Regulatory T cells (Tregs) adoptive immunotherapy is emerging as a viable treatment option for both autoimmune and alloimmune diseases. However, numerous challenges remain, including limitations related to cell number, availability of target-specific cells, stability, purity, homing ability, and safety concerns. To address these challenges, cell engineering strategies have emerged as promising solutions. Indeed, it has become feasible to increase Treg numbers or enhance their stability through Foxp3 overexpression, post-translational modifications, or demethylation of the Treg-specific demethylated region (TSDR). Specificity can be engineered by the addition of chimeric antigen receptors (CARs), with new techniques designed to fine-tune specificity . The introduction of B-cell targeting antibody receptor (BAR) Tregs has paved the way for effective regulation of B cells and plasma cells. In addition, other constructs have emerged to enhance Tregs activation and function, such as optimized chimeric antigen receptors constructs and the use of armour proteins. Chimeric antigen receptor expression can also be better regulated to limit tonic signaling. Furthermore, various opportunities exist for enhancing the homing capabilities of CAR-Tregs to improve therapy outcomes. Many of these genetic modifications have already been explored for conventional CAR-T therapy but need to be further considered for CAR-Tregs therapies. This review highlights innovative CAR-engineering strategies that have the potential to precisely and efficiently manage immune responses in autoimmune diseases and improve transplant outcomes. As these strategies are further explored and optimized, CAR-Treg therapies may emerge as powerful tools for immune intervention. Autoimmune and alloimmune diseases are major public health challenges. Autoimmune diseases affect millions of individuals worldwide and can lead to severe disability and even death . More thImmunosuppressive drugs are currently the standard of care for managing these diseases . While t+ T lymphocytes characterized by high expression of IL-2Rα (CD25) and the transcription factor forkhead box p3 (Foxp3)  have emerged as key players in the maintenance of immune tolerance . Tregs a (Foxp3) , as well (Foxp3) . Tregs u (Foxp3) ).Preclinical models and early-phase clinical trials using adoptive Treg immunotherapy to promote tolerance have yielded promising results, underscoring the safety and potential effectiveness of this approach . HoweverIn contrast to effector CAR T cells, CAR constructs designed for Tregs (CAR Tregs) represent a distinct branch within the field of CAR T cell therapies. While effector CAR T cells are primarily engineered to target and eliminate specific disease-related cells, such as cancer cells, by inducing cytotoxicity and releasing pro-inflammatory cytokines (reviewed in ), CAR Tret al. . Howeveet al. pioneered this approach, inducing Foxp3 expression in CD25−, CD45RO− CD4+ lymphocytes by retroviral transduction. The transduced cells exhibited a regulatory phenotype similar to CD25+ CD4+ Tregs, demonstrated in vitro suppressive capabilities, decreased proliferation, cytokine production reduction, and heightened expression of Treg-associated molecules, such as CD25 and CTLA4   to induce Foxp3 expression (in vitro and in vivo (Bacchetta  (xGvHD) . Howeverfunction , their rpression . The tar in vivo . However in vivo . These o in vivo .+ T cells has also been combined with CAR expression . Indeedgulators . There agulators . Pharmacgulators . However in vivo . There iiewed in ). HoweveThree conserved non-coding DNA sequence (CNS) elements located at the FOXP3 locus have significant implications for the fate of Tregs. Specifically, CNS1 serves as a TGF-β-sensitive enhancer element crucial for induced Treg generation, CNS2 plays a critical role in stabilizing Foxp3 expression within dividing Treg cells, and CSN3 can modulate Treg frequency in the thymus and periphery but becomes dispensable once Foxp3 is expressed . NotablyCurrently, three clinical trials are registered using monospecific CAR-Tregs in humans . Howeveret al. developed this novel receptor and demonstrated that TanCARs retain the cytolytic capacity of T cells, even when one of the target molecules is lost. This approach also results in a more controlled response against tumors by recognizing both targets in an animal disease model  of the nuclear protein LA/SS-B  were developed . In summn grafts . Bachman LA/SS-B , and the LA/SS-B . The advet al., in cytotoxic T cells, to destroy autoantigen-specific hybridoma in a pemphigus vulgaris model (in vitro (human) and in vivo (mouse). The same team then tested their FVIII BAR in Tregs and demonstrated their ability to suppress the memory antibody response in spleen cultures from FVIII-immunized mice in vitro and completely prevented the development of anti-FVIII antibodies following FVIII immunization  as the targeted protein  were ineffective in promoting CAR-Tregs function, while CD28-CAR-Tregs exhibited highest effectiveness. PD-1-CAR Tregs displayed ineffective activation, and TNFR2 CARs resulted in a marked increase in TSDR methylation  in effector T cells. Their strategy involved designing a gRNA targeting the 5′end of the first exon of TRAC and employing an adeno-associated virus (AAV)-based repair vector encoding a self-cleaving P2A peptide followed by the CAR-CD19 cDNA. The resulting CAR-T cells not only demonstrated uniform CAR-CD19 expression, but also exhibited a reduction in tonic signaling, exhaustion and an increase in antitumour efficacy . The resulting cells demonstrated uniform CAR expression, retained their phenotype , and exhibited A2-specific suppressive functionality in vitro and in vivo. These cells demonstrated a delay in GvHD development, exclusively in the presence of HLA-A2, whether expressed by simultaneously injected peripheral blood mononuclear cells or the recipient mice themselves. These outcomes effectively illustrated that these genetically engineered CAR-Tregs display antigen-dependent in vivo suppression, independent of TCR expression , which fuses the scFv to the N terminus of the TCRε subunit . This apShaping Tregs for better homing abilities to specific inflammation sites has been a topic of interest since the early 2000s. Studies investigating homing patterns of Tregs using UV irradiation showed migration to lymph nodes upon injection . The indIt should also be considered that antigen-specific Tregs inherently exhibit more efficient homing abilities than expanded Tregs generated from polyclonal pools . The homStudies in conventional CAR-T cell therapy have demonstrated that chemokine-specific CARs can more efficiently inhibit the growth of solid and immunosuppressive cancers. Human solid tumors xenograft models have been evaluated for chemokines such as CXCL1-2. The intra-tumoral presence of CAR-T cells increased when modified for CXCR1-2 surface expression . Transdu+ Tregs enhances recruitment to sites of intestinal inflammation (+Foxp3+ T helper (Th)1-Tregs, improves migration towards CXCL9, 10 and 11 . HoweveAnother strategy involves the use of enzyme inhibitors, which can reversibly lead to CAR degradation. This requires the addition of a bromodomain or protease domain on the CAR, which can be modulated using a proteolysis-targeting chimera compound or a protease inhibitor (reviewed in ). None oPermanent elimination of CAR-T cells can be achieved by integrating suicide genes into these cells. For example, metabolic suicide gene systems can be employed with a non-toxic prodrug therapy administered systematically. Genetically-modified cells possess the gene necessary to convert the prodrug into a toxic compound (reviewed in ). AlternThe development of advanced generations of CARs, such as TanCARs, UCARs, SynNotch CARs, or BARs, holds great promise for CAR-Treg therapy by overcoming challenges related to immunogenicity and production limitations. Furthermore, the incorporation of armour proteins and safety mechanisms like suicide genes, as well as enhancements of homing abilities through chemokine receptors, have the potential to revolutionize the targeting precision and stability of CAR-Tregs. The use of these innovative constructs brings us closer to achieving precise, safe, and efficient treatments for autoimmune and alloimmune diseases. However, further progress requires a rigorous evaluation of these novel CAR-Tregs in preclinical models prior to their introduction into clinical trials.


Heterocyclic aromatic amine molecularly imprinted polymer nanospheres with surface-bound dithioester groups (haa-MIP) were firstly synthesized via reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization. Then, a series of core-shell structural heterocyclic aromatic amine molecularly imprinted polymer nanospheres with hydrophilic shells (MIP-HSs) were subsequently prepared by grafting the hydrophilic shells on the surface of haa-MIP via on-particle RAFT polymerization of 2-hydroxyethyl methacrylate (HEMA), itaconic acid (IA), and diethylaminoethyl methacrylate (DEAEMA). The haa-MIP nanospheres showed high affinity and specific recognition toward harmine and its structural analogs in organic solution of acetonitrile, but lost the specific binding ability in aqueous solution. However, after the grafting of the hydrophilic shells on the haa-MIP particles, the surface hydrophilicity and water dispersion stability of the polymer particles of MIP-HSs greatly improved. The binding of harmine by MIP-HSs with hydrophilic shells in aqueous solutions is about two times higher than that of NIP-HSs, showing an efficient molecular recognition of heterocyclic aromatic amines in aqueous solution. The effect of hydrophilic shell structure on the molecular recognition property of MIP-HSs was further compared. MIP-PIA with carboxyl groups containing hydrophilic shells showed the highest selective molecular recognition ability to heterocyclic aromatic amines in aqueous solution. Molecularly imprinted polymers (MIPs) are artificial receptors with permanent template cavities (imprinted binding sites) in the network, which are complementary to the shape, size, and functionality of the template ,2. Due tThere have been many works focused on the MIPs-based solid-phase extraction of target analytes in real matrices ,15,16,17Heterocyclic aromatic amines (HAAs), which are usually formed by a complex Maillard reaction and/or thermal pyrolysis from amino acids, proteins, reducing sugars, and other precursors, are highly concerned toxicants widely found in fried food, coffee, and cigarette smoke ,27,28. MIn this study, HAA molecularly imprinted polymer nanospheres with surface-bound dithioester groups (haa-MIPs) were firstly synthesized via RAFT precipitation polymerization. Subsequently, surface grafting of hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA), poly (Itaconic acid) (PIA), and poly(diethylaminoethyl methacrylate) (PDEAEMA) outer shells was performed via on-particle RAFT polymerization, using the “living” character of the haa-MIP to construct a series of core-shell structural HAA molecularly imprinted polymer nanospheres with hydrophilic shells (MIP-HSs). The water dispersion stability and molecular recognition of HAAs by haa-MIP and MIP-HSs nanospheres were studied by equilibrium binding analysis to demonstrate the efficient HAA molecular recognition in aqueous solution.Well-defined HAA molecularly imprinted polymer nanospheres with surface-bound dithioester groups (haa-MIPs) were firstly synthesized by RAFT precipitation polymerization of methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) with harmine as template. Then, using the haa-MIP with “living” dithioester end groups as the immobilized RAFT agent, on-particle RAFT polymerization of hydrophilic monomers, such as 2-hydroxyethyl methacrylate (HEMA), itaconic acid (IA), and diethylaminoethyl methacrylate (DEAEMA), resulted in a series of core-shell structural HAA molecularly imprinted polymer nanospheres with hydrophilic shells (MIP-HSs) .