpoints for meta-analysis to ensure the minimum generalizability of the pooled odds ratio. The odds ratio and corresponding 95% confidence interval (CI) were calculated using random-effects models. ur study . We usedur study .P = .030) and neutropenia (P = .048).The systematic literature search identified 23 386 records, from which 34 potentially eligible studies were collected after intensive screening. The duplicate citations and papers without full text were removed. Only studies registered in ClinicalTrials.gov were included to be able to analyze adverse events comprehensively. Ultimately, 20 randomized controlled trials comprising 10 075 patients met the predefined criteria were included in this meta-analysis. We included 5745 patients who had received any antibody-drug conjugate  and 4330 patients who did not (control arm) . DetailsThe baseline characteristics of the included studies are presented in First, we analyzed a core set of 12 clinically relevant symptoms recommended by the US National Cancer Institute for cross-study comparisons of symptomatic effects . Of thesFigure 2;Antibody-drug conjugates were associated with a statistically significantly lower risk of all-grade febrile neutropenia , leukopenia , lymphopenia , and neutropenia  compared with control groups . AntibodAntibody-drug conjugates were associated with a statistically significantly higher risk of all-grade increased alanine aminotransferase  and increased aspartate aminotransferase  than antibody-drug conjugate–free therapy Figure 2;.Antibody-drug conjugates were associated with a statistically significantly higher risk of cataract , eye pain , photophobia , and blurred vision  than seen in control groups Figure 2;.Antibody-drug conjugates were associated with a statistically significantly decreased risk of hot flush  compared with the control groups . There wWith regard to adverse events related to the kidney and urinary system, there was no statistically significant difference in the incidence of all-grade and high-grade adverse events between groups with and without antibody-drug conjugates .Antibody-drug conjugates were associated with a statistically significantly increased risk of all-grade epistaxis  . There wAntibody-drug conjugates were associated with a statistically significantly increased risk of all-grade abdominal pain , upper abdominal pain , dry mouth , vomiting , gingival bleeding  Figure 2;.Antibody-drug conjugates were associated with a statistically significantly increased risk of all-grade headache  and peripheral motor neuropathy  . There wI2 > 50%), we conducted subgroup analyses by type of cancer  and antibody-drug conjugate regimen (antibody-drug conjugates vs antibody-drug conjugates plus chemotherapy). The risk of all-grade and high-grade febrile neutropenia, all-grade and high-grade neutropenia, and all-grade leukopenia was higher in patients with hematologic malignancies. The risk of all-grade headache was higher in patients with solid tumors, whereas the risk of all-grade peripheral neuropathy was lower in patients with solid tumors. Otherwise, a similar toxicity profile was observed between solid tumor and hematologic malignancy groups , including pinatuzumab vedotin  and polaConversely, there was a statistically significantly higher incidence of thrombocytopenia in patients who received antibody-drug conjugate–based therapy than in those treated with comparator therapy. The higher incidence of epistaxis and gingival bleeding in antibody-drug conjugate–treated patients may be attributable to the thrombocytopenia associated with antibody-drug conjugates. The increased risk of thrombocytopenia was prominent in patients treated with maitansine- and monomethyl auristatin F (MMAF)–conjugated antibody-drug conjugates ,40. ThroConsistent with a previous report showing a clear payload association with ocular toxicities typically induced by antibody-drug conjugates containing DM4 and MMAF4 , ocular In contrast to the ocular adverse events, which were observed only in DM4- or MMAF-conjugated antibody-drug conjugates, hepatic-related adverse events, such as increases in alanine aminotransferase and aspartate aminotransferase levels, were associated with antibody-drug conjugates of different payloads. These results should raise caution among clinicians considering antibody-drug conjugate–based regimens for patients with preexisting liver disease, regardless of malignancy type and antibody-drug conjugate type ,49.Several limitations of the present study should be noted. First, the sample sizes of the included studies varied statistically significantly, which may explain the heterogeneity in some results. In addition, small sample size may potentially affect the precision of some estimates. Second, the number of studies was still insufficient to conduct subgroup analysis. For example, only 1 trial evaluated the safety of antibody-drug conjugates such as mirvetuximab soravtansine, and sacituzumab govitecan, making it impossible for subgroup analysis based on each antibody-drug conjugate. In addition, of 20 clinical trials included in our meta-analysis, 16 had an open-label design, which may raise the risk of ascertainment bias. A recent report, however, indicated that there is no evidence of statistically significant bias for patient-reported outcomes based on the absence of blinding in oncology clinical trials . Thus, aTo the best of our knowledge, this is the first study to comprehensively compare the tolerability of antibody-drug conjugate–based regimens with other, standard treatments across multiple malignancies. Our analysis suggests that antibody-drug conjugate–based therapy resulted in a higher incidence of various types of all-grade treatment-related adverse events, including cardiovascular, hepatic, gastrointestinal, neurologic, and ocular toxicity, but a statistically significantly lower risk of all-grade hematologic adverse events, except for thrombocytopenia, which were observed in the antibody-drug conjugate–based groups, compared with the groups not using antibody-drug conjugates. Although our results provide valuable information for clinicians to balance the benefits and risks of treatment options in their decision making, it is crucial to consider the limitations of our study, particularly the heterogeneity of included studies, when applying these findings to clinical practice.pkad069_Supplementary_DataClick here for additional data file.


