Vector-borne diseases (VBDs), such as dengue, Zika, West Nile virus (WNV) and tick-borne encephalitis, account for substantial human morbidity worldwide and have expanded their range into temperate regions in recent decades. Climate change has been proposed as a likely driver of past and future expansion, however, the complex ecology of host and vector populations and their interactions with each other, environmental variables and land-use changes makes understanding the likely impacts of climate change on VBDs challenging. We present an environmentally driven, stage-structured, host–vector mathematical modelling framework to address this challenge. We apply our framework to predict the risk of WNV outbreaks in current and future UK climates. WNV is a mosquito-borne arbovirus which has expanded its range in mainland Europe in recent years. We predict that, while risks will remain low in the coming two to three decades, the risk of WNV outbreaks in the UK will increase with projected temperature rises and outbreaks appear plausible in the latter half of this century. This risk will increase substantially if increased temperatures lead to increases in the length of the mosquito biting season or if European strains show higher replication at lower temperatures than North American strains. In 2017nce 1990 , though nce 1990 –7. Numernce 1990 . For exance 1990 ,9 to shaAedes albopictus, a vector of dengue, chikungunya and Zika, established in Italy in 1990 and has since spread across much of the Mediterranean basin, with rising temperatures, trade and travel implicated in its introduction and subsequent dispersal [Disentangling the impacts of climatic and non-climatic drivers on expansion of VBDs is challenging, creating difficulties in forecasting potential effects of climate change ,10. For ispersal . Historiispersal . Howeverispersal  and estaispersal .et al. [A recent review by Sadeghieh et al.  found thtability . Novel atability ,19. In dtability .et al. [et al. [R0 values for WNV in Europe could change by a factor of approximately 6 across the range of predicted vector–host ratios. Similarly, temperature-dependence of vector biting and mortality, and pathogen latency, resulted in an approximate threefold increase in R0 values of WNV across a temperature gradient of 18–28°C. Given this, there is a clear need for modelling approaches which capture the seasonal nature of drivers of VBDs.Despite their wide applicability, existing mathematical models of VBDs generally make simplifying assumptions regarding vector or pathogen dynamics, as summarized by Reiner et al.  in a revf models ). This if models . Likewiss et al.  estimateflavivirus primarily transmitted in a cycle between Culex pipiens mosquitoes and birds [We propose a novel approach that explicitly models temperature effects on the timing and seasonal coincidence of events in the pathogen, host and vector life cycle. In doing so, we aim to improve prediction of the risk of VBD establishment in marginal temperate environments under climate change. Using delay-differential equations (DDEs) with environmentally driven delays we incorporate realistic, climate-dependent representations of vector vital rates and pathogen latency, thus capturing the impacts of climate on seasonal variations in vector–host ratios on transmission risk. We apply our modelling approach to the prediction of establishment risk of WNV in the UK, as WNV is a VBD that exhibits high seasonality and which is currently expanding its distribution into Northern Europe, having recently been reported in both Germany and the Netherlands ,23,24. Wnd birds  that cannd birds . As WNV nd birds ,28. Howe. 2Cx. pipiens seasonal dynamics [We developed a susceptible–exposed–infectious–recovered (SEIR) vector–host mathematical model with compartments for each life stage of the vector population and for each possible infection status of hosts and adult vectors . The moddynamics , which hdynamics . It is bdynamics ,30). Adudynamics . Diseasedynamics . For simdynamics . Insteaddynamics .Figure The explicit temperature-dependence in vector development and survival rates and pathogen replication rates enables predictions of whether projected temperatures are sufficiently high to sustain transmission cycles. By coupling the seasonal vector model with a seasonal host model, we can control the timing of pathogen introduction and make predictions about how timing of the pathogen introduction will affect outbreak risk. The use of temperature-dependent, stage-structured DDEs allows us to capture developmental lags in both the vector population dynamics and the transition of exposed to infectious mosquitoes, facilitating understanding of whether the time between pathogen introduction and the cessation of the active vector season is sufficient to allow amplification of the pathogen to levels which may cause spillover into humans. The flexibility of our mechanistic modelling framework, which is based upon the fundamental biological processes, means that it is straightforward to change parameters, such as the season start and end timings, to understand how risk may change under future climate. While this model is applied to WNV in this case, the underlying mechanistic model framework could readily be adapted to study other pathogens.Cx. pipiens is substantially less than 12 km [We simulated the WNV model under various warming scenarios provided as part of the UK Climate Projections 2018 (UKCP18) report produced by the Met Office . We usedan 12 km . Each siet al. [Risk maps showing MIR for each grid square for