−1 (C=O stretching vibration), 1260 cm−1, and 1160 cm−1 (C−O−C stretching vibration) belonged to the ester groups in the EGDMA units, which verified the existence of EGDMA units. Meanwhile, the peaks at around 3580 cm−1 (O-H stretching vibration), as well as the 1730 cm−1 peak (C=O stretching vibration), confirmed the existence of MAA units in both the un-grafted haa-MIP and the grafted MIP-HSs particles. The small peak around 1630 cm−1 in the FTIR spectra is ascribed to the C=C stretching vibration, which is from the un-crosslinked EGDMA units in the MIPs. The characteristic band appeared at around 1565 cm−1 (C–N stretching vibration band) in the FTIR spectrum of MIP-PDEAEMA, indicating the successful surface grafting of PDEAEMA shells onto the precursor of haa-MIP beads. It should be noted that the characteristic absorption peaks of PHEMA and PIA chains were overlapped by the haa-MIP core in the FTIR spectra of MIP-PHEMA and MIP-PIA.The obtained un-grafted haa-MIP and grafted MIP-HSs nanospheres were characterized by Fourier transform infrared (FTIR) spectra. As seen in To improve the surface hydrophilicity of MIPs beads, grafting of hydrophilic shells was achieved by on-particle RAFT polymerization of hydrophilic monomers. The resultant core-shell structural MIP-HSs were then characterized by laser diffraction particle size analyzer, and the results are shown in The morphology of MIP-HSs was observed by SEM. As shown in To verify the molecular selectivity of the haa-MIP nanospheres toward HAA, equilibrium binding of the haa-MIP and the NIP nanospheres toward the template of harmine in acetonitrile were firstly compared, and the results are shown in To further study the selectivity of haa-MIP nanospheres toward other HAAs that are structural analogs of harmine, the binding property of haa-MIP and NIP nanospheres toward harman and AαC was further investigated, which is shown in It has been well demonstrated that the solvent, such as water, could play an important role in the MIPs binding process because the solvent can not only affect the MIPs’ surface properties, but also can have great influence on the interactions (such as hydrogen bond) between the MIPs and the target analytes. To reduce the non-specific binding and improve the specific binding of harmine in aqueous solution, hydrophilic polymer shells were grafted onto the haa-MIP to obtain core-shell structural HAA molecularly imprinted polymer nanospheres with hydrophilic shells (MIP-HSs).To further study the selectivity of MIP-PIA nanospheres to HAAs that are structural analogs of harmine, the binding of MIP-PIA and NIP-PIA nanospheres to two other HAAs, harman and AαC, was further investigated. Methacrylic acid (MAA), Ethylene glycol dimethacrylate (EGDMA), 2-Hydroxyethyl methacrylate (HEMA), Itaconic acid (IA), diethylaminoethyl methacrylate (DEAEMA), 2,2′-azobisisobutyronitrile (AIBN), 4,4’-Azobis (V501), harmine, harman, and 2-amino-9H-pyrido [2-3-b]indole (AαC) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTP) was purchased from Sigma-Aldrich . MAA, HEMA, and DEAEMA were purified by vacuum distillation before use. AIBN and V501 were recrystallized from ethanol before use. MilliQ deionized water  was used. Other materials were used as received.v/v), and methanol successively until no template could be detected in the washing solution. After being dried at 40 °C under vacuum for 48 h, light pink haa-MIP nanospheres were obtained. The none-imprinted polymer (NIP) nanospheres were also prepared and purified as control under the identical conditions, except that no template was added to the polymerization solution.The haa-MIP nanospheres with surface-bound dithioester groups were synthesized via RAFT precipitation polymerization by using harmine as template. Typically, MAA , harmine , EGDMA , CTP , AIBN , and acetonitrile (80 mL) were placed in a dry glass ampule equipped with a magnetic stirring bar, and then the solution was degassed under reduced pressure by triple freeze–pump–thawing cycles. After being purged with argon, the reaction mixture was sealed and stirred at 25 °C for another 2 h in order to allow the self-assembly of the functional monomer and template. Then, the polymerization was carried out at 70 °C for 16 h. The reaction was stopped by rapid cooling with liquid nitrogen, and the resulting polymer particles were obtained by centrifugation and then washed thoroughly with acetonitrile, methanol/acetic acid  were added into a dry glass ampule equipped with a magnetic stirring bar. After being degassed by three freeze–pump–thaw cycles and purged with argon, the reaction mixture was sealed and polymerization at 70 °C for 12 h. The resultant polymer particles were collected by centrifugation and then washed thoroughly with methanol/water (3:1 v/v) and methanol successively, before drying at 40 °C under vacuum. The none-imprinted polymer with different hydrophilic shells (NIP-HSs) were also prepared as control using the same polymerization procedure, except for replacing the haa-MIP nanospheres by NIP particles.A series of core-shell structural MIP-HSs with hydrophilic shells were prepared by on-particle RAFT polymerization of hydrophilic monomers  using the above obtained “living” haa-MIP nanospheres as the immobilized RAFT agent and a certain amount of sacrificial RAFT agent of CTP . The typFourier transform infrared (FTIR) spectra were measured on a Bruker Tensor 27 spectrometer by pressing the sample to be tested into a potassium boride (KBr) disk. The size distribution of the particle was determined by laser diffraction on a Mastersizer 3000 particle size analyzer. The microscopic morphology was observed by a Zeiss Gemini 300 field emission electron scanning microscope (SEM) with an accelerating voltage of 10 kV.To 5.0 mL of solution of HAA , 5.0 mg of polymer particles  was added. The mixture was then stirred on a rocking table at 25 °C for 12 h. Supernatant (3.0 mL) was collected from each sample by centrifugation separation, and the concentration of the un-bound HAA was subsequently measured using a Cary-100 UV-vis spectrophotometer. The bound amount of the HAA was calculated by the difference between the initial and the un-bound HAA after the binding procedure.HAA molecularly imprinted polymer nanospheres with surface-bound dithioester groups (haa-MIPs) were firstly synthesized via RAFT precipitation polymerization. With the “living” haa-MIP nanospheres as the immobilized RAFT agent, a series of core-shell structural HAA molecularly imprinted polymer nanospheres with hydrophilic shells (MIP-HSs) were subsequently prepared by on-particle RAFT polymerization of hydrophilic monomer of HEMA, IA, and DEAEMA. The haa-MIP showed high affinity and specific recognition toward harmine and its structural analogs in pure acetonitrile, but nearly lost the specific binding ability in aqueous solution. After the grafting of the hydrophilic shells on haa-MIP particles, the binding of harmine by MIP-HSs in aqueous solutions is about two times higher than that of NIP-HSs, showing an improved molecular recognition in aqueous solutions. The effect of the hydrophilic shell structure on the molecular recognition property of MIP-HSs was also compared. The highest selective molecular recognition toward HAA was achieved by MIP-PIA with carboxyl groups containing hydrophilic shells. These MIP-HSs may be used in the solid phase extraction procedure for heterocyclic aromatic amines analysis in food and biological samples and/or as chemical sensor building blocks for heterocyclic aromatic amines.


Advanced prostate cancer remains a clinical challenge, requiring novel therapeutic treatments and biomarkers for patient-risk stratification. From a metabolic perspective, primary prostate cancer shows unique features with respect to most solid tumors, being mainly characterized by an oxidative/lipogenic phenotype. However, increased aerobic glycolysis with enhanced lactate production (Warburg effect) is frequently observed in high-risk disease and during the onset of castration resistance. In this review, we summarize the key role of lactate in prostate cancer progression. We also highlight the clinical implication of lactate as a predictive/prognostic biomarker and the therapeutic potential of targeting lactate metabolism in prostate cancer.Advanced prostate cancer represents the fifth leading cause of cancer death in men worldwide. Although androgen-receptor signaling is the major driver of the disease, evidence is accumulating that disease progression is supported by substantial metabolic changes. Alterations in de novo lipogenesis and fatty acid catabolism are consistently reported during prostate cancer development and progression in association with androgen-receptor signaling. Therefore, the term “lipogenic phenotype” is frequently used to describe the complex metabolic rewiring that occurs in prostate cancer. However, a new scenario has emerged in which lactate may play a major role. Alterations in oncogenes/tumor suppressors, androgen signaling, hypoxic conditions, and cells in the tumor microenvironment can promote aerobic glycolysis in prostate cancer cells and the release of lactate in the tumor microenvironment, favoring immune evasion and metastasis. As prostate cancer is composed of metabolically heterogenous cells, glycolytic prostate cancer cells or cancer-associated fibroblasts can also secrete lactate and create “symbiotic” interactions with oxidative prostate cancer cells via lactate shuttling to sustain disease progression. Here, we discuss the multifaceted role of lactate in prostate cancer progression, taking into account the influence of the systemic metabolic and gut microbiota. We call special attention to the clinical opportunities of imaging lactate accumulation for patient stratification and targeting lactate metabolism. Prostate cancer (PCa) is the second leading cause of cancer death in men in the US and the third in Europe ,2. Bone Patients with metastatic CRPC are commonly treated with second-generation AR antagonists , intra-tumor androgen synthesis inhibitors , radium 223 chloride (approved in PCa patients with bone metastasis), taxane or platinum-based chemotherapy, poly(ADP-ribose)polymerase (PARP) inhibitors (approved in PCa patients with mutations in homologous recombination repair genes), and immune checkpoint inhibitors (approved in PCa patients with metastatic PCa harboring microsatellite instability or mismatch repair defects) . While t18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) in detecting primary PCa [13C] pyruvate to lactate in patient-derived xenografts (PDX) from AR-driven compared to AR-negative CRPC, suggesting AR-mediated modulation of aerobic glycolysis  and LDH5 or LDHA [A4]), and three heterotetrameric isoforms . All isoforms are able, with variable efficacy, to catalyze both the reduction of pyruvate to lactate and the oxidation of lactate to pyruvate . LDHA haertility . LDHA prertility . Furtherertility . The autertility .Several meta-analyses have been conducted to validate serum LDHA as a prognostic biomarker by examining the correlation of serum LDH with overall survival, cancer-specific survival, and progression-free survival in patients with metastatic PCa. A meta-analysis of 59 studies  analyzed serum LDH levels in patients with metastatic CRPC and metastatic hormone-sensitive PCa . The autTo avoid intracellular lactate accumulation, cancer cells export lactate in symport with protons into the TME through monocarboxylate transporters (MCTs). MCT-1, which has a higher affinity for lactate, is mainly involved in the uptake and import of lactate into