The global increase in cancer incidence presents significant economic and societal challenges. While chimeric antigen receptor-modified T cell (CAR-T) therapy has demonstrated remarkable success in hematologic malignancies and has earned FDA approval, its translation to solid tumors encounters faces significant obstacles, primarily centered around identifying reliable tumor-associated antigens and navigating the complexities of the tumor microenvironment. Recent developments in single-cell RNA sequencing (scRNA-seq) have greatly enhanced our understanding of tumors by offering high-resolution, unbiased analysis of cellular heterogeneity and molecular patterns. These technologies have revolutionized our comprehension of tumor immunology and have led to notable progress in cancer immunotherapy. This mini-review explores the progress of chimeric antigen receptor (CAR) cell therapy in solid tumor treatment and the application of scRNA-seq at various stages following the administration of CAR cell products into the body. The advantages of scRNA-seq are poised to further advance the investigation of the biological characteristics of CAR cells in vivo, tumor immune evasion, the impact of different cellular components on clinical efficacy, the development of clinically relevant biomarkers, and the creation of new targeted drugs and combination therapy approaches. The integration of scRNA-seq with CAR therapy represents a promising avenue for future innovations in cancer immunotherapy. This synergy holds the potential to enhance the precision and efficacy of CAR cell therapies while expanding their applications to a broader range of malignancies. Chimeriin vivo, tumor immune evasion, the impact of different cellular components on clinical efficacy, the development of clinically relevant biomarkers, and the creation of new targeted drugs and combination therapy approaches. Ultimately, this review aims to enhance the clinical effectiveness of CAR cell therapy while minimizing any adverse reactions.This review focuses on the progress of chimeric antigen receptor (CAR) cells in solid tumor therapy and the role of scRNA-seq in CAR cell therapy at various stages following the administration of CAR cell products into the body. Advancements in scRNA-seq will further promote the investigation of the biological characteristics of CAR cells 2Adoptive cell therapy (ACT) employs patient-derived immune cells, particularly diverse T cell variants, which are cultured and genetically modified outside the body before being reintroduced to the patient as a therapeutic intervention for the identification and precise targeting of cancerous cells. Among the diverse ACT approaches, CAR-T therapy has gained prominence . CAR-T tHowever, CAR-T cell therapy faces numerous challenges and potential setbacks, including the advancement of illnesses in individuals, constraints in T cell collection, setbacks in CAR cell production, inadequate generation of CAR cells, inherent T cell deficiencies, and various additional factors. The TME plays a crucial role in CAR-T therapy . When CACurrently, multiple ongoing clinical trials are investigating the use of CAR-T cell therapy in the management of solid tumors, including lung cancer, liver cancer, and other tumor types –17. ThisIn recent times, CAR-natural killer (CAR-NK) and CAR-macrophage (CAR-M) therapies have gained increasing attention as potential supplements or alternatives to CAR-T cell therapy, specifically for addressing solid tumors , 6. FigCAR-NK cells offer unique advantages compared to CAR-T cells, as they can be generated from established cell lines or using allogeneic natural killer (NK) cells with different major histocompatibility complexes (MHC), broadening their range of applications. These cells can eliminate cancer cells using both CAR-mediated and CAR-independent methods while exhibiting reduced toxicity. CAR-NK cell therapy has been supported by clinical trials, demonstrating its efficacy and tolerability –28.Macrophages, abundant within TMEs and highly skilled in penetrating tumors and regulating immune responses, present a promising avenue for CAR engineering. CAR-engineered macrophages have the potential to overcome various challenges associated with CAR-T and CAR-NK cell therapies, particularly in the context of solid tumors . HoweverCAR-T cell therapy, while demonstrating remarkable success in hematological malignancies, faces considerable challenges when applied to solid tumors. These challenges arise from the unique microenvironment of solid tumors, characterized by immunosuppressive factors, limited T cell infiltration, and spatial heterogeneity. In order to address these challenges, several innovative strategies have emerged:(1) Targeting the Tumor Microenvironment: One approach involves modifying CAR-T cells to secrete cytokines or factors that can remodel the TME, making it more conducive to immune cell activity.(2) Enhancing T Cell Infiltration: Another strategy is to enhance T cell homing and infiltration into solid tumors include the engineering of CAR-T cells to include chemokine receptors specific to the TME.(3) Combination Therapies: Finally, another possible strategy is the combination of CAR-T cell therapy with other immunotherapies or conventional treatments to create a synergistic effect.In summary, the obstacles in solid tumor CAR-T cell therapy are indeed substantial, but ongoing research and innovative approaches are continuously advancing our ability to tackle these challenges effectively.3Single-cell RNA sequencing (scRNA-seq) is a powerful technique for deep sequencing of cellular mRNA, enabling high-throughput, quantitative profiling of gene expression and transcriptome signatures . HoweverIn the past, researchers generally considered cellular uniqueness and traits of a group to be identical. However, advances in oncological research have revealed significant heterogeneity among individual tumor cells, emphasizing the importance of single-cell analysis. Intra-tumor heterogeneity was first described in the 1800s by pathologist Rudolf Virchow . Due to ScRNA-seq offers enhanced resolution of transcriptomes, providing a deeper understanding of cellular phenotypes . This te3.1ScRNA-seq has played a pivotal role in understanding the efficacy, development, and mechanisms of CAR-T cell therapy. Long-term studies by Melenhorst et al. have demonstrated the enduring impact of CAR-T cell therapy in treating chronic lymphocytic leukemia (CLL), particularly in tracking the persistence and development of CD4+ CAR-T cells over a decade . Further3.2ScRNA-seq has played a critical role in addressing the ongoing issue of scarce safe target antigens for CAR-T cell therapy. Analysis of more than 500,000 cells, including those from acute myeloid leukemia (AML) patients and healthy controls, has led to the identification of potential CAR-T cell targets like CSF1R and CD86 . These t3.3ScRNA-seq has been a vital tool for comprehending on-target and off-tumor effects in CAR-T cell therapy , 47. Thr3.4The interaction between CAR-T cell subclusters and the TME is crucial for the success of CAR-T cell therapy, with scRNA-seq being an instrumental part of studying this dynamic interaction. Using a mouse model of B cell lymphoma treated with anti-CD19 CAR-T cell therapy, significant changes in the TME during therapy were observed . CAR-T c3.5Relapses are a common challenge in CAR-T cell therapy, both in cases of CD19-positive and CD19-negative disease. However, scRNA-Seq has been a valuable tool in uncovering the mechanisms behind relapse. By examining single-cell data obtained from infusion products, researchers have identified factors linked to relapse, including a lack of TH2 activity in CD19-positive cases . TIGIT e4While this mini-review has aimed to provide an overview of the current applications of single-cell RNA sequencing (scRNA-seq) in CAR therapy, it is essential to consider the potential future directions in CAR cell therapy and how scRNA-seq might contribute to these developments.4.1Looking ahead, CAR cell therapy holds promise in several key areas:(1) Personalized Medicine: The integration of scRNA-seq data with patient-specific genomic information can lead to the development of personalized CAR therapies, tailored to the unique molecular characteristics of a patient’s tumor.(2) Next-Generation CARs: The engineering of novel CAR constructs, such as universal CAR-T cells, bi-specific CARs, or armored CARs, will expand the applicability of CAR therapies to a broader spectrum of cancers.(3) Reducing Toxicity: Efforts to mitigate the side effects and cytokine release syndrome associated with CAR-T cell therapy are ongoing, and future therapies are likely to be safer and more targeted.4.2As we envision the future of CAR cell therapy, scRNA-seq will continue to play a pivotal role:(1) Mapping Tumor Heterogeneity: ScRNA-seq enables in-depth characterization of the heterogeneous cell populations within a tumor, aiding in the identification of new target antigens and the development of more effective CAR constructs.(2) Tracking CAR-T Cells: ScRNA-seq can be employed to monitor CAR-T cell behavior within the patient, helping to optimize dosing, predict responses, and evaluate long-term effects.(3) Understanding Resistance Mechanisms: By analyzing single-cell transcriptomes, we can uncover the resistance mechanisms that solid tumors may employ against CAR-T cell therapy, thereby guiding the development of strategies to overcome them.By combining scRNA-seq with CAR therapy, it offers an exciting avenue for future innovations in cancer immunotherapy. This synergy holds the potential to enhance the precision and efficacy of CAR cell therapies while expanding their applications to a broader range of malignancies.5Despite certain limitations, such as the challenge of accurately linking gene transcription to protein expression, scRNA-seq is increasingly recognized for its potential in CAR-T cell therapy. ScRNA-seq has been invaluable for investigating the molecular complexities and diversity of cell populations engineered with a CAR design. Nevertheless, the potential of scRNA-seq to analyze intricate cell populations suggests an exciting opportunity for concurrent screening of diverse CAR constructs. This approach could enable the association of specific CAR cell transcriptomic profiles with optimal applications or guide the personalized design of CARs tailored to individual patients’ distinct requirements, thereby enhancing the potential of immunotherapy as a form of personalized healthcare.ZP: Conceptualization, Writing – review & editing. TW: Writing – original draft. LM: Conceptualization, Writing – original draft, Writing – review & editing.