each of the 12 sets of predicted temperature data were produced for the last year of each decade from 2019 until 2079. Under our model, the vector seasonal abundance patterns and pathogen replication rates will vary temporally across years and spatially across the UK as a result of differences in input temperature and photoperiod (the number of daylight hours per day). Three different times of WNV introduction times were considered: introduction at the end of March, the end of April and the end of May. These timings are consistent with the arrival times of a range of potentially competent migratory hosts such as the swallow, chiffchaff and willow warbler , among oet al. . We alsoet al. . To undeet al. [Details of the mathematical model used to simulate vector and host population dynamics and on the assumptions regarding WNV transmission between the mosquito and bird populations are given in full in electronic supplementary material, S1. The following further information is given in electronic supplementary material: parameterization of the extrinsic incubation period/viral replication rate S2.1) , parametet al. . In the .1 , para. 3. 3.1The predicted MIR following WNV introduction via infected birds arriving at the end of March was never projected to exceed 0.25 because this was earlier than termination of mosquito diapause. Likewise, the predicted risk for late April introduction was low for all climate projections with seven of the 12 simulations in the year 2079 predicting MIR values below 0.25 across the entire UK . In those runs for which the MIR exceeds 0.25 in some areas risks are isolated to a very small area and the predicted MIR is generally only very slightly above 0.25. Risks are predicted to be low for these earlier introduction times because we assume that adult females exiting diapause only take one blood meal before laying an egg raft and dying due to the energetic demands of diapause . TransmiThe predicted MIR following introduction via migratory birds at the end of May is substantially higher, as there has been sufficient time for mosquitoes to exit diapause and for the first spring generation to develop prior to arrival of the virus. Of the 12 sets of climate projections available, 11 of them resulted in predicted MIR values above 0.25 in at least one year in a least one location in the UK. In Tmin. Due to a lack of data linking temperature to WNV replication rates in a European context, we use a laboratory-derived relationship based on a North American strain of WNV, increasing the importance of understanding the sensitivity of this parameter in particular. In Tmin for viral replication inside the vector, which is 7.3°C  [A sensitivity analysis of the more uncertain parameters regarding WNV transmission between vectors and hosts is presented in electronic supplementary material, S2.8. However, one parameter which is likely to be particularly influential in the determination of WNV risk under future climate scenarios is the thermal minimum for WNV replication,  11.4°C) . It is c. 3.2The general pattern of increase in WNV risk and the prediction that outbreaks will be unlikely until the middle of the twenty-first century stems from changes to a range of processes affecting virus transmission across the mosquito biting and development season. Specifically, three of the main processes known to drive WNV transmission  are the biting rate, the vector–host ratio and the viral replication rate . In To understand the relative importance of each of the modelled transmission processes  and the interactions between these processes in determining the MIR, a series of Besag–York–Mollié 2 (BYM2) spatial models were fitted to the results of all model runs using INLA   to larger more permanent sites  [We have made the simplifying assumption in this work that there is no spatial variability in the extent of vector breeding habitat. This is clearly untrue, however translating the effect of changing rainfall, a known predictor of habitat , into a r butts) . Human br butts) , as theyr butts) . Indeed,r butts) . Given tr butts) , should We highlight that our modelling framework, combining environmentally forced stage-structured DDEs capturing the vector life cycle with equations representing host and pathogen dynamics, could be applied to study establishment risk of a wide range of VBDs across temperate regions under projected climate scenarios. While the model presented here focusses specifically on WNV, modification of the pathogen replication rate would readily allow other mosquito-borne diseases of risk in Europe, such as Usutu, to be studied  and smalThe model presented predicts that WNV outbreaks in the UK are unlikely given current temperatures, though the outbreak risk is predicted to increase as temperatures warm throughout the century. Nonetheless, these predictions are based upon temperatures simulated under RCP8.5, which is intended to describe a worst-case scenario with regards to continued investment in fossil fuels and no effort to reduce greenhouse gas emissions . Further