the cell, whereas MCT-4 is mostly involved in lactate efflux. Immunohistochemical analyses of MCT-1 and MCT-4 in a well-annotated specimen collection including PCa/adjacent non-neoplastic tissues, prostatic intraepithelial neoplasia lesions, and normal prostatic tissues uncovered a significant increase in MCT-4 and decrease in MCT-1 in PCa cells compared to adjacent normal epithelium. MCT-4 expression was positively correlated with higher PSA levels, Gleason score, pT stage, perineural invasion, and biochemical recurrence .While accumulating evidence supports lactate metabolism as an actionable metabolic adaptation and LDHA/MCTs as predictive and prognostic biomarkers, their clinical translation has only recently been actively investigated. We believe that the implementation of real-time, non-invasive metabolic imaging during diagnostic workup and therapy follow-up will be crucial in this context.Aerobic glycolysis and lactate synthesis/transport are fine-tuned by AR signaling and alterations in oncogene and tumor suppressors that usually occur in CRPC. These metabolic adaptations support PCa progression, as discussed below.Early evidence that AR signaling directly modulates the expression of glycolytic genes emerged from the work of Massie and coworkers in 2011 . Using a13C]pyruvate, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS) performed in AR-dependent CRPC PDX models  [In vivo confirmation of the AR- and AR-V7-dependent regulation of lactate synthesis in CRPC was independently obtained from data of hyperpolarized (HP) [1-nd 133-4 ) and AR-nd 133-4 ) 30]. A. A13C]pye above) ,29.13C glucose/13C glutamine flux analysis and HP [1-13C]pyruvate MRSI were performed together with ex vivo measurements of steady-state metabolites, enzymatic activities, and oxygen consumption/extracellular acidification rates. Castration-resistant TRAMP tumors  showed increased levels of U-13C glucose-derived lactate, increased lactate/reduced citrate steady-state levels, and a higher LDHA activity compared to androgen-sensitive TRAMP tumors. These metabolic features were associated with increased HP [1-13C]pyruvate-to-lactate conversion (high HP Lac/Pyr ratio) (13C]pyruvate can noninvasively capture enhanced aerobic glycolysis in CRPC . In. In+/NADtabolism  92]. In. In+/NADtabolism . In humaFinally, new data have uncovered CAF-derived lactate shuttling as a mediator of ECM remodeling. According to this recent study, CAF-derived lactate is taken up by PCa cells and oxidized to α-KG, which is used as a co-factor for the activity of the enzyme collagen prolyl-4-hydroxylase, resulting in increased collagen hydroxylation and deposition. Increased collagen deposition is involved in the activation of the discoidin-domain collagen receptor 1 and its downstream signaling via STAT3, which significantly enhances PCa cells’ invasive properties. CAF-derived lactate has also been shown to also boost the formation of anchorage-independent tumor clusters and transendothelial migration in vitro and in vivo, suggesting a potential role of collagen in circulating tumor cells and the metastatic dissemination of lactate-reprogrammed PCa cells .Altogether, these findings have uncovered lactate shuttling as a metabolic fuel, immune/epigenetic modulator, and ECM remodeler to sustain PCa progression. Disrupting PCa–CAFs interaction holds potential as a therapeutic approach.Diet and metabolic diseases, including obesity, affect the TME and disease progression  95,96].,96.95,96Metabolic diseases , diet , and drug exposure  also induce gut microbiota dysbiosis, which is associated with the promotion of PCa progression  101,102102,103.The gut microbiota generates metabolites that circulate within the human host and affect cancer risk. For example, increased levels of the serum gut-microbiota-derived metabolites choline, trimethylamine N-oxide, phenylacetylglutamine have been associated with a diagnosis of aggressive PCa and increased risk of lethal PCa . FurtherHowever, confirmatory studies using multiracial populations with different lifestyles are strongly needed. Improvements in the integration of shotgun metagenome and metabolic analyses will be instrumental in clarifying the role of lactate-metabolizing gut bacteria in PCa progression.In addition to the gut microbiome, the prostate and bladder microbiome, which can be easily assessed in the urine and prostatic fluid, can also affect PCa development and progression ,111,112.18F-FDG PET in metastatic CRPC and on developing HP 13C pyruvate MRSI to monitor pyruvate-to-lactate conversion in real time. The use of HP MRSI has significantly expanded our knowledge on lactate as a potential diagnostic, prognostic, and predictive biomarker.Given the evidence for a metabolic switch during PCa progression, extensive efforts have been dedicated to exploiting glucose metabolism rewiring and lactate levels for diagnostic and therapeutic purposes. These efforts have focused on repurposing 18F-FDG PET measures neither the glycolytic flux nor the lactate synthesis per se. However, it can assess the uptake and accumulation of the non-metabolizable glucose analogue 18F-FDG, which often correlates with glycolytic flux and lactate synthesis in cancer. 18F-FDG PET shows a limited usefulness in the detection and staging of primary PCa, whose oxidative/lipogenic phenotype is better captured using precursor radionucleotides such 11C/18F-choline and 11C-acetate  pyruvate MRSI has enabled the direct and non-invasive investigation of lactate as an early prognostic and predictive biomarker in preclinical/ex vivo studies. HP [1-13C] magnetic resonance (MR) is a powerful metabolic imaging method that uses a dynamic nuclear polarization technique to provide signal enhancements of over 10,000-fold for 13C-enriched, endogenous, non-radioactive compounds suitable for clinical translation for the real-time monitoring of enzyme kinetics. Contrary to 18F-FDG, which assesses only glucose uptake and phosphorylation, HP 13C MRSI using [1-13C]pyruvate detects the rate of conversion for [1-13C]pyruvate to [1-13C]lactate . The earliest application of this technology in an animal model of PCa, namely the TRAMP, demonstrated an increased pyruvate-to-lactate conversion in aggressive PCa pyruvate MRSI  and were able to differentiate clinically significant PCa from indolent disease in a low/intermediate-risk population. The authors also uncovered a correlation between the [1-13C]lactate signal with both LDHA  and MCT-4 (ratio of epithelium-to-stroma), highlighting the potential of HP 13C MRSI to stratify patients based on metabolic differences in the epithelial and stromal tumor compartments [13C]pyruvate MRI in monitoring PCa patients under active surveillance (NCT03933670).Over the last two decades, the work of several groups worldwide has been groundbreaking in translating the application of HP pyruvate MRI performance in monitoring patient responses to treatment, including chemotherapy and ADT.Consistent efforts have been devoted to evaluating HP pyruvate MRI acquisition into the standard-of-care multiparametric MRI for guiding transrectal ultrasound fusion prostate biopsies pyruvate-to-lactate conversion predicts clinically relevant disease and holds potential as: (i) a non-invasive approach to stratify high-risk PCa patients, and (ii) a tool to monitor residual/significant disease in patients given treatment (including treatments with LDHA and MCT-4 inhibitors) or on active surveillance. Given the pleiotropic oncogenic effects of lactate on both PCa and cells in the TME, targeting lactate metabolism represents a strategy to both halt cancer cell dissemination and promote cancer immune surveillance. Reverting the immune suppressive phenotype using lactate metabolism inhibitors may enhance the response to ICIs, which has thus far been poor for PCa. Furthermore, the AR-dependent regulation of metabolic pathways offers opportunities for combined treatments of lactate metabolism inhibitors with AR-signaling inhibitors, including enzalutamide and abiraterone. The non-invasive measurement of [1-


DNA-polymer hybrids have been attracting interest asadaptablefunctional materials by combining the stability of polymers with DNAnanotechnology. Both research fields have in common the capacity tobe precise, versatile, and tunable, a prerequisite for creating powerfultools which can be easily tailored and adapted for bio-related applications.However, the conjugation of hydrophilic DNA with hydrophobic polymersremains challenging. In recent years, polymerization-induced self-assembly(PISA) has attracted significant attention for constructing nano-objectsof various morphologies owing to the one-step nature of the process,creating a beneficial method for the creation of amphiphilic DNA-polymernanostructures. This process not only allows pure DNA-polymer-basedsystems to be produced but also enables the mixture of other polymericspecies with DNA conjugates. Here, we present the first report ofa DNA-PEG corona nano-object’s synthesis without the additionof an external photoinitiator or photocatalyst via photo-PISA. Furthermore,this work shows the use of DNA-macroCTA, which was first synthesizedusing a solid-support method resulting in high yields, easy upscaling,and no need for HPLC purification. In addition, to the formation ofDNA-polymer structures, increasing the nucleic acid loading of assembliesis of great importance. One of the most intriguing phenomena of DNAis the hybridization of single-stranded DNA with a second strand,increasing the nucleic acid content. However, hybridization of DNAin a particle corona may destabilize the nanomaterial due to the electrostaticrepulsive force on the DNA corona. Here, we have investigated howchanging the DNA volume fraction in hybrid DNA-polymer self-assembledmaterial affects the morphology. Moreover, the effect of the coronacomposition on the stability of the system during the hybridizationwas studied. Additionally, the hybridization chain reaction was successfullyapplied as a new method to increase the amount of DNA on a DNA-basednano-object without disturbing the morphology achieving a fluorescencesignal amplification. Recently, to overcome theselimitations the direct extension of DNA strands with polymers hasbeen reported using reversible addition-fragmentation chain transfer-mediatedpolymerization-induced self-assembly (RAFT-mediated PISA) has beendeveloped.6 PISA allows the direct constructionof polymeric nanoparticles (NP) of various morphologies, normallyspheres, vesicles, or worms.7 Control overparticle composition, morphology, and functionality can be achievedby varying the monomer, macromolecular chain transfer agent (macroCTA),and concentration.1 In 2020, Luckerathet al. suggested that DNA–polymer conjugates can self-assembleto nano-objects by thermal RAFT PISA through the grafting from approach.6 This reaction was performed in a low volume system,relying on enzyme degassing using glucose, glucose oxidase, and sodiumpyruvate to get rid of oxygen in the system which would hinder thepolymerization. This resulted in high monomer conversions in a systemwhere the monomer to initiator ratio could be precisely controlled,allowing the manipulation of architectures formed. Yang et al. modifieda photochain transfer agent (photo-CTA) to perform the first aqueousRAFT photo-PISA of functional DNA-polymer nanostructures under anenzyme-assisted approach.8 2-hydroxypropylmethacrylate (HPMA) was used as a monomer to manipulate the DNA-polymernanostructure. This work showed that DNA-poly(2-hydroxypropyl methacrylate)(DNA-PHPMA) particles had enhanced nuclease resistance and increasedcellular uptake.DNA-polymer hybrids are adaptable functionalmaterials with numerousapplications ranging from nanotechnology to biomedicine.9 The concept behind HCR is a multihybridizationevent between two