Chimeric antigen receptor T (CAR-T) cells have shown promising efficacy in treating hematological malignancies, particularly CD19 CAR-T for B-cell acute lymphoblastic leukemia with a 70~94% complete remission rate , and usiChen et al.) has crucial implications for screening new targets for CAR-T therapy. The use of multidimensional omics data advanced CAR-T cell therapy . Additionally, B7-H3 CAR-T cells have been shown to have potent preclinical activity against pediatric solid tumors and brain tumors , early-onset gastric cancer , lung cancer , and gliomas , based on clinical information and high-throughput data of patients. These prediction models have significant reference values for predicting the efficacy of CAR-T treatment in solid tumors.Although new targets are constantly being discovered, there is still a need of effective prediction methods for their clinical efficacy. Several researches have constructed prognostic models of CD19 CAR-T therapy in B cell acute lymphoblastic leukemia , 20 or lCAR-T cells provide remarkable opportunities and are expected to become an effective means of treating multiple tumors in the future. However, current challenges include antigen escape relapse resulting from selective immune pressure of CAR-T cells and the lack of ideal targets in solid tumors. Moreover, the lack of clinical efficacy prediction means of CAR-T therapy also hinders its application and promotion to some extent. Our Research Topic provides valuable references for screening new targets of CAR-T and predicting its efficacy. We invite you to read each of these enlightening articles.All authors have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


GATA2 deficiency is an inborn error of immunity (IEI) characterized by infectious susceptibility and increased risk of myelodysplasia leading to acute myeloid leukaemia (AML). Oral anomalies already described in this disorder include recurrent viral and fungal infections and oral ulcers.We report a 9-year-old girl presenting with AML with myelodysplasia-related changes, monosomy 7 karyotype on marrow aspirate, numerous flat warts on her hands and multiple dental caries at oral cavity inspection. Dental evaluation and genetic testing (Sanger sequencing) for GATA2 were carried out considering the peculiar clinical presentation.Dental evaluation showed extensive caries and periodontal disease, while genetic studies revealed a known c.1009 C>T (p.Arg337X) mutation in GATA2. After multidisciplinary discussion, affected teeth were extracted before chemotherapy, in general anaesthesia, together with scaling and root planning of the alveolar sockets. Subsequently, the patient underwent hematopoietic stem cell transplantation (HSCT) from her HLA-matched GATA2 wild-type sibling, who did not bear any dental anomalies. No dento-alveolar infections were encountered during post-chemotherapy aplasia. This case first describes the association between GATA2 deficiency and extensive dental caries with periodontal disease, highlighting the importance of an early dental evaluation and intervention in children with leukaemia. Key words:GATA2 deficiency, Inborn errors of immunity, teeth, dental decay, multidisciplinary approach. GATA2 deficiency MIM#614172) determines a syndrome characterized by a wide range of clinical manifestations including myelodysplasia leading to acute myeloid leukaemia (AML), monocytopenia and mycobacterial infections (MonoMAC) syndrome, diffuse warts and lymphedema 4172 dete,4. GATA2A 9-year-old girl was referred to our Haematology-Oncology Unit from a foreign country for severe anaemia  with low monocytes and reticulocytes. Family history revealed peculiar clinical features on the mother’s side  mutation, already known as pathogenic  and due to the deep scaling and root planning carried out during the operation, the bleeding had been quite considerable.The patient underwent 2 cycles of Idarubicin-Cytarabine-Etoposide chemotherapy according to local AML treatment protocol. Marrow evaluations after chemotherapy showed poor response to treatment, therefore – despite the persistence of myeloblasts at marrow re-evaluation –, the family accepted to undergo HSCT from the patient’s HLA-matched, GATA2 wild-type sibling. Of note, no dento-alveolar infections were encountered during chemotherapy and her healthy brother did not bear any teeth abnormalities.HSCT was performed upon myeloablative conditioning (Thiotepa-Treosulfan-Fludarabine) using bone marrow as stem cell source. No severe infections took place during post-induction aplasia, and engraftment was successfully attained with full donor chimerism. Post-transplant follow-up showed grade I upper gastro-intestinal Graft-versus-Host Disease, treated with topical steroids. Unfortunately, AML recurrence occurred and the patient eventually deceased 4 months after HSCT.To our knowledge, this is the first case of extensive caries and periodontal disease in a patient with GATA2 deficiency. Therefore, this report expands the spectrum of the oral manifestations of this disorder, which already include oral ulcers and recurrent fungal, HSV and HPV infections, that may eventually lead to squamous cell carcinoma ,5,12,13.The dental involvement of our patient was diagnosed at the onset of AML, which is notably associated with immune suppression and oral manifestations . HoweverDespite the severity of the dental Tableau, an early surgical approach with extraction of the affected teeth with scaling and root planning of the alveolar sockets allowed to reduce the risk of life-threatening oral infections, especially throughout the prolonged phase of post-induction aplasia during HSCT. This suggests that pre-transplant work-up should regularly include a dental evaluation and eventually a preventive surgical intervention, especially in children more prone to develop oral disease, such as those affected by IEI ,6,8.In conclusion, we report the first case of GATA2 deficiency associated with dental caries and periodontal disease, expanding the clinical spectrum of this disorder.


Patients with stage IIB-IV melanoma who have had surgery are recommended to receive systemic adjuvant therapy, intended to target the residual micro-metastatic disease and reduce the risk of melanoma relapse and death from melanoma. Multiple adjuvant therapy regimens have been tested over the past three decades leading to regulatory approvals. Due to significant improvements in relapse-free survival, programmed cell death protein 1 (PD-1) blockades and BRAF-MEK inhibitors (for BRAF mutant melanoma) are currently used as the standard-of-care adjuvant treatments for surgically resected stage III-IV melanoma. In the US, pembrolizumab has achieved regulatory approval as an adjuvant therapy for resected stage IIB-IIC melanoma, while nivolumab is currently under FDA review for this indication. This review examines melanoma’s phase III adjuvant treatment trials, focusing on the latest updates. It also reviews the role of biomarkers in potentially individualizing adjuvant therapy and summarizes the main limitations and future directions of adjuvant therapy options for high-risk melanoma.The consideration of systemic adjuvant therapy is recommended for patients with stage IIB-IV melanoma who have undergone surgical resection due to a heightened risk of experiencing melanoma relapse and mortality from melanoma. Adjuvant therapy options tested over the past three decades include high-dose interferon-α, immune checkpoint inhibitors , targeted therapy (dabrafenib-trametinib for BRAF mutant melanoma), radiotherapy and chemotherapy. Most of these therapies have been demonstrated to enhance relapse-free survival (RFS) but with limited to no impact on overall survival (OS), as reported in randomized trials. In contemporary clinical practice, the adjuvant treatment approach for surgically resected stage III-IV melanoma has undergone a notable shift towards the utilization of nivolumab, pembrolizumab, and BRAF-MEK inhibitors, such as dabrafenib plus trametinib  due to the significant enhancements in RFS observed with these treatments. Pembrolizumab has obtained regulatory approval in the United States to treat resected stage IIB-IIC melanoma, while nivolumab is currently under review for the same indication. This review comprehensively analyzes completed phase III adjuvant therapy trials in adjuvant therapy. Additionally, it provides a summary of ongoing trials and an overview of the main challenges and future directions with adjuvant therapy. Melanoma arises from the uncontrolled proliferation of melanocytes in the skin ,2. In thNRAS, neurofibromin 1 (NF1), KIT proto-oncogene receptor tyrosine kinase (KIT), telomerase reverse transcriptase (TERT) gene, cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, and phosphatase and tensin homolog (PTEN) gene, contributing to melanoma progression and metastasis  antagonist and nivolumab versus nivolumab monotherapy in patients diagnosed with stage III or IV melanoma who have undergone a complete removal of their tumor(s). Additionally, KEYVIBE-010 (NCT05665595) is a new study that is assessing the RFS in patients with adjuvant pembrolizumab + vibostolimab (MK-7684A) as anti-TIGIT (T cell immunoglobulin and ITIM domain) compared to pembrolizumab alone . Lastly,® (ACCR) annual meeting this year and showed that mRNA-4157/V940 + pembrolizumab showed a significant 44% reduction in the risk of recurrence or death