Here, we demonstrate the supreme adjuvant effect of the recently developed and pharmaceutically defined double-stranded (ds)RNA adjuvant Riboxxim especially when incorporated into PLGA particles. Encapsulation of Riboxxim together with antigens potently activates murine and human dendritic cells, and elevated tumor-specific CD8+ T cell responses are superior to those obtained using classical dsRNA analogues. This PLGA particle vaccine affords primary tumor growth retardation, prevention of metastases, and prolonged survival in preclinical tumor models. Its advantageous therapeutic potency was further enhanced by immune checkpoint blockade that resulted in reinvigoration of cytotoxic T lymphocyte responses and tumor ablation. Thus, combining immune checkpoint blockade with immunotherapy based on Riboxxim-bearing PLGA particles strongly increases its efficacy.With emerging supremacy, cancer immunotherapy has evolved as a promising therapeutic modality compared to conventional antitumor therapies. Cancer immunotherapy composed of biodegradable poly(lactic- PLGA based cancer immunotherapy incorporating antigen and TLR ligands has resulted in enhancement of the anti-tumour response. Here, the authors explore the use of a defined double stranded RNA adjuvant, Riboxxim, and test its incorporation with PLGA immunotherapy in the context of in vivo tumour models and show enhanced induction of the anti-tumour response. Due to the unique ability of dendritic cells (DCs) to prime and activate naive T cells, effective antigen charging and stimulation of DCs are key goals in immunotherapy. We and others have established the use of PLGA-MP (microparticles) and NP (nanoparticles) as efficient antigen delivery systems for targeting DCs in situ2. The aliphatic copolymer PLGA is extensively used for optimizing controlled and joint release of immune stimulants and antigens in cancer immunotherapy in preclinical settings3. Due to its biodegradability and proven safety profile, PLGA and its formulations have been licensed by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for pharmaceutical applications via parenteral and mucosal routes4. Especially, PLGA-MP exhibit ideal properties for facilitated uptake by antigen-presenting cells (APCs). Concomitant delivery of antigens and adjuvants to the same APC leads to efficient DC or macrophage activation and simultaneous activation of both, CD4+ T helper cells and CD8+ cytotoxic T lymphocytes (CTL), via cross-presentation5.Cancer still represents one of the most prevalent and challenging malignancies worldwide as a failure of standard therapies results in tumor relapse and metastasis formation. Hence, there is an urgent need for the development of alternative therapies such as immunotherapy with cancer vaccines6. The dsRNA analog poly(I:C) (polyinosinic:polycytidylic acid) is the most widely used TLR3 agonist but also engages RIG-I and preferably MDA57. Its adjuvant properties result from induction of type I interferons and pro-inflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF), and interleukin (IL)−1β8 thus enabling an improved efficacy of cancer immunotherapy in murine tumor models11. However, its ill-defined macromolecular structure and heterogeneous composition prevented polyI:C from extensive clinical use due to unpredictable pharmacokinetics and toxicity issues. The TLR3/RIG-I ligand Riboxxim™, in contrast, is characterized by its well-defined chemical structure as double-stranded RNA and nucleotide composition of 100 bp for effective TLR3 triggering. An uncapped triphosphate moiety at the 5′ end of Riboxxim also enables the activation of RIG-I12. Riboxxim exhibits very good solubility in water, prolonged stability in solution at 4 °C and in serum.Successful immunotherapy against cancer also requires an ideal adjuvant. Multiple immunostimulatory RNA duplexes with agonistic activity for pattern-recognition receptors (PRR) have been reported as candidates for adjuvants in cancer immunotherapy. Double-stranded RNA molecules classically trigger endosomal TLR3 (Toll-like receptor 3). Depending on the structural characteristics and length of the viral RNA mimetic, it may also activate cytosolic RIG-I-like receptors (RLRs), including retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5). TLR3 and RIG-I downstream signaling results in nuclear translocation of the key transcription factors nuclear factor κ/light-chain enhancer of activated B cells (NF-κB) and interferon regulatory factor (IRF)3/7 culminating in secretion pro-inflammatory cytokines and of type I interferons (IFN), respectively. Co-encapsulation of TLR3 ligands with antigen greatly enhanced PLGA particle-mediated cancer vaccine efficacy by inducing reliable maturation of DCs13. Monoclonal antibodies directed against these checkpoint receptors have achieved efficient remission of several tumor types, which resulted in FDA and EMA approval of immune checkpoint blockade for adjuvant treatment of advanced melanoma, head and neck squamous cell carcinoma, and metastatic bladder carcinoma14. However, the majority of cancer patients did not benefit from this therapy with only up to 20% complete responses in patients with solid cancer. The therapeutic success of immune checkpoint blockade necessitates the preexistence of cancer-specific CTLs at the tumor site. Thus, there is an urgent medical need to develop defined immunotherapeutic approaches that would elicit tumor-specific CTLs and boost the success of checkpoint blockade.Cancer immunotherapy has been greatly improved and promoted by pharmacological blockage of so-called immune checkpoint receptors such as CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), PD-1 (programmed cell death protein 1), and its ligand PD-L1. These molecules can be co-opted as immune evasion mechanism by tumors to inhibit CTL activity and suppressing antitumor immune responses18. With respect to a possible clinical translation, this study demonstrates the immune-potentiating capacity of GMP-grade Riboxxim contained in PLGA-MP and the synergistic therapeutic effect of combining this approach