species of DNA hairpins which are initiated by aninitiator ssDNA, yielding a nicked DNA duplex with repeating units x. In the past, HCR products were obtained with a varietyof functional moieties such as fluorophores,10 gold NPs,11 and electrochemical reagents12 to achieve biosensing, signal transduction,or transforming input molecules. As we know, corona modification ofNPs influences the surface characteristics and properties of the nano-objects.To the best of our knowledge, HCR on DNA-polymer nano-objects hasnot been investigated before and represents an important step towardresponsive DNA-polymer hybrids. Hence, here we aim to apply HCR toDNA-particles to prove the possibility of building larger DNA constructson top of a short starting sequence covalently bound to the particle.The presence of the initiator DNA to gives a fluorescence read-outand signal amplification. We believe this represents not only keyknowledge to achieve interactive DNA particles to act as biosensor,but moreover we show that DNA-polymers can be used as foundation forlarger DNA constructs without disturbing particle morphology.Most natural DNA forms a double helix structurethrough hybridization,which is used in vivo for storing and transmitting genetic information.Hybridization is promoted by the formation of hydrogen bonds betweenthe nucleobases of single-stranded DNA (ssDNA) with its complementaryDNA (cDNA). Due to the high sequence-specificity of DNA hybridization,it is possible to construct even large and complicated DNA structures.Thus, precise DNA sequence design is the key to unlocking structureand/or sequence driven DNA-based materials in nanoscience. The advantageof an increased cellular uptake and nuclease resistance make DNA-polymerhybrids the perfect candidate for the development of a new class ofa responsive biocompatible material. However, not much is known aboutthe ability to build DNA structures in situ after DNA-polymer assemblyand how DNA hybridization impacts the size and/or morphology of theDNA-polymer particles. Over the past decade, signal amplificationusing nucleic acids has become an attractive tool in biotechnology.Among DNA signal amplification techniques, hybridization chain reaction(HCR) is a simple yet powerful molecular tool with various applicationsin biosensing, bioimaging, bioanalysis, and biomedical research.14-macroCTA by solid support synthesis, resultingin high yields, scalability, and high purity without the need forfurther high-performance liquid chromatography (HPLC) purification.This macroCTA has been combined with trithiocarbonate-based poly(ethyleneglycol) (PEG) macroCTA for the novel fabrication of binary mixturesof PEG113-PHPMA400 and DNA14-PHPMA400 nanomaterials. No additionl external photoinitiator orphotocatalyst was required and the synthesis was undertaken via low-volumeRAFT-mediated photo-PISA using enzyme-assisted degassing. Systematicvariation of the relative proportions of PEG113 and ssDNA14 macroCTAs resulted in the formation of diblock copolymerspheres, lumpy rods, or vesicles, where increasing the ratio of PEG113 led to the formation of higher order morphologies and morestable particles in salt/buffer solutions. Moreover, the resultingparticles were further hybridized with TAMRA-cDNA. In addition, HCRwas first applied to hybrid NPs to enhance the amount of DNA on theparticle surface without disturbing particle morphology obtaininga fluorescence signal  was formed by the conjugation of ssDNA14-NH2 (5′-5AmMC12-TGTAGCGTTGTTGC-3′)with a 4-cyano-4-(dodecylsulfanylthiocarbonyl) sulfanyl pentanoicacid group (CEPA) at its 5′ terminus via solution or solidsupport approaches  and is finally eluted from the solid supportwithout the need for further purification. The solid support-mediatedsynthesis is separated into four stages. During stage 1, ssDNA14-NH2 was bound to the solid support. In stage2, the small molecule was added as activated ester to the adsorbedDNA in DMF. In stage 3, the excess small molecules including degradedside products were removed by washing with DMF, while the modifiedDNA remained adsorbed to the solid support. During the final stagethe modified DNA was eluted using a water-based elution buffer.13In order to synthesize the DNA-loadedparticles, a high yielding,easily scalable, and simple method for synthesizing DNA-macroCTA wasneeded. The ssDNAproaches 1a. By us14-macroCTA wasconfirmed byusing HPLC equipped with a UV–vis detector monitoring the elutedDNA solution at 260 and 309 nm as the maximum absorbance of the DNAand trithiocarbonate group, respectively. High conversion and purity(95%) of the targeted ssDNA14-macroCTA strand was achievedwith the solid support approach, represented by the occurrence ofone DNA-species signal at both detector wavelengths 260 and 309 nm. LC/MS furthermore confirmedthe successful formation of the desired product. Meanwhile, the solutionapproach shows a second signal at 309 nm, indicating an impurity fromthe trithiocarbonate-group-containing starting material. AdditionalHPLC purification and extensive purification steps including spinfiltration and ethanol precipitation were required to obtain a comparablepurity. Such time-consuming purification steps are not feasible especiallyif high amounts of ssDNA14-macroCTA are required . Our results show that the solid support-mediatedsynthesis of the desired macroCTA-modified DNA is not only superiorin terms of yield and purity but is moreover easy to apply to large-scaleDNA reactions (up to 100 nmol) without the need of further purification,while the solution method is limited to 2 nmol DNA in each reactionbatch. Thus, we accomplished the synthesis of the ssDNA14-macroCTA in a few hours with the large quantities, high yields,and purity, needed for further utilization in a DNA nano-object PISAreaction.The purity of the ssDNA113-PHPMA400 andDNA14-PHPMA400 diblock copolymer NPs were synthesizedby systematicallyvarying the PEG113 and ssDNA14 macroCTAs molefractions via low-volume RAFT-mediated photo-PISA. These CTAs arephotoiniferters, which can be cleaved by light at 405 nm to generatea thiocarbonylthio radical.16 This radical subsequently servesthe dual roles of initiating the polymerization of a monomer and actingas a RAFT agent, eliminating the requirement for additional initiatorsor catalysts. Thus, this is the first report of DNA-polymer nanostructuresproduced via low-volume PISA (50 μL) without adding an externalphotoinitiator or photocatalyst. PEG113-macroCTA was chosendue to its biocompatible properties being extensively studied in vivo to protect DNA from degradation, and its similarmolecular weight to ssDNA14-macroCTA.17 HPMA was chosen due to the literature precedent for forminga hydrophobic polymer block with hydrophilic polymer chain such asPEG.18 Low-volume aqueous RAFT-mediatedphoto-PISA of HPMA was undertaken under 405 nm visible light irradiationat 37 °C using an enzyme degassing approach, i.e., glucose oxidase.20 Mineral oil was added on top of the 50 μL solution to preventdiffusion of residual oxygen into the solution, which inhibits thepolymerization.A series of PEGTable S1 summarizesthe targeted diblockcopolymer compositions, HPMA monomer conversions, molecular weightdata, dynamic light scattering (DLS) data, and morphological assignments.High monomer conversions (>70%) were achieved after 2 h of reaction,as determined by 1H NMR spectroscopy. It is important tonote that the targeted degree of polymerization for PHPMA is 400.However, incomplete polymerization conversion might have an effecton the resulting morphology of the self-assembled nanostructure. PHPMAwas successfully polymerized with a significant increase in molecularweight confirmed by size exclusion chromatography analysis (in DMF). Zeta potentials have beenapplied to measure the surface electrical charge of particles. Thezeta potential of 100% DNA-PHPMA400 was found to be around−32.4 ± 9.7 mV revealing the presence of negative chargefrom DNA on the outer surface of particles, whereas pure PEG113-PHPMA400 exhibited a nearly neutral surface charge (−0.6± 3.1 mV). The 50% DNA-PHPMA400 displayed a negativecharge of approximately −28.6 ± 7.0 mV, which falls betweenthe values of the 100% DNA and PEG113-PHPMA400 systems. This rationally indicated that the negative charge fromDNA on the particle surface decreases when the fraction of DNA isreduced.S3, respectively.When 100% ssDNA14–PHPMA400 diblock copolymerchains self-assembled only spherical particles formed . Interestingly, for all systems with 50% DNA copolymer or lessDNA content, only vesicles were observed. This morphology transitionwas observed because increasing the mole fraction of the PEG113-macroCTA reduces the electrostatic repulsion from DNA chains inthe corona. Thus, as the proportion of the PEG113-macroCTAis increased, the volume fraction of electrostatic chains in the coronallayer is gradually reduced, which means that the volume fraction ofthe hydrophobic PHPMA block required to access the lumpy rods or vesiclesis correspondingly lower. DLS was used to measure the hydrodynamicdiameter (Dh), where the 100% ssDNA14–PHPMA400 copolymer displayed a unimodalparticle size distribution with Dh approximately90 nm , the 90% DNA systemhad a multimodal particles size distribution . In the 50, 10, and 0% DNA systems, unimodal particlesize distributions were observed with the Dh increasing from 150 to 460 nm as the DNA content decreased . This increase in hydrodynamic diameter is consistent with theformation of higher order polymeric morphologies, as the bilayer structureof the vesicles increases the size of the nanomaterial.The morphology of the self-assembled nanostructureswas determinedby transmission electron microscopy (TEM) and cryogenic TEM (Cryo-TEM)images of each diblock composition was used to assign the copolymermorphology, as shown in s formed 1c. This 2+) from thesalt solution on the self-assembly process .22 As expected, when the molefraction of DNA on particles decreased, the screening effect had lessof an effect, and particles are more stable. Subsequently, the DNAfunctionality for hybridization of NPs with varying DNA content (50and 10%) was probed by DLS using a complementary DNA strand (cDNA)(5′-GCA ACA ACG CTA CA-3). For NPs with 50% DNA, the size ofparticles gradually increased until a cDNA ratio = 0.4 at which pointthe quality of the DLS data deteriorated, indicating aggregation asalso confirmed by TEM . For DNA containing NPs alone, particles had no observablefluorescence . To confirm thatcDNA was not entangled but hybridized on the surface of DNA-polymerparticles, we mixed PEG113-b-PHPMA400 with TAMRA-cDNA. PEG113-b-PHPMA400 particle were not observed in the fluorescent field; thereforeconfirming that there is no interaction between PEG and cDNA . Thus, confocal imaging confirmedthe presence of DNA at the particle surface and its ability to hybridizewith a complementary strand. This means DNA-polymer particles cansimply be functionalized after the assembly process by adding fluorophore-labeledcDNA with a maximum 0.4:1 mol ratio of cDNA to the 50% DNA coronacontaining NPs. However, if greater loadings of nucleic acid are requiredfor larger DNA constructs the system becomes destabilized and alternativestrategies are required.To confirm that hybridization of cDNA was occurringon the surfaceof the NPs, confocal fluorescence microscopy was used; this methoddirectly images the fluorescent cDNA-particles hybridized on the DNA-coronawhich means the turbidity or the effect from photobleaching of fluorophoresis negated. The 50% DNA system was selected to study hybridizationwith fluorescent TAMRA-cDNA by confocal fluorescence microscopy, asit has a high DNA mole fraction but is still stable. A mixture ofTAMRA-cDNA and DNA containing NPs were clearly observed as stabledots in the fluorescence images 2j. ConveNagg) for the 