in patients than pembrolizumab alone . Similarly, DMFS was lower in the pembrolizumab group than mRNA-4157/V940 + pembrolizumab . Interestingly, the trAEs number was similar among both groups. As a next step, a phase III trial is planned, which will be an active-comparator-controlled clinical trial called “V940-001” (NCT05933577) among participants with high-risk stage II-IV melanoma , activate the immune system to target specific cancer antigens. Despite limited efficacy in advanced metastatic disease, trials of 6-MHP in patients with resected stage IV melanoma have shown significantly increased median survival compared to controls . The phamelanoma .The outcomes of clinical trials for melanoma and cancer vaccines, in general, have been largely unsatisfactory in cases of advanced disease, with ORRs < 5% . HoweverThe impact of adjuvant anti-PD-1 immunotherapy on OS in high-risk melanoma patients after surgical removal is unclear. Despite improvements in RFS in major randomized trials like KEYNOTE-054 , KEYNOTEThe choice of adjuvant treatment for individuals with surgically excised cutaneous melanoma depends on multiple determinants, including tumor stage, BRAF mutation status, the probability of relapse, comorbid conditions and patient wishes. Patients presenting with high-risk BEAF WT melanoma are typically prescribed adjuvant anti-PD-1 immunotherapy in the absence of limiting comorbid conditions such as symptomatic autoimmunity. Those with BRAF-mutant melanoma have the options of anti-PD-1 monotherapy or dabrafenib–trametinib where the choice depends on patients wishes for oral agents versus intravenous therapy, the adverse event profiles, and comorbid conditions. Despite adjuvant ipilimumab showcasing favorable overall survival benefits when compared to placebo (EORTC 18071)  and to i


Improvement of psychosocial-spiritual well-being in patients with life-threatening or life-limiting illness is desirable. Resilience and mindfulness are considered to be helpful for enhancing psychosocial-spiritual well-being. Mindfulness-based interventions have been shown to promote resilience to stress and enhance well-being. However, in medical patients, evidence for the associations between mindfulness and resilience is lacking. We hypothesize patients with higher levels of psychosocial-spiritual well-being demonstrate greater resilience and mindfulness.200 patients  with serious and or life-limiting illnesses were recruited from the NIH Clinical Center. Patients completed a demographic questionnaire, the NIH-HEALS measure of psychosocial-spiritual well-being, the Connor-Davidson Resilience Scale (CD-RISC-10), and the Mindful Attention Awareness Scale (MAAS). The demographic questionnaire also included a question on current stress level.s=0.44, p < 0.001) and MAAS . These findings were consistent across all three NIH-HEALS factors. Additionally, CD-RISC-10 and MAAS demonstrated a meaningful relationship to each other . All three constructs were inversely related to current stress level.The NIH-HEALS was positively correlated to CD-RISC-10 (rFindings suggest that there is a meaningful relationship between psychosocial-spiritual well-being, mindfulness, and resilience. Mindfulness and resilience are positively correlated in a medical population. Clinical interventions aimed at enhancing psychosocial-spiritual well-being through mindfulness and resilience can be highly promising for patients with severe and or life limiting illness. Psychosocial-spiritual well-being is a multifaceted concept including psychological, social, and spiritual determinants of health . It is cPsychosocial-spiritual well-being outcomes can be enhanced by resilience and mindfulness . ResilieResilience has been shown to improve quality of life in patient with hypertension , glaucomMindfulness has been identified as an essential protective factor that promotes resilience against psychological distress , 34. MinInterestingly, the relationship between mindfulness and resilience has not been well-studied in clinical populations . To our The present study investigates the relationship between psychosocial-spiritual well-being, with two constructs of resilience and mindfulness in patients with severe and or life-limiting illnesses. We predict (1) a strong relationship between psychosocial-spiritual well-being and resilience; (2) a strong relationship between psychosocial-spiritual well-being and mindfulness; and (3) a strong association between resilience and mindfulness. We further hypothesize that these constructs will be related to current stress level in patients.The present study is part of a larger study that validated the National Institutes of Health (NIH) Healing Experiences of All Life Stressors (NIH-HEALS), a measure of psychosocial-spiritual healing developed by the Pain and Palliative Care Service (PPCS) at the NIH Clinical Center (CC). Additional information about the study design is detailed elsewhere .The studTwo hundred (200) patients from the NIH Clinical Center (CC) participated in the study, aged 18–89 years. All patients included in the study who were recruited from both the inpatient and outpatient clinics at the NIH CC, had serious and/or life-limiting medical illnesses and were already participating in experimental treatments and research projects related to their specific diseases. The majority of patients had advanced/metastatic cancer in one or several organs, while other conditions such as blood dyscrasias, graft vs. host disease (GVHD), and rare genetic conditions were also present  or median . Data were assessed for the assumption of normality, and appropriate methods were used as indicated. Continuous data between two groups were compared using Wilcoxon rank sum tests, and among three or more categories using mixed models. Post-hoc pairwise comparisons were corrected for multiple comparisons using the Bonferroni method, and corrected p-values are reported. Spearman’s rho statistics were used for assessing correlations between measures. Results were interpreted based on the magnitude of effects  along with p-values. Data were analyzed using SAS v9.4 .The sample included 200 patients with an age range of 18–89 years and mean of 50.2 ± 15.5 years. Of the 193 participants who indicated their gender, 53% identified as women and 47% identified as men. No other gender identification was made. Patients were Caucasian (72%), Black or African American (16%), or Asian (7%). Of the whole sample, 7% identified their ethnicity as Hispanic or Latinx. Subjects reported their religious affiliations as Christian (66%), Not Affiliated (12%), or Atheist (6%). The vast majority, 70%, of patients, were diagnosed with advanced/metastatic cancers. Other medical diagnoses included severe/rare non-genetic conditions (11%), severe/rare genetic conditions (9%), blood dyscrasias (9%), and acquired immunodeficiency syndrome or AIDS (1%). Patients described their illness severity as extremely severe (43%), severe (36%), moderate (14%), and mild to not severe (7%). Further description of participant demographics and condition profiles are detailed in Table The median (IQR) total NIH-HEALS score was 134 (120–145)  and MAAS total score   , CD-RISC-10 , and MAAS .Additionally, NIH-HEALS, CD-RISC-10, and MAAS demonstrated an inverse correlation with current stress level. Patients reported their current level of stress as Extreme (3%), Severe (11%), Moderate (47%), Mild (29%), or No Stress (10%). Current level of stress was inversely related to NIH-HEALS , and there is a similar positive correlation between psychosocial-spiritual well-being and mindfulness . Additionally, resilience and mindfulness were positively correlated in this patient cohort . All three constructs were inversely related to current stress level.The results of the current study indicate a relationship between psychosocial-spiritual well-being, resilience, and mindfulness in patients with severe or life limiting illness. Our results demonstrate that psychosocial-spiritual well-being is positively related to resilience  and the Mindfulness-Based Cognitive Therapy (MBCT) have been shown to reduce psychological stress and improve well-being in several clinical populations –56. SimiThe study limitations include reliance on self-rated measures and a correlational study design. More in-depth studies may be conducted to further characterize psychosocial-spiritual well-being, resilience, and mindfulness using clinical interviews or clinician-administered measures. Due to the correlational nature of the present study, the relationships found among study constructs do not establish cause and effect. Experimental designs are needed to further evaluate the associations among these and other related constructs in controlled settings. Nonetheless, the current findings suggest that interventions aimed at enhancing mindfulness and resilience may improve psychosocial-spiritual well-being and quality of life in patients with severe and life limiting illness. It should be noted, however, that generalization of results to other patient groups require additional investigations. Moreover, the NIH-HEALS is a newly validated scale. Future studies should investigate its various components in different cultures and subject groups. Additionally, the NIH-HEALS is a 35-item tool, a shorter version of this measure might be more appropriate for patients with severe illness and in other clinical populations.