50% DNA coronaNPs. Here, for this mixed DNA and PEG based system, each particleconsists of approximately 2700 chains . Therefore, DNA constitutes about 1350 chains per particle.From the hybridization study, approximately 500 cDNA chains were ableto hybridize with DNA on the surface of particle without negativelyimpacting the colloidal stability of the system. This means that lessthan half of the DNA chains on the surface of the particle can behybridized before the system destabilizes.To quantify the amount of DNA thatcould be functionalized on eachparticle, static light scattering (SLS) was used to obtain aggregationnumbers  in step 1, followed by the addition of hairpin 1 (H1) and hairpin 2 (H2) representing 1 cycle ofHCR  can only hybridizein the presence of the initiator DNA (I) on the particlesurface  noting that only theone addition of I is necessary. To increase the numberof performed HCR cycles, H1 and H2 wereadded stepwise up to 10 cycles, forming linear duplex DNA chains onthe particle surface. Successful hybridization on the toehold of theadded hairpin triggers the unfolding or opening of the hairpin structurerevealing the next available single stranded DNA for hybridizationin a staggered pattern. HCR study was investigated by DLS, Cryo-TEM,and confocal fluorescence microscopy. DLS data show Dh increase significantly from approximately 150 to 300nm  and FAM-labeled hairpin 2 (FAM-H2) were usedand investigated by confocal fluorescent spectroscopy in merged fluorescentchannel. As expected, the starting material particles did not showany fluorescence . Particle fluorescence changed to yellow when H1 and FAM-H2 were hybridized to particle-I after 1 HCR cycle caused by a combination of the fluorescence emissionfrom TAMRA-I (λem: 580 nm) with thefluorescence emission from FAM-H2 (λem: 517 nm) at an equal ratio (H2 (Figure S10; fluorescence values were calculated for each particlefrom the CLSM images in To increase the amountof nucleic acid loading on the particlecorona, we investigated using HCR to increase DNA content while alsoproducing a fluorescence read-out signal, which is desired for theadaptation of DNA-polymers as biosensors. HCR is an interesting approachbecause of its enzyme-free nature, use of isothermal conditions, uncomplicatedprotocols, and exceptional amplification efficiency.e ofHCR 3a, wheresurface S8 notingsurface S8 notingsurface S8 notingsurface S8 notingsurface S8 noting14-macroCTA by solid support synthesis, resultingin high yields, easy upscaling, and purity without the need for furtherpurification. With the use of low-volume RAFT-mediated photo-PISAunder enzyme-assisted degassing conditions, this ssDNA14-macroCTA has been combined with trithiocarbonate-based PEG113-macroCTA to fabricate the first mixture of PEG and a DNA hybrid.Crucially, the particles were further applied with hybridization andHCR techniques with the simple addition of complementary DNA strands.This provides a convenient route for the synthesis of ssDNA-macroCTA,performing simple PISA in the construction of complex DNA–PEGpolymer architectures, and straightforward applications with hybridizationfor bioimaging. HCR was first applied with DNA–PEG polymerarchitectures as a tool obtaining a large DNA construct on the particlesurface without disturbing the particle’s morphology whileproducing a fluorescence read-out. These methods could address thechallenges of applying DNA nanotechnology to engineer on polymer surfacesfor constructing responsive nanomaterials.In conclusion, we have introduced the firstsynthesis of trithiocarbonate-basedssDNA


Negative mental well-being may hinder, and positive mental well-being facilitate physical activity (PA) when one’s daily routines are compromised. COVID-19 posed challenges to both mental well-being and PA, in Finland especially for those over 70, who faced the strictest restrictions. Therefore, the aim was to investigate the relationships of mental well-being with PA during the COVID-19 in two cohorts of different ages.Data came from two population-based studies. Participants of the longitudinal TRAILS study  were representative of their age cohort. Participants of the randomized controlled trial PASSWORD  had attended a year-long multicomponent physical training intervention during 2017-2019. Self-reported changes in PA  and PA frequency  during the COVID-19 were collected from April 2020 to June 2020 (PASSWORD) or July 2021 (TRAILS). Positive mental well-being was assessed by positive affect from the International Positive and Negative Affect Schedule Short Form . Negative mental well-being indicators were negative affect  and depressive symptoms . Relationships between mental well-being and PA were analyzed using logistic and linear regression models, adjusted by sex and, in PASSWORD, age.Positive affect was positively associated with increased PA (odds ratio (OR)=1.751-2.661, p = 0.013-0.034) and PA frequency  in both studies, and inversely with decreased PA in the PASSWORD . Higher negative affect and depressive symptoms were associated with decreased PA in the PASSWORD . Additionally, depressive symptoms were associated with lower PA frequency in both studies .Positive mental well-being was consistently and positively associated with PA during COVID-19 in older adults. Higher negative mental well-being was more clearly associated with poor PA behaviors in the older cohort facing stronger restrictions. Supporting positive mental well-being may be as important as reducing negative mental well-being to facilitate physically active lifestyle among older adults during exceptional circumstances.The Academy of Finland .


Fibrosis is a common healing process that occurs during stress and injury in cardiovascular diseases. The evolution of fibrosis is associated with cardiovascular disease states and causes adverse effects. Fibroblast activation is responsible for the formation and progression of fibrosis. The incipient detection of activated fibroblasts is important for patient management and prognosis. Fibroblast activation protein (FAP), a membrane-bound serine protease, is almost specifically expressed in activated fibroblasts. The development of targeted FAP-inhibitor (FAPI) positron emission tomography (PET) imaging enabled the visualisation of FAP, that is, incipient fibrosis. Recently, research on FAPI PET imaging in cardiovascular diseases increased and is highly sought. Hence, we comprehensively reviewed the application of FAPI PET imaging in cardiovascular diseases based on the state-of-the-art published research. These studies provided some insights into the value of FAPI PET imaging in the early detection of cardiovascular fibrosis, risk stratification, response evaluation, and prediction of the evolution of left ventricular function. Future studies should be conducted with larger populations and multicentre patterns, especially for response evaluation and outcome prediction. Fibrosis is a common response and an important mediator of various types of injuries involved in cardiovascular diseases. Although fibrosis may be sometimes beneficial and crucial for preventing dysfunction of the involved organs in some circumstances , as the disease evolves, fibrosis gradually becomes uncontrolled and excessive, contributing to adverse cardiovascular remodelling and development of adverse cardiovascular events ,2. There18F]F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT, none of which are specific to fibrosis [18F]F-FDG PET/CT focuses on viable myocardium rather than fibrosis F-FF-18F]F-68Ga]Ga-FAPI-04 imaging in oncologic cohorts found an association between FAPI uptake and cardiac risk factors [68Ga]Ga-FAPI uptake was also reported after chemoradiotherapy in oncologic patients F-FDG, FAPI presents a better TBR under many conditions and does not require fasting or blood glucose restriction. Moreover, radiolabelled FAPI enables radioligand therapy using a theranostic method. Currently, the application of FAPI tracers in cardiovascular diseases raised significant concerns. [68Ga]Ga-FAPI-04, [68Ga]Ga-FAPI-46, and [18F]AlF-NOTA-FAPI-04 are mainly used FAPI tracers in cardiovascular diseases, including myocardial infarction, coronary artery disease (CAD), heart failure (HF), secondary myocardial injury such as oncotherapy-induced myocardial injury and iatrogenic myocardial injury, cardiac amyloidosis, cardiomyopathy, and vascular diseases such as atherosclerosis, thrombosis, and arteritis. These studies provide the value of FAPI PET imaging in the preliminary evidence, early detection of cardiovascular fibrosis, risk stratification, response evaluation, and prediction of the evolution of LV function through pre-clinical studies, case reports, retrospective studies, and prospective studies, as presented in FAP targeting radiopharmaceuticals attracted considerable attention. The first clinical investigation of FAP targeting radiopharmaceuticals was a radiolabelled FAP antibody in 1994, followed by a series of studies on radiolabelled FAP targeting antibodies and peptides ,38,39. AThe main excretory system for the FAPI PET tracer is the urinary system. Hence, there is robust accumulation of the FAPI tracer in the urinary bladder and urinary tract ,80,81. I68Ga]Ga-FAPI-02, benign lesions present constant or slightly decreased uptake, whereas malignant lesions tend to present slight increase over time F-FAPI-42 was obvious in the gallbladder and common bile duct, impeding its use in detecting biliary diseases [68Ga]Ga-FAPI-46 demonstrated the most intensive accumulation in all pathologies, implying a better performance in malignant and non-malignant detection.The uptake patterns of the different FAPI analogues are similar, but with subtle discrepancies. The accumulation of [diseases . [68Ga]G68Ga]Ga-FAPI and [18F]F-FDG, FAPI uptake was lower than FDG uptake in the brain, oral mucosa, parotid gland, myocardium, blood pool, liver, pancreas, spleen, kidney, spinal cord, and gastrointestinal tract. The uptakes of the two tracers were nearly equal in terms of the fat content. FAPI-avid showed a more significant increase than FDG-avid in the lungs, thyroid, and skeletal muscles. In summary, [68Ga]Ga-FAPI showed a better detection rate owing to the high TBR in lesions, especially those with high [18F]F-FDG background activity. Notably, [68Ga]Ga-FAPI accumulation in the mediastinal region, especially in the cardiac muscle and blood pool, was lower than that of [18F]F-FDG, implying that [68Ga]Ga-FAPI PET has a better diagnostic value than [18F]F-FDG in cardiovascular diseases [In a head-to-head comparative study of the biodistributions of [diseases .68Ga]Ga-FAPI uptake correlated with cardiovascular risk factors such as certain chemotherapies , chest radiation history , and metabolic disease Ga-FAPI-46 PET in a retrospective study of 12 patients after pulmonary vein isolation [68Ga]Ga-FAPI-04 and [68Ga]Ga-FAPI PET imaging [68Ga]Ga-FAPI uptake in the right ventricle (RV) was observed in pressure overload cardiac diseases, such as idiopathic pulmonary arterial hypertension (IPAH) and hypertensive heart disease Ga-FAPI-04 PET showed that FAPI uptake in the infarct and adjacent regions increased, peaking at 6 days and gradually decreasing to the baseline level by 2 weeks, at different time points post-MI [68Ga]Ga-FAPI-04 PET/CT imaging in monitoring activated fibroblasts in the early post-MI stage and in evaluating reparative fibrosis [68Ga]Ga-FAPI-04 uptake in the corresponding myocardium after acute MI, implying that the non-invasive assessment tool, FAPI PET imaging, may provide a great opportunity for the early identification of fibrosis Ga-FAPI-04 in the heart was higher in the AAC group than in the control group after the second week, whereas an echocardiographic abnormality appeared in the fourth week. In this comparative study, [68Ga]Ga-FAPI-04 PET has advantages in identifying the early stages of fibroblast activation and assessing the extent of cardiac fibrosis compared with echocardiography, which only reflects established cardiac fibrosis.The latest pre-clinical studies further explored the capacity of FAPI PET for the early detection of fibrosis in HF. Song et al. established an HF model using isoproterenol (ISO)-induced HF rats to collect sequential [T images . The phoy stages b. This p surgery . The acc68Ga]Ga-FAPI-04 PET/CT, echocardiography, and right heart catheterisation within 7 days to assess cardiac function and pulmonary haemodynamics, respectively Ga-FAPI-04 PET/CT in the early detection of RV remodelling in patients with PAH, achieving better management of HF.Pulmonary arterial hypertension (PAH) is a preliminary etiology of HF, which is characterised by overload pressure in the RV. Early detection with a novel strategy for optimal assessment of RV is warranted. A single-centre cohort enrolled 16 patients with PAH who underwent Ga-FAPI tracer was observed in the LV myocardium of a patient with pancreatic ductal cancer, with no clinical signs of cardiac discomfort, indicating secondary myocardial injury during chemotherapy [18F]AlF-NOTA-FAPI-04 PET imaging study based on a retrospective analysis of patients with oesophageal squamous cell carcinoma and animal models confirmed that FAPI uptake in the damaged myocardium began to increase from the second week after radiation exposure, reaching a maximum on the fifth week [Totzeck et al. reported a case in which the accumulation of [otherapy . This cafth week . Additio18F]F-FDG PET/CT showed diffuse FDG accumulation in the mediastinum and could not distinguish the lesion from the adjacent background. [68Ga]Ga-FAPI PET/CT indicated the lesion with intense FAPI uptake located in the main and bilateral proximal pulmonary arteries. Vascular involvement of Rosai–Dorfman disease was identified by histopathology of the resected lesion. Hence, [68Ga]Ga-FAPI PET/CT may outperform [18F]F-FDG PET/CT in the early detection of vascular lesions with a high TBR ratio through visualising fibroblast activation and fibrosis. FAPI PET could achieve early detection of vascular diseases by visualising fibroblast activation and fibrosis . A 28-ye68Ga]Ga-FAPI-04 PET/CT to detect arterial fibroblast activation [68Ga]Ga-FAPI-04 accumulation in 800 sites. Among these sites, 377 (47.1%) had vessel wall calcification that matched. Both the number of FAPI accumulation sites and the corresponding TBR had a significant correlation with the number, the thickness, and the circumference of calcified plaques. Therefore, [68Ga]Ga-FAPI-04 may be a potential tracer for early detection of arterial wall lesions and is correlated to marked calcification and overall calcified plaque burden.A retrospective study with 69 oncologic patients was conducted to assess the feasibility of Ga-FAPI-04 PET imaging in risk stratification in CAD AlF-NOTA-FAPI uptake was analysed according to intensity, extent, and amount. All HCM patients presented with increased and heterogeneous FAPI uptake in the LV myocardium compared with normal controls . FAPI uptake regions were more than hypertrophic regions, as detected by CMR in HCM ; 84%  of non-hypertrophic segments presented with FAPI uptake. Log-transformed FAPI amount had a negative correlation with LVEF z-score . FAPI uptake was positively correlated with the 5-year sudden cardiac death (SCD) risk score . Therefore, FAPI PET may be a promising modality for risk stratification in HCM because it reflects myocardial fibrosis.Only one study investigated FAPI PET in patients with HCM . In thisLight-chain cardiac amyloidosis (AL-CA) is a life-threatening disease involving multiple organs and is characterised by plasma cell dyscrasia with insoluble immunoglobulin AL fibril deposition in multiple organs and systems, leading to organ dysfunction . The hea68Ga]Ga-FAPI-04 LV uptake (patchy [n = 4] vs. extensive [n = 20]). SUV ratio and LV molecular volume were significantly higher in the PET-extensive group than in the PET-patchy group  Ga-FAPI-04 imaging in 1177 arterial segments from 41 patients without cardiovascular disease symptoms, correlating it with the degree of calcification and cardiovascular risk factors Ga-FAPI PET/CT outperforming [18F]F-FDG PET/CT in evaluating disease activity and extent in a patient with TA Ga-FAPI-04 images of 13 patients with CTEPH and 13 controls analysed the correlation between FAPI uptake of the PA and remodelling parameters derived from right heart catheterisation (RHC) F-FAPI PET scan was performed to evaluate fibrosis before discharge, showing heterogeneous FAPI uptake in the myocardium and indicating underlying fibrosis with cardiac involvement. After 2 months of treatment, follow-up FAPI PET revealed that tracer uptake in the corresponding area was significantly reduced, implying mitigated and ameliorated fibrosis and favourable therapy response (Eosinophilic leukaemia (EL) is a haematological disease characterised by persistent eosinophilia with variable manifestations . Eosinopresponse . Based o18F]AlF-NOTA-FAPI PET imaging study based on 14 first-time ST-segment elevation myocardial infarction (STEMI) patients after primary percutaneous coronary intervention (PPCI) identified the prognostic evaluation of FAPI PET imaging, post-MI, for the first time Ga-FAPI-46 PET/CT in thirty-five patients with acute MI (AMI) [68Ga]Ga-FAPI-46 signal did not match the manifestation of other imaging findings, but could predict the progression of cardiac dysfunction, complementary to current imaging techniques. Interestingly, the FAPI uptake volume was not correlated with LVEF measured simultaneously at baseline , whereas a significant inverse correlation between FAPI uptake volume and follow-up LVEF was observed . A recent prospective study quantitatively evaluated changes in the intensity and extent of myocardial fibroblast activation after AMI, using [68Ga]Ga-DOTA-FAPI-04 PET/MRI AlF-NOTA-FAPI uptake was associated with myocardial injury biomarkers’ T2-weighted imaging (T2WI), ECV, and LGE derived from CMR at both patient and segmental levels  [68Ga]Ga-FAPI-46 uptake markedly exceeded the infarcted area that was detected on CMR, suggesting that the cell-based FAPI uptake of fibroblast activation differed from CMR-derived interstitial features [18F]AlF-NOTA-FAPI accumulation regions were more than the hypertrophic regions detected by CMR ; and 84%  in non-hypertrophic segments that presented with FAPI uptake AlF-NOTA-FAPI-04 uptake in the damaged myocardium began to increase from the second week after radiation exposure and reached a maximum at the fifth week, whereas LVEF measured by CMR decreased from week eight onwards Ga-DOTATATE recently attracted significant interest for the visualisation of inflammation in cardiovascular diseases Ga-DOTATATE and [68Ga]Ga-FAPI-04 were obtained on two separate days. [68Ga]Ga-DOTATATE PET manifested scarce, diffused, and homogenous tracer accumulation at 87% of the LV . On the contrary, FAPI PET showed intense, focal, and heterogeneous accumulation in the septal, anterior, and lateral wall rather than in the inferior wall at 52% of the LV . The mismatched images between [68Ga]Ga-DOTATATE and [68Ga]Ga-FAPI-04 indicate the potential of FAPI PET to distinguish certain pathophysiological processes other than inflammation in ICI-associated myocarditis.A patient with advanced upper tract urothelial carcinoma treated with ICIs was enrolled in a dual-tracer imaging modality study because of suspected cardiac dysfunction . The ima13N-ammonia ([13N]NH3) is a classic tracer used in cardiac PET imaging to evaluate blood flow in the myocardium and coronary arteries [13N]NH3 PET and [68Ga]Ga-FAPI PET [68Ga]Ga-FAPI was observed in the anterior and inferior walls (arteries ,117,118.FAPI PET . The foror walls b. The miFibrosis is a common response to stress and injury in cardiovascular diseases. The evolution of fibrosis contributes to adverse cardiovascular remodelling and adverse cardiovascular events. Fibroblast activation is the key point for the formation and progression of fibrosis. The incipient detection and evaluation of activated fibroblasts is essential for patient management and prognosis. FAP, as a biomarker of activated fibroblasts, was targeted and visualised by FAPI PET to reflect and evaluate fibrosis in cardiovascular diseases. The state-of-the-art applications and the performance of FAPI PET in various cardiovascular diseases were summarised and discussed. Initially, FAPI PET in cancer therapy-associated myocardial injury, iatrogenic myocardial injury, myocardial injury involved in systemic diseases such as cardiac amyloidosis and systemic sclerosis, and vascular diseases such as thrombosis and atherosclerosis were incidentally discovered and preliminary investigated. Significantly, the potential of FAPI PET in tracing fibroblast activation and accomplishing early detection of cardiovascular fibrosis was evaluated and identified in MI, myocardial remodelling in HF, secondary myocardial injury, and vascular diseases. Furthermore, FAPI PET could assist in risk stratification on CAD, cardiomyopathy, atherosclerosis, arteritis, and CTEPH through appropriate evaluation of the activity and severity of cardiovascular disease. Furthermore, the role of FAPI PET in response evaluation of cardiac disease was also demonstrated in a patient with EL and EM. In addition, the prognostic value of FAPI PET was also investigated in MI, and supposed as a recommendable modality for prediction of the evolution of LV function.Comparative and complementary studies on multimodal imaging were conducted. The mismatch between FAPI uptake and CMR imaging demonstrates the potential of FAPI imaging to complement CMR imaging in predicting remodelling in MI, depicting the extent of fibrosis apart from hypertrophy in HCM, and earlier detection of radiation-induced myocardial injury. Moreover, FAPI PET combined with other imaging modalities is beneficial for distinguishing certain pathophysiological processes and differential diagnosis.Although FAPI PET is considered as a promising tool for evaluating cardiovascular fibrosis, there are still gaps in knowledge and studies needing filling. Firstly, the potential of FAPI PET for early detection of vascular disease is little tapped. Secondly, given the limited number and volume of studies on FAPI PET imaging in early detection and risk stratification for cardiovascular diseases, large-population and multicentre studies are warranted to maximise the optimal capacity of FAPI PET imaging in these fields. Thirdly, future research perspectives on FAPI PET imaging in cardiovascular diseases could lay particular emphasis on response evaluation and prediction of the evolution of LV function, as there are merely a few related studies with small-volume and single-sort disease to date. Thirdly, the comparison and relationship between FAPI PET imaging and other imaging modalities should be further investigated to better understand the advantages and optimised application scenarios of FAPI PET imaging. Lastly, due to the current application of FAP inhibitors in cancer treatments , FAPI-PEFibrosis is a response to various cardiovascular pathologies that manifests as ECM remodelling and superfluous collagen deposition mediated by activated fibroblasts. FAP is specific for identifying activated fibroblasts, which are biomarkers of the early stages of cardiovascular fibrosis. The FAP-targeting radiotracer FAPI PET imaging may be used for specific imaging of incipient fibrosis in cardiovascular diseases. This approach may facilitate the identification of early fibrosis, distinguish early-stage vs. end-stage diseases, evaluate therapeutic responses, and predict cardiac function in cardiovascular diseases.