Staphylococcus aureus (HKSA) in young (aged 2–4 months) and old (aged 12–18 months) mice, organotypic culture of precisely cut lung slices, and endothelial cells (mLEC) isolated from young and old mice. HKSA and Tr-OxPL combination caused a higher degree of vascular leak, the accumulation of inflammatory cells and protein in bronchoalveolar lavage, and inflammatory gene expression in old mice lungs. HKSA caused a greater magnitude of inflammatory gene activation in cell and ex vivo cultures from old mice, which was further augmented by Tr-OxPLs. L37pA peptide targeting CD36 receptor attenuated Tr-OxPL-induced endothelial cell permeability in young and old mLEC and ameliorated KOdiA-PC-induced vascular leak and lung inflammation in vivo. Finally, CD36 knockout mice showed better resistance to KOdiA-PC-induced lung injury in both age groups. These results demonstrate the aging-dependent vulnerability of pulmonary vasculature to elevated Tr-OxPL, which exacerbates bacterial lung injury. CD36 inhibition is a promising therapeutic approach for improving pneumonia outcomes in aging population.Truncated phospholipid oxidation products (Tr-OxPL) increase in blood circulation with aging; however, their role in the severity of vascular dysfunction and bacterial lung injury in aging groups remains poorly understood. We investigated the effects of six Tr-OxPL species: KOdiA-PC, POVPC, PONPC, PGPC, Paz-PC, and Lyso-PC on endothelial dysfunction and lung inflammation caused by heat-killed  OxPL in non-alcoholic steatohepatitis (NASH) in vivo [Phospholipids are major building blocks of cell membranes and constituents of circulating lipoproteins. These lipid molecules undergo enzymatic oxidation via lipoxygenases and cyclogeneses or reactive oxygen species (ROS)-mediated nonenzymatic reactions, resulting in the generation of a wide variety of oxidized phospholipid (OxPL) products with profound biological activities ,2. Incre in vivo ,9. From  in vivo ,11,12.Elevated oxidative stress and inflammation are common features of aging, making the elderly population more vulnerable to various infections, including respiratory diseases ,14. For Staphylococcus aureus in developing lung endothelial dysfunction in young and old mice. We also evaluated the protective effects of CD36 inhibitory peptide L37pA against Tr-OxPLs in vivo using mouse models of ALI and also in vitro using the cultures of precisely cut lung slices (PCLSs) and lung endothelial cells from both young and old mice. Cluster of differentiation 36 (CD36) is a class B scavenger receptor that is primarily involved in the uptake of OxPLs . ECs havsn-glycero-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine (PGPC), 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (Paz-PC), O-1-O-Palmitoyl-2-O--L-glycero-3-phosphocholine (KOdiA-PC), 1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) and lysophosphocholine (Lyso-PC) were purchased from Cayman chemicals . L37pA was obtained from Pepmic Co. Ltd. . Heat-killed Staphylococcus aureus was purchased from InvivoGen . Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Dynabeads, type I collagenase, and penicillin/streptomycin were purchased from Thermo Fisher Scientific . CD31 and ICAM-2 antibodies were from BD Biosciences  and VE-cadherin antibody was from Santa Cruz Biotechnology . Endothelial cell growth medium EGM-2 was obtained from Lonza . 1-Palmitoyl-2--8, bacterial particles) was injected intranasally, followed by the injection of the indicated Tr-OxPLs (10 mg/kg) into the jugular vein. L37pA  was applied 30 min prior to Tr-OxPLs treatment where indicated. The control groups received the same volume of sterile saline. After 20–24 h, animals were sacrificed under ketamine  anesthesia, and bronchoalveolar lavage (BAL) fluid was collected to analyze total cells and protein content as described earlier [All the protocols involving animal care and treatments were approved by the University of Maryland Institutional Animal Care and Use Committee. Young (aged 2–4 months) and old aged 18–24 months) C57/B6J mice from Jackson Laboratories  were used for the experiments. The breeders of CD36 knockout mice in C57B6 background were also purchased from Jackson Laboratories , and the offspring were used for experiments at ages 2–4 months and 18 months. HKSA , cDNA was synthesized using the iScript cDNA synthesis kit . Gene expression changes were determined by employing the ΔΔCt method after Ct values were normalized to GAPDH. The primers used for PCR are listed in An electric cell–substrate impedance sensing system  was used to measure endothelial permeability by monitoring transendothelial electrical resistance (TER) over time, as described previously . Brieflyt test and one-way ANOVA followed by post-hoc Fisher’s test, which was used for multiple groups. p < 0.05 was considered to be statistically significant.Data were presented as means ± S.D. For TER studies, six to eight independent measurements were performed for each condition. For qRT-PCR and Western blot analyses, three independent experiments were performed with each stimulation. The comparison between the control and the stimulated group was performed using unpaired Student’s Staphylococcus aureus (HKSA) and Tr-OxPLs in the young and old groups. Old mice (aged 18–24 months) developed more severe ALI in response to HKSA as compared to young (aged 2–4 months) counterparts, reflected by higher cell counts and protein content in BAL  from young and old mice. mRNA expression of a senescence marker gene cyclin-dependent kinase inhibitor 1 p21The incubation of mLEC with KOdiA-PC, PONPC, Paz-PC, POVPC, Lyso-PC, or PGPC also induced the transcriptional activation of TNFα, IL-6, KC, and IL-1β, although to a much lesser extent than HKSA B. ImportWe investigated whether the phenomenon of age-dependent exacerbation of inflammation and lung barrier dysfunction observed in animal models can also be detected in lung EC cultures isolated from young and old mice. Primary cultures of ECs from young and old lungs were exposed to increasing concentrations of KOdiA-PC, PONPC, Paz-PC, POVPC, Lyso-PC, and PGPC. A dose-dependent decline in TER reflecting increased permeability was observed in both young and old lung ECs. However, Tr-OxPLs-induced permeability response was more pronounced in lung ECs from the old group as compared to young EC counterparts . Of noteWe next investigated the effects of combined Tr-OxPL and HKSA treatment on permeability response by lung ECs isolated from young and old mice. Tr-OxPLs and HKSA exhibited synergistic effects on endothelial permeability in lung ECs from both age groups. However, the magnitude of barrier dysfunction caused by the combination of Tr-OxPLs and HKSA was much greater in ECs isolated from old mice . All sixConsidering CD36 as the principal receptor interacting with Tr-OxPLs, we investigated whether the inhibition of CD36 offers protection against Tr-OxPL-mediated endothelial dysfunction. Published studies show that CD36 inhibitory peptides may rescue ALI caused by bacterial lipopolysaccharide . PrimaryTo investigate whether the protective effects of L37pA against Tr-OxPL-induced EC dysfunction can be recapitulated in vivo, we utilized KOdiA-PC as an example of a Tr-OxPL compound inducing lung dysfunction in young and old mice. L37pA (20 mg/kg) was injected intravenously 30 min following KOdiA-PC  challenge. In agreement with the results described above, KOdiA-PC-treated old mice displayed elevated BAL total and PMN cell counts and protein content in comparison to the young group A. More iAn increase in the incidence, morbidity, and mortality of infectious respiratory diseases in older populations is well documented ,17. The Staphylococcus aureus in inducing lung injury and inflammation [The present study demonstrates that aging directly contributes to the severity of ALI caused by bacterial infection and can be further exacerbated via the elevation of circulating Tr-OxPLs. The elevation of various Tr-OxPL species exhibited deleterious effects on lung vascular ECs in a dose-dependent manner. However, bacterial particles caused much more potent inflammatory activation and lung injury. Despite the high magnitude inflammation caused by HKSA, submaximal doses of Tr-OxPLs were able to further augment lung injury and EC dysfunction. These effects are dependent on CD36 signaling and can be controlled via the modulation of CD36 activity. Furthermore, this study identifies, for the first time, CD36 as a major receptor mediating the deleterious effects of Tr-OxPLs on endothelial function. We utilized a clinically relevant model of dose-controlled ALI caused by HKSA, which closely mimics the presence of killed bacteria after antibiotic treatment. Multiple studies have already established the potency of HKSA or heat-killed methicillin-resistant ammation ,36,37.Our results also demonstrate that Tr-OxPLs augmented EC dysfunction caused by Gram-positive bacterial particles and other inflammatory agonists. Similarly, Tr-OxPLs augmented HKSA-induced lung injury in vivo. Such synergy between circulating Tr-OxPLs and infectious agonists in the induction of lung EC inflammation and barrier dysfunction may be a cause of more severe bacterial lung inflammation in aging populations. These results also emphasize the role of pulmonary endothelium as a direct effector and target of plasma Tr-OxPLs. It is also important to note that “low” Tr-OxPL doses that did not cause EC barrier dysfunction alone, still augmented deleterious effects of HKSA. The synergistic effects of HKSA and Tr-OxPLs may have important clinical implications as they document that even the modest elevation of circulating Tr-OxPL may have significant adverse effects on the health of old individuals due to impaired immune function and increased susceptibility to bacterial infections. One can argue the physiological relevance of higher doses of Tr-OxPLs applied in this study but there are no published data on accurate levels of circulating OxPLs in human except the general consensus that these lipid mediators are elevated during various pathological states ,38. In jOur published studies demonstrated elevated production of Tr-OxPLs in aging mice challenged with LPS , suggestOur results show that old ECs retained this feature even during a few passages in culture. In addition, experiments with PCLS from adult and old mice that were challenged with HKSA in organotypic culture demonstrate similar age-dependent augmentation of HKSA-induced inflammation and accelerated senescence reflected by the higher induction of senescence markers by HKSA in PCLS and ECs isolated from old mice. Interestingly, the augmenting effects of various tested Tr-OxPLs in young and old animals were not uniform, with some species such as Paz-PC, KOdiA-PC showing stronger variations, while POVPC and PGPC showing a mild increase, and a minimal difference was observed with Lyso-PC. The varying potency of Tr-OxPLs in exacerbating bacteria-induced lung injury warrants further investigation aimed at the development of more selective therapies to mitigate the effects of the most harmful Tr-OxPLs.S. aureus particles can be recognized by CD36 in addition to canonical TLR2/6 receptor mechanism. This interaction likely occurs via the recognition of lipoteichoic acid present on the bacterial wall, which contains phospholipid moieties. In addition, CD36 expressed by macrophages regulates phagocytosis. These factors: CD36 expression by other inflammatory cells and possible interaction of CD36 both with Tr-OxPLs and S. aureus add a complexity to the interpretation of animal experiments, which demonstrated the attenuation of Tr-OxPL/HKSA-induced endothelial dysfunction and ALI via the pharmacological and molecular inhibition of CD36.In order to investigate the cellular mechanisms of Tr-OxPL-induced EC dysfunction in ALI models, we focused on CD36, one of the principal receptors that directly targets oxidized LDL and phospholipids. Surprisingly, the potential role that CD36 has in the modulation of Tr-OxPLs-induced regulation of endothelial function has not been explored so far. To fill this gap in knowledge, we utilized CD36 inhibitory synthetic peptide L37pA to examine its protective effect against Tr-OxPLs-induced augmentation of ALI. Our earlier study showed that chemically synthesized amphipathic helical peptides targeting CD36 rescued lung injury and inflammation caused by Gram-negative endotoxin LPS . In addiOur new data show the strong attenuation of endothelial barrier dysfunction by L37pA caused by several Tr-OxPLs, except for Lyso-PC, where only a marginal protective effect was observed . More stWhile the results presented in this study firmly support CD36 as a critical receptor mediating the deleterious effects of Tr-OxPLs on lung endothelium, one important question remains unanswered: how do CD36 inhibitors protect against Tr-OxPLs-induced endothelial dysfunction? Possible scenarios include the blocking of Tr-OxPLs internalization by L37pA, thus minimizing the bioavailability and toxic effects of the lipid products, additional anti-inflammatory activities of the inhibitor via interactions with other receptor(s), and interference with barrier-disruptive signaling cascades activated by Tr-OxPLs. These mechanisms warrant further investigations.In conclusion, this study demonstrated, for the first time, that the deleterious effects of elevated Tr-OxPL associated with aging affect the functional stability of lung ECs and exacerbate ongoing infectious lung injury. These age-dependent complications may be alleviated by L37-pA, a synthetic peptide antagonizing CD36 function. Such protective effect was more pronounced in old animals. These results, together with data showing less severe Tr-OxPL-induced lung dysfunction in CD36 KO mice, further support the CD36-dependent mechanism of L37-pA-mediated lung protection in aging mice. Collectively, our findings suggest that synthetic peptides targeting CD36 hold strong therapeutic potential in mitigating aging-associated endothelial dysfunction and improving lung inflammatory syndromes in aging populations.


The COVID-19 pandemic caused by the SARS-CoV-2 virus is still a global health concern. Several spike (S) protein-based vaccines have been developed that efficiently protect the human population against severe forms of COVID-19. However, some SARS-CoV-2 variants of concern (VOCs) have emerged that evade the protective effect of vaccine-induced antibodies. Therefore, efficient and specific antiviral treatments to control COVID-19 are indispensable. To date, two drugs have been approved for mild COVID-19 treatment; nevertheless, more drugs, preferably broad-spectrum and ready-to-use therapeutic agents for new pandemics, are needed. Here, I discuss the PDZ-dependent protein-protein interactions of the viral E protein with host proteins as attractive alternatives for the development of antivirals against coronavirus. In the past 20 years, three highly pathogenic coronaviruses (CoVs) have emerged as threats to humans: severe acute respiratory syndrome coronavirus SARS-CoV-1) in 2002; Middle East respiratory syndrome coronavirus (MERS) in 2012; and the currently circulating SARS-CoV-2, the causative agent of the recent COVID-19 pandemic  in 2002;. Howeverhttps://covid19.who.int/ accessed on 14 April 2023) ,5756,57]Recombinant SARS-CoVs with mutated PDZbm in the E protein or lacking the full-length protein generally restore the PDZbm sequence or generate a novel chimeric gene to restore the absence of PDZbm after several cell passages ,60. ThisIn addition, although PDZbm is conserved between SARS-CoV-1 and 2, it appears that the SARS-CoV-2 E protein binds more host PDZ proteins than SARS-CoV-1, probably with higher affinity, as has been suggested for PALS1. This may imply that amino acid substitutions/deletions near PDZbm might affect the structure, and IC activity and/or confer more flexibility to the SARS-CoV-2 E protein, thus defining its PDZ interactome  61,62].,62.61,62PDZ-dependent PPIs have been widely studied as therapeutic targets, and both endogenous and pathogen-derived PDZbms have shed light on important functional interactions that are useful for drug design. Most inhibitors are directed to the interaction interface between the PDZ hydrophobic cavity and the PDZbm ligand, acting as competitive agonists to prevent the PDZ-PDZbm interaction . NotablyMapping virus–host PPIs is important for understanding viral pathogenicity and revealing the fundamental host proteins hijacked for successful viral replication. The E protein of CoVs is one of the most abundantly expressed in infected cells and is an important virulence factor of highly pathogenic CoVs. Furthermore, within the E protein sequence, the PDZ-binding motif has been recognized as a virulence determinant by itself; hence, identification of its target proteins in infected cells is mandatory to gain insight into the viral pathogenic mechanisms and, concomitantly, to document potential therapeutic targets. Targeting host PDZ proteins seems to contribute significantly to the highly pathogenic CoVs life cycle; thus, targeting these virus–host interactions may help control airway epithelial damage and viral dissemination during COVID-19. Interfering the host–virus interface could be a comprehensive strategy to reduce resistance emergence, as the virus is less likely to mutate at such specific interacting moieties. It is tempting to consider the blockage of PDZ-dependent interactions of the highly pathogenic CoV E protein with host PDZ proteins as a promising method for the current COVID-19 pandemic control. Additionally, since PDZbm is a conserved motif among distinct CoV strains, it would be appealing to consider the generation of pan-CoV inhibitors using viral PDZbm-based PPIs.