Previous researches have not distinguished between-person effects from within-person effects when exploring the relationship between self-esteem, coping styles, and anxiety symptoms among adolescents.To address this gap, this study investigated reciprocal associations among self-esteem, coping styles, and anxiety symptoms in a three-wave longitudinal panel survey, using an analytical strategy that disaggregates within-person and between-person variance.The data was drawn from the Longitudinal Study of Adolescents’ Mental and Behavioral Well-being Research study conducted in 10 public schools in the Guangdong province of China. All participants had a baseline visit  and follow-up interviews at one-year intervals for 3 years. A random intercept cross-lagged panel model combined with mediation analysis was performed.At the within-person level, following results were observed. (1) Low self-esteem and anxiety symptoms bidirectionally predicted each other. (2) Low self-esteem and negative coping style bidirectionally predicted each other. (3) Anxiety symptoms predicted subsequent negative coping style but not vice versa. At the between-person level, we obtained the following main results. (1) Significant predictive effects on the random intercept were found among all three study constructs. (2) There were sex differences regarding the association between self-esteem and anxiety symptoms and the correlation strength of females was greater than that of males. (3) Self-esteem mediated the reciprocal relations between coping styles and anxiety symptoms.These results could be an important advance by elucidating the reciprocal relationships among self-esteem, coping styles, and anxiety symptoms at the within-person level, suggesting that interventions targeted at promoting self-esteem and cultivating positive coping style may help reduce adolescent anxiety.None Declared


Moreover, mixing small quantities of enantiomers ofglutamic acid-BTA and glutamate methyl ester-BTA in the presence ofthe achiral alkyl-BTAs leads to deracemization and inversion of thehelical structures in solution via the insitu reaction toward a thermodynamic equilibrium. Theoreticalmodeling suggests that the observed effects are caused by enhancedcomonomer interactions after the chemical modification. Our presentedmethodology enables on-demand control over asymmetry in ordered functionalsupramolecular materials.Amplification of asymmetry in complex molecular systemsresultsfrom a delicate interplay of chiral supramolecular structures andtheir chemical reactivity. In this work, we show how the helicityof supramolecular assemblies can be controlled by performing a non-stereoselectivemethylation reaction on comonomers. By methylating chiral glutamicacid side chains in benzene-1,3,5-tricarboxamide (BTA) derivativesto form methyl esters, the assembly properties are modulated. As reactedcomonomers, the methyl ester-BTAs induce a stronger bias in the screw-senseof helical fibers predominantly composed of stacked achiral alkyl-BTAmonomers. Hence, applying the  Mechanisms that could cause symmetry breaking andasymmetry amplification are widely discussed, yet experimental substantiationis lacking.32 One supramolecular polymer platform consists of benzene-1,3,5-tricarboxamide(BTA), which is widely studied due to its modularity.35 These discotic molecules form helical supramolecular polymers inapolar solvents through π–π stacking and intermoleculartriple hydrogen bonding. A helical bias, i.e., thepreference to adopt P or M helicity, is often introduced via the intrinsic molecular chirality of the monomers. Thus, supramolecularpolymer systems demonstrate how molecular chirality is translatedinto macroscopic asymmetry. Consequently, the inherent asymmetry ofsupramolecular polymer systems can be transferred into reaction products via asymmetric metal and organocatalysis.44Supramolecular polymers are of interest in amplificationof asymmetrystudies due to the ability to form dynamic chiral M or P helices thatchange handedness in response to external factors.51 The first method is the Sergeant-and-Soldiers experiment, in whichthe majority of the system consists of achiral monomers that can adopta preferred M or P helicity by doping the system with chiral monomers.The second method is the Majority-Rules experiment, in which the nethelicity is governed by mixtures of the two enantiomers. The principlerelies on the mismatch penalty, an intrinsic value that describesthe penalty for a monomer to enter a stack of its unfavored helicity.The combination of the two methods is a Diluted Majority-Rules experiment,in which an enantiomeric excess (ee) is introducedin the sergeant fraction of a Sergeant-and-Soldiers experiment.54 The abovementioned experiments result in nonlinear translation ofmolecular chirality into macroscopic asymmetry and fully rely on thermodynamicequilibria that result from mixing of (a)chiral comonomers. So far,two reports have demonstrated the combination of supramolecular polymerswith covalent reactions to amplify the supramolecular asymmetry.23 Herein, in situ reactivity is combined with theSergeant-and-Soldiers principle to trap the supramolecular chiralityinto molecular chirality, enhancing the ee of supramolecularmonomers. As a consequence, the asymmetry of the aggregated speciesis amplified. We propose that performing the reaction directly onthe sergeant instead of the soldier would lead to a more effectiveand robust amplification of asymmetry on the supramolecular structuresin thermodynamic equilibrium.Methods to study the amplification of asymmetry in macromoleculeswere originally developed by Green and co-workers and were later translatedto supramolecular polymer systems.in situ covalentmethylation . We chose the asymmetric design to promote the formationof elongated one-dimensional (1D) structures and decrease the morphologicaltransition from fibers to dimers, which has been demonstrated forsymmetric ester-BTAs.55 The carboxylicacid moiety in the glutamic acid side chain was expected to interferewith the intermolecular hydrogen bonding and retard supramolecularpolymerization, as shown by kinetic evolution experiments .57 Moreover,this carboxylic acid was identified as a target for chemical modification,of which the reaction product was expected not to interfere with theintermolecular hydrogen bonding. Thus, we aimed to in situ methylate the carboxylic acid to promote supramolecular polymerizationand found that (trimethylsilyl)diazomethane ((TMS)CHN2)was reactive and selective in the desired assembly conditions . Methylation with (TMS)CHN2 yielded the stable product Glu(OMe)-BTA -BTA 1a,58 whiGlu-BTA and Glu(OMe)-BTA in methylcyclohexane (MCH) were characterizedat room temperature by Fourier transform infrared (FTIR) and CD spectroscopy.The FTIR spectra show absorption bands at 3236 cm–1 (bonded N–H stretch), 1643 cm–1 , and 1562 cm–1  that are typical forthreefold hydrogen bonding between amides of the BTA core in supramolecularpolymers . Furthermore, the band at 1747 cm–1 can be ascribedto an unbound ester C=O, indicating no formation of dimericaggregates.55 The shoulder at 1716 cm–1 in the spectrum of Glu-BTA could beascribed to bonded carboxylic acid moieties, indicating a bifurcatedhydrogen bond of the amide C=O in the aggregated state.60Homopolymerizations of polymers 1a.59 TheGlu-BTA and Glu(OMe)-BTA exhibit Cotton effectswith positivemaxima at 222 nm (g-factor) at room temperature is larger forhomopolymers of Glu(OMe)-BTA compared to Glu-BTA . We observed elongation temperatures(Te) of 42 and 57 °C for Glu-BTA and Glu(OMe)-BTA, respectively, by conducting variable-temperatureCD measurements .63 The extracted thermodynamic parameters and the resulting bindingconstants at 20 °C in the elongation phase of homopolymerization(Ke) from these two methods were comparable.The calculated Ke values of Glu-BTA and Glu(OMe)-BTA were 2 × 105 and 5× 105, respectively, confirming that polymers of Glu(OMe)-BTA are more stable.The CD spectra of 50 μM samples of both t 222 nm 1b. These Glu-BTA S5. We obGlu-BTA and Glu(OMe)-BTA inSergeant-and-Soldiers experiments to explore the potential for asymmetryamplification by in situ chemical modification ofthe sergeant. In separate experiments, the sergeants Glu-BTA and Glu(OMe)-BTA were mixed with a-BTA soldiers in ratios between 1 and 20 mol % (Glu-BTA and Glu(OMe)-BTA -BTA into polymers of a-BTA was further corroborated by FTIR spectroscopy, as bothcopolymer mixtures exhibited identical spectra of polymeric aggregates. These spectra did not demonstratea significant proportion of chain ends, indicating that neither Glu-BTA or Glu(OMe)-BTA is effective as a chaincapper. The CD signal at 222 nm is plotted over the sergeant rangefor both experiments to demonstrate the net helicity of the thermodynamicequilibrium state at each composition (Glu(OMe)-BTA compared to Glu-BTA. Temperature-dependentCD and ultraviolet–visible (UV–vis) measurements providemechanistic details on the intercalation of sergeants into stacksof soldiers. In the presence of 5 mol % Glu(OMe)-BTA,the helical bias is introduced at a temperature of 73 °C, correspondingto the Te of a-BTA supramolecularpolymerization. Hence, the intercalation of this sergeant occurs simultaneouslywith supramolecular polymerization of the soldiers -BTA. This was further corroborated by conductinga Sergeant-and-Soldiers experiment at variable temperature with the tert-butyl protected precursor BTA as the sergeant . The temperature-dependent data perfectly overlaps withthat of Glu(OMe)-BTA as the sergeant , despite the sterically demanding tert-butyl moiety.We studiedthe role of 20 mol % 1a. The COMe)-BTA 2a.50 ThegregatesS8a. ThesgregatesS8a. ThesGlu-BTA and Glu(OMe)-BTA as sergeants prompted us to investigate the effectof sergeant ee on the asymmetry of copolymers. Forthis purpose, weconducted Diluted Majority-Rules experiments, where we vary the enantiomericcomposition of the sergeant while keeping the sergeant fraction constantat 5 mol % -BTA sergeants indicates a Majority-Rules behavior, i.e., the enantiomers in minority can intercalate into stacks of theunfavored helicity.The differences between  5 mol % 3a. The C63 These simulationsallowed us to examine the effect of the binding constant (Ke) and mismatch penalty (MMP) of both chiralsergeants Glu-BTA and Glu(OMe)-BTA on theasymmetry of copolymers. The simulated Sergeant-and-Soldiers  and Diluted Majority-Rules experiments-BTA . These experimental results showeda stronger asymmetry amplification for Glu(OMe)-BTA comparedto Glu-BTA, indicating either a higher Ke or a lower MMP for homopolymerization of Glu(OMe)-BTA with respect to Glu-BTA. Comparing these findings withthe Diluted Majority-Rules experiments revealed that the experimentalresults are in line with simulations, in which we vary Ke, but in contradiction with simulations, in which wevary MMP. Therefore, the combination of experiments and simulationselucidates that methylation of Glu-BTA into Glu(OMe)-BTA results in an increase in Ke, i.e., stronger binding between sergeant and soldiers. Thisstronger interaction leads to an enhanced translation of molecularchirality into supramolecular asymmetry in thermodynamically stablecopolymers.We used mathematical mass-balance models of two- and three-componentcopolymerization to simulate the Sergeant-and-Soldiers and DilutedMajority-Rules experiments.Soldiers S10 and DSoldiers S10 and DKe for Glu-BTA and Glu(OMe)-BTA to bind tosoldier stacks was exploited for the purpose of on-demand amplificationof asymmetry. The in situ reaction procedure wasoptimized as described in the SI . The expected amplification can be expressed as the ΔCD,which is calculated as the difference in CD value at 222 nm betweenthe copolymer systems with Glu-BTA versus Glu(OMe)-BTA as the sergeant . First, weoptimized the in situ reaction conditions to be compatiblewith the conditions for supramolecular polymerization as describedin the Supporting Information. Next, applyingour optimized reaction procedure to Sergeant-and-Soldiers mixturesof Glu-BTA and a-BTA copolymers resultedin amplification of asymmetry -BTAvia their distinct effect on the asymmetry of primarily soldier-containingcopolymers.The difference in sergeant S9. Firstsergeant S9. Firstsergeant S9. Firstin situ reaction on the macroscopic asymmetry were realizedby mixing enantiopure(R)-Glu-BTA with (S)-Glu(OMe)-BTA in a Diluted Majority-Rules system with95 mol % a-BTA (R)-Glu-BTA / (S)-Glu(OMe)-BTA from1/1 to 1/0 and observed drastically shifted CD values compared tothe Diluted Majority-Rules experiments that included only enantiomersof either Glu-BTA or Glu(OMe)-BTA (S)-Glu(OMe)-BTA sergeant even up to 80% excess of (R)-Glu-BTA sergeant, where a racemic state of the helices in solution is obtained.In these mixtures, the addition of the reagents for in situ methylation will selectively convert enantiomers of Glu-BTA into Glu(OMe)-BTA, as illustrated in Glu(OMe)-BTA. Accordingly, the arrows in in situ chemical modification: inverting the asymmetry of the major screw-sense(orange arrow) and deracemization of helices in solution (black arrow).Addition of the reagents in a mixture containing 70 mol % (R)-Glu-BTA in the sergeant fraction (an excessof 40%) indeed resulted in an inversion of the CD signal. Here, anabsolute CD change of 92 mdeg within 8 h was obtained . Similarly, the introduction of the reagents in the CD-silent mixturecontaining 90 mol % (R)-Glu-BTA in thesergeant fraction (an excess of 80%) resulted in deracemization. Here,an absolute CD change of 62 mdeg was observed within 8 h -BTA 6b. Now, obtained S9b. Simii.e., deracemization), amplification,and inversion of asymmetry by the addition of chemical reagents.We have demonstrated that covalent andnoncovalent chemistry canbe combined to precisely control the asymmetry in supramolecular polymersunder thermodynamic equilibrium. When mixed with achiral comonomers,the covalent methylation of the carboxylic acid side chain of chiralmonomers enhanced the intermolecular interactions and thus participationin supramolecular polymerization. The observed macroscopic effectis a stronger helical bias upon chemical conversion of the chiralmonomers. Variations in the enantiomeric composition of the chiralcomonomer fraction enabled controlled effects on the asymmetry, whichare initiation (in situ can be employed as a mean to control asymmetric features in molecularsystems. We propose that such simple systems can greatly contributeto our understanding of mechanisms toward homochirality in complexsystems.The presented work is an illustration of how a multistep approachof covalent and noncovalent reaction steps