Distinguishing whether and how pre-existing characteristics impact maternal responses to adversity is difficult: Does prior well-being decrease the likelihood of encountering stressful experiences? Does it protect against adversity’s negative effects? We examine whether the interaction between relatively uniformly experienced adversity (due to COVID-19 experience) and individual variation in pre-existing  distress predicted mothers’ pandemic levels of distress and insensitive caregiving within a country reporting low COVID-19 death rates, and strict nationwide regulations.Fifty-one Singaporean mothers and their preschool-aged children provided data across two waves. Pre- pandemic onset maternal distress  was captured via self-reports and maternal sensitivity was coded from videos. Measures were repeated after the pandemic’s onset along with questionnaires concerning perceived COVID-19 adversity  and pandemic-related objective experiences . Regression analyses (SPSS v28) considered pre-pandemic onset maternal distress, COVID-19 stress, and their interaction upon post-pandemic onset maternal distress. Models were re-run with appropriate covariates  when significant findings were observed. To rule out alternative models, follow up analyses (PROCESS Model) considered whether COVID-19 stress mediated pre- and post-pandemic onset associations. Models involving maternal sensitivity followed a similar data analytic plan., p < 0.01) but not pandemic assessed maternal sensitivity. Perceived COVID-19 stress significantly contributed to post-pandemic onset maternal distress for mothers with pre-pandemic onset distress scores above , but not below , the median. Objective COVID-19 adversity did not account for findings. Post-hoc analyses did not suggest mediation via COVID-19 stress from pre-pandemic to pandemic maternal distress.Pre-pandemic maternal distress moderated the association between COVID-19 perceived stress and pandemic levels of maternal distress (β = 0.22Pre-existing risk may interact with subsequent perceptions of adversity to impact well-being. In combination with existing research, this small study suggests prevention programs should focus upon managing concurrent mental health and may highlight the importance of enhanced screening and proactive coping programs for people entering high stress fields and/or phases of life.The online version contains supplementary material available at 10.1186/s12888-023-04867-w. The overall indirect effect was not significant Β = 0.04 , thus not supporting a mediational model.Figure p < 0.001), and there were significant effects of pre-pandemic maternal distress and COVID-19 stress, as well as their interaction. First, there was an inverse relation between higher levels of pre-pandemic maternal distress and increased distress from pre-pandemic to pandemic . Second, higher levels of COVID-19 stress were positively associated with increased maternal distress from pre-pandemic to during the pandemic . Finally, higher levels of pre-pandemic distress in combination with higher levels of COVID-19 stress predicted more positive  change scores in pre-to-pandemic distress . However, for mothers with pre-pandemic distress scores below one standard deviation under the mean, COVID-19 stress did not predict pre-to-pandemic maternal distress . In sum, while higher levels of pre-pandemic distress associated with less increase in pre-to-pandemic distress, mothers with higher pre-pandemic distress who also experienced higher levels of COVID-19 stress exhibited higher levels of change in pre-to-pandemic distress.Difference scores were obtained by subtracting the pre-pandemic maternal distress composite score from the pandemic assessed maternal distress composite score. Table and felt high levels of COVID-19 stress. In fact, greater amounts of COVID-19 stress were positively associated with higher change scores only amongst mothers with higher levels of pre-pandemic distress. To our knowledge, no other group has examined such an interaction, which may be important in determining the complex way in which past and concurrent perceptions influence mental health.The purpose of this study was to investigate the interactive effects of concurrent adversity and prior maternal distress on later maternal distress and parenting quality. Specifically, we examined the interactive effect of COVID-19 related adversity and maternal distress assessed prior to the pandemic upon maternal distress and parenting assessed during the pandemic. A composite of maternal distress consisting of psychological distress, parenting stress and trait anxiety was used in this study, while parenting quality was examined using an observational measure of maternal sensitivity. As hypothesized, COVID-19 stress was directly associated with maternal distress assessed during the pandemic, but this was qualified by an interaction effect. Amongst mothers with higher levels of pre-pandemic maternal distress, COVID-19 related stress predicted greater maternal distress assessed during the pandemic, but this pattern was not observed amongst mothers with lower levels of pre-pandemic distress. This effect was robust, after accounting for maternal education, and the interaction remained significant when objective COVID-19 related stressors were considered. Importantly these results extend, but do not contradict, past research indicating that there was less of an increase in mental health symptoms amongst those who had higher levels of pre-pandemic distress . As withIn contrast, with regards to observed parenting, COVID-19 stress was not associated with maternal sensitivity assessed during the pandemic and there were no interactive effects with pre-pandemic maternal distress, even when pre-pandemic maternal sensitivity and maternal education were considered. Maternal sensitivity assessed during the pandemic was also not associated with maternal distress assessed during the pandemic. Hence, while results support our hypothesis concerning maternal distress, the hypothesis that parenting quality would be differentially affected by the interaction between pre-pandemic maternal distress and COVID-19 stress was not supported.The current research’s study design helps to rule out a potential confound, present in much research examining the impact of prior mental health factors on the relation between stress and trauma on later functioning. That is, exposure to COVID-19 governmental restrictions was “assigned” to the entire study group – not just those with pre-existing risks – minimizing the likelihood that relations between pre-existing risk and post-adversity outcomes are due to an increased likelihood of risk begetting risk, as opposed to risk leading to greater vulnerability in the face of adversity. Moreover, the current work also considered relations between self-reported perceptions of COVID-19 distress and variation in objective stressors during the same time period and still uncovered a significant interaction between pre-pandemic maternal distress and COVID-19 stress . In addition, there is some reason to consider that reliance on social support as a buffer against stress may vary by ethnicity , 48, furSecond, it is possible that although all participants were exposed to the COVID-19 pandemic and its associated restrictions, individuals had unique experiences in their exposure to specific COVID-19 stressors that could have influenced levels of perceived stress from COVID-19. Still, analyses incorporating additional data about the COVID-19 experiences continued to reveal a significant interaction between pre-pandemic distress and perceptions of COVID-19 stress, though post-hoc tests were no longer significant , and Post-Circuit Breaker.Additional file 3: Comparisons Between Mothers Who Completed the First Visit Only vs. Both Visits. A description of the tests comparing both groups of mothers and the results, including Supplementary Table 1.Additionalfile 4: COVID-19 Questionnaire. A copy of the questionnaire used to assess stress and stressors related to COVID-19.Additional file 5: Moderation Analyses Controlling for Covariates. A description of the moderation analyses with covariates, including Supplementary Tables 2 and 3.Additional file 6: Secondary Analyses of Objective COVID-19 Experiences. A description of the measures and of objective COVID-19 experiences and secondary analyses controlling for them, including Supplementary Tables 4 and 5.Additional file 7: Results of Moderation Analyses Without Outliers. Results of the main moderation analyses for pandemic maternal distress without three outliers, and Supplementary Table 6.Additional file 8: Post-hoc Analyses Examining Potential Memory Bias. A description of analyses to investigate whether retrospective reports of COVID-19 stress could have biased results.