SARS-CoV-2 breakthrough infections, associated with waning immunity, increase systemic antibody levels. In this study, we analyzed the impact of the infection timing on the magnitude of the systemic humoral response and whether breakthrough infections also boost antibody levels in the salivary compartment. We observed that the combination of infection plus vaccination, regardless of infection timing, produced a sharp increase in systemic antibodies, which were higher in subjects infected after third doses. Moreover, despite high systemic antibody levels, breakthrough infections after dose three occurred and boosted antibody levels in the salivary compartment. These results suggest that current vaccination strategies against COVID-19 should be improved. Results also showed that determination of salivary antibodies against SARS-CoV-2 could be a valuable tool in disease prevalence studies, for the follow-up of vaccinated individuals, and to assist vaccination strategies against COVID-19, especially in settings where blood sampling cannot be fulfilled. SARS-CoV-2 breakthrough infections in fully vaccinated individuals were associated with low systemic antibody levels, suggesting that third dose administration might reduce the risk of infection . It was The systemic humoral response against SARS-CoV-2 has been extensively studied, while less attention has been paid to the specific salivary antibodies. Some studies in SARS-CoV-2-infected subjects have shown a correlation between the systemic and salivary anti-SARS-CoV-2 IgG antibody levels ,7, suggeIn this work, we analyzed the impact of SARS-CoV-2 infection on the magnitude of the systemic humoral response in subjects infected before vaccination or after the first, second, or third vaccine doses. Salivary antibody levels before and following SARS-CoV-2 breakthrough infections were also investigated.Results presented in this report are part of an ongoing observational prospective cohort study started at the beginning of the pandemic among healthcare workers from the Academia Nacional de Medicina, in order to study the humoral immune response against SARS-CoV-2 after vaccination and/or infection.n = 18), and after the first , second , and third vaccine dose . They all had mild COVID-19 based on the World Health Organization classification    and in uninfected control subjects vaccinated with three doses (CG), before (T3) and following breakthrough infections after third doses (T30.0001)] B, remain0.0001)] B. A posi 0.0001) C.The systemic antibody concentrations following vaccination in subjects with prior SARS-CoV-2 infection have been widely studied . A stronIn this study, we analyzed the impact of infection timing on the magnitude of the systemic humoral response. We observed a sharp increase in systemic antibodies following infections after the first, second, or third vaccine doses, reaching GMCs 64-, 20-, and 6.8-fold higher than before infection. These results showed that SARS-CoV-2 infection at any timepoint of vaccination boosts systemic antibody levels. Moreover, antibody levels following breakthrough infections after dose three were higher than in subjects vaccinated and infected at any previous time point. These results agree with a study showing that increased number of exposures to SARS-CoV-2 antigens, through vaccination or infection, enhance serum-specific, antibody-binding titers [Antibody detection in saliva has been used to monitor exposure to pathogens, since the antibody profiles in saliva match well with those found in blood . AlthougIn this report, we analyzed salivary antibody levels before and after SARS-CoV-2 breakthrough infections in cases and in uninfected vaccinated control subjects. We observed that most subjects showed IgG-specific salivary antibodies at the time of infection, albeit at much lower levels than those in blood. Following infection, salivary antibody levels increased in cases, being significantly higher than before infection or in uninfected controls. These results show that SARS-CoV-2 breakthrough infections boost IgG antibody levels both in blood and saliva. Whether this boost of specific salivary antibodies could confer immune protection for future exposures remains to be determined. As previously reported ,9, we obLimitations of this study include the small number of subjects within groups, lack of information on neutralization titers, cellular immune responses, viral variants involved in the infections, and their associations with the antibody levels reached. Moreover, although salivary antibodies were not measured from the beginning of the study, our preliminary results show that four months after the third dose, salivary antibody concentrations in subjects infected at different time points of vaccination are higher than in uninfected vaccinated subjects (unpublished data).While an enhanced neutralizing activity against the omicron variant was demonstrated after the administration of third doses , infectiSaliva collection is an attractive alternative to blood testing as it is non-invasive and allows home-based self-collection. This is important for studies in pediatric populations or where blood sampling is not possible. However, one limitation for the detection of specific salivary antibodies is that their levels are lower than in serum. In this report, we showed that salivary antibody titers increased after SARS-CoV-2 breakthrough infections. Therefore, determination of salivary antibodies against SARS-CoV-2 could be a valuable tool in disease prevalence studies, for the follow-up of vaccinated individuals, and to assist vaccination strategies against COVID-19, both in adult and pediatric populations, especially in settings where blood sampling cannot be fulfilled.In summary, our results showed that the combination of SARS-CoV-2 infection plus vaccination, regardless of infection timing, produced a sharp increase in antibody levels, which was reflected both in the blood and the salivary compartment.


Clinical neurosciences were heavily influenced by the Spanish neuropathologist Santiago Ramon y Cajal (1852–1934) for ~80 years. Cajal, credited with discovering the axonal growth cone, conducted meticulous silver nitrate stainings, which revealed the ability of adult central nervous system (CNS) neurons to sprout and form new axonal connections during learning and memory processes . Similar to a previous Research Topic that explored the significance of persistent neuroinflammation in chronic neurodegeneration  in drinking water for 5 days, on formalin-induced inflammatory pain, pain hypersensitivity, and locomotor dysfunction after thoracic spinal contusion. The authors also studied TrkB, ERK1/2, and phosphorylated ERK1/2 expression in the lumbar spinal cord and trunk skin. Their study showed that formalin-induced pain responses were attenuated in 1NMP-treated mice. Weekly assessment of tactile sensitivity with the von Frey test showed that treatment with 1NMP immediately after spinal cord injury (SCI) blocked the development of mechanical hypersensitivity up to 4 weeks post-SCI. Contrastingly, when treatment started 2 weeks after SCI, 1NMP reversibly and partially attenuated hind-paw hypersensitivity. Locomotor scores were improved in the early-treated 1NMP mice compared to late-treated or vehicle-treated SCI mice. 1NMP treatment attenuated SCI-induced increases in TrkB and phosphorylated ERK1/2 levels in the lumbar cord but failed to exert similar effects in the trunk skin. These results highlight the significance of appropriate timing of plasticity-promoting therapies. They also suggest that plasticity promotion in some cases may also have detrimental maladaptive effects, as shown for TrkB activation which leads to spinal pain hypersensitivity and impaired locomotor function when initiated early after SCI.Synaptic plasticity is promoted by neurotrophic factors, among which brain-derived neurotrophic factor (BDNF), which signals through high affinity tropomyosin receptor kinase B (TrkB), has a particularly long history of studies in models of brain injury in view of its ability to promote functional neurological recovery  the need of thorough mechanistic studies, which besides offering the mode-of-action framework for plasticity-promoting treatments may disclose surrogate markers for subsequent proof-of-concept trials in human brain-injured patients, and (ii) the importance of the appropriate timing of treatments, which circumvents the risk of maladaptive plasticity resulting in unfavorable outcomes or treatment complications. There are still challenges ahead in the clinical implementation of plasticity-promoting treatments. This Research Topic helped define valuable research avenues to follow.DH: Writing—original draft, Writing—review and editing. MB: Writing—review and editing. LP-J: Writing—review and editing.


Coronary microvascular dysfunction (CMD) is common in patients hospitalized with SARS-CoV-2 infectionThree nonrandomized studies in non-infected patients are showing the beneficial effects of statin treatment on CMDEffective cholesterol-lowering medication during and after SARS-CoV-2 infection, especially in hypercholesterolemic COVID-19 patients, is of great significancePatients with hypercholesterolemia often have coronary microvascular dysfunction (CMD). Viral infections, such as the SARS-CoV-2 infection, may also result in CMD. Three non-randomized studies have shown significant beneficial effects of statins on CMD in non-infected patients. Similarly, in SARS-CoV-2 - infected patients one beneficial mechanism of action of statins may be the amelioration of endothelial dysfunction, which is a major driver of CMD. Apart from statins, lipoprotein apheresis and PCSK9 inhibitors can also improve or even reverse CMD. The potential reversal of CMD by using effective cholesterol-lowering medications during and after COVID-19 infection, especially in hypercholesterolemic COVID-19 patients, is important. Coronary microcirculation refers to the coronary vessels with a diameter of less than 500 µm which, in individuals without epicardial atherosclerosis, vasodilate during exercise and thereby augment myocardial perfusion . CoronarIn addition to hypercholesterolemia, viral infections may also result in CMD. More than a decade ago CMD was reported in patients with acute respiratory distress syndrome and influenza A (H1N1) . This yeAs statins improve endothelial dysfunction and can reduce vascular inflammation, they were included in the list of secondary prevention strategies depicted in the recent European Society of Cardiology Working Group on Coronary Pathophysiology and Microcirculation position paper on "coronary microvascular dysfunction in cardiovascular disease" . It is nApart from statins, lipoprotein apheresis and PCSK9 inhibitors as cholesterol-lowering strategies can also reverse CMD ,28. The Regarding SARS-CoV-2–infected patients, the microvascular angiopathic consequences on the myocardium may lead to myocardial ischemia and dysfunction with grave consequences .Therefore, it is of paramount importance to aim at preventing the development of CMD in COVID-19 patients.Both severe hypercholesterolemia and SARS-CoV-2 infection cause endothelial dysfunction and the associated CMD. Therefore, it is appropriate to attempt to mitigate or reverse CMD in hypercholesterolemic COVID-19 patients by using effective cholesterol-lowering medications, particularly statins. This reminder is particularly important since many hypercholesterolemic patients, including those with familial hypercholesterolaemia in whom the hypercholesterolemia can be severe, remain undertreated or even untreated before, during, and after the infection.