N gene concentration by analyzing samples from several INSTAND external quality assessment (EQA) schemes starting in March 2021. The absolute N gene concentration was measured using reverse transcriptase digital PCR (RT-dPCR) as reference value. Moreover, the performance of five commercial ELISA tests using an EQA inactivated SARS-CoV-2 sample at different concentrations was assessed on the basis of these reference values.In routine clinical laboratories, severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the COVID pandemic, a wide range of antigen detection tests were also in high demand. We investigated the correlation between SARS-CoV-2 NCap antigen and Quantitative ELISA and RT-dPCR results showed a good correlation between SARS-CoV-2 NCap antigen and RNA concentration, but this correlation varies among SARS-CoV-2 isolates. A direct correlation between SARS-CoV-2 NCap antigen concentration and genome concentration should not be generally assumed.Further correlation studies between SARS-CoV-2 RNA and NCap antigen concentrations are needed, particularly in clinical samples and for emerging SARS-CoV-2 variants, to support the monitoring and improvement of antigen testing. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first reported in December 2019 in Wuhan, China . This diN gene and NCap antigen concentrations by analyzing dilution series of samples from the INSTAND External Quality Assessment (EQA) scheme for SARS-CoV-2 antigen detection (Program 410). For this purpose, we analyzed three quantitative ELISA assays for SARS-CoV-2 NCap antigen quantification and, for comparison, two qualitative ELISAs widely used in SARS-CoV-2 diagnostics. We used serial dilutions of one INSTAND EQA SARS-CoV-2 antigen sample from March 2021. Moreover, the quantitative ELISA kit with the best performance in terms of linearity, limit of detection (LOD) and limit of quantification (LOQ) was consequently used for correlation studies and for testing the samples from the cited EQA schemes of March, September and November 2021 and March 2022.The correlation of SARS-CoV-2 NCap antigen concentration with cycle threshold (Ct) values such as RNA concentration in clinical samples has been investigated . Howeverhttps://www.instand-ev.de/en/instand-eqas/eqa-program/offer/virus-antigen-detection-sars-cov-2-ag/). INSTAND  has been designated as a German reference institution for quality assurance in medical laboratories by the German Medical Association and is accredited according to DIN EN ISO/IEC 17043:2010. The samples investigated in this study are shown in Table The samples derived from EQA schemes were provided by INSTAND e.V (The Roche Elecsys SARS-CoV-2 (product number: 09 345 272 190) and the Euroimmun SARS-CoV-2 (product number Eqs. 2606–9601) antigen tests were performed for the qualitative detection of SARS-CoV-2 NCap. The ProteinTech (KE30007), Abcam (Ab274341), and GeneTex (GTX535824) tests were used for quantitative measurements of the SARS-CoV-2 NCap. To compare the dilution linearity of the three quantitative ELISA tests, a highly concentrated SARS-CoV-2 sample  and a series of five dilutions  were examined in duplicate on three different days. For the qualitative ELISAs, just five dilutions  were examined. Linear regression analysis was performed using Gen5 analysis software. LODs were determined as the concentration of recombinant nucleocapsid that provides a signal at least three times the standard deviation above the assay background generated using the correspondent kit assay diluent as a blank sample. For the LOQ, ten standard deviations were added to the background signal. For the background measurements of the different ELISA kits, 20 replicates were conducted.N gene quantification, a duplex assay was performed using China N and SarE genes with primers and probe concentrations of 400 nM and 200 nM, respectively, and optimized by varying the annealing temperature. The sequence of China N and SarE primers is described in [After RNA extraction with the Qiagen QiaAmp RNA kit, one step RT-dPCR reactions were carried out as described in . For N gribed in –11, Dropribed in . The PCRWe participated in the Virus Antigen Detection SARS-CoV-2 EQA scheme Program 410) of INSTAND in September/November 2021 and March 2022 together with 200 laboratories and used the Abcam ELISA kit for NCap antigen testing. Each program covers chemically inactivated samples from three different sources . The results of data analysis are presented as descriptive statistics by mean and standard deviation as appropriate. As a non-normal distribution was confirmed by a Kolmogorov–Smirnov test, a non-parametric Mann–Whitney U test was used to compare the SARS-CoV-2 nucleocapsid concentration between the ELISA kits. For all statistical analyses, A highly concentrated SARS-CoV-2 EQA sample distributed in March 2021, EQA Scheme Prog. 410 , was used to detect and measure the NCap by using different ELISA tests. NCap quantification was significantly lower when using the GeneTex assay (965 pg/ml) as compared to Abcam (2509 pg/ml) and Proteintech (2213 pg/ml). To verify the accuracy of the results from the samples investigated, we assessed the linearity at different levels of dilution in the diluent buffer of each ELISA kit  than in the delta isolate   isolate and between 220.75 pg/ml and 1481.05 pg/ml for the samples containing VOCs. The RNA concentrations ranged from 9.1 × 102 = 0.999) as well as the lowest LOD and LOQ. As such, it is the preferred assay for quantifying NCap antigen. The performance of the Proteintech kit is in terms of dilution linearity and LOD/LOQ less satisfactory than that of the Abcam kit. Moreover, this study indicates that GeneTEX protein quantification is less efficient and accurate than the other quantitative ELISA kits used here, showing poor dilution linearity and very high LOD and LOQ for protein measurements. Therefore, GeneTEX does not appear to be useful for NCap quantification in EQA samples.The direct correlation of RNA and protein concentrations in SARS-CoV-2 as measured by diagnostics tests is still poorly understood. To date, RT-qPCR is considered the gold standard for SARS-CoV-2 detection . But thiIn principle, if we compare the results of RT-dPCR and ELISA measurements using the dilution series of EQA samples, we expect to find a perfect one-to-one correlation. However, our study indicates no such one-to-one correlation of SARS-CoV-2 RNA and NCap antigen among the dilution series of the studied EQA samples. Hence, the amount of SARS-CoV-2 NCap antigen cannot be predicted based on the samples’ genome concentration. There are many factors affecting the correlation, including the nature of the SARS-CoV-2 isolate.In conclusion, our data suggest that constant evaluation of the sensitivity of ELISA for detecting new SARS-CoV-2 variants is vital to support monitoring and antigen testing. Further analysis including clinical samples and RT-dPCR absolute quantification will also help us to better understand the correlation between RNA and NCap antigen.The limitation of this study is the reduce number of SARS-CoV-2 variants of concern and non-variants of concern samples used; additionally clinical samples should be also tested to better understand the correlation between RNA and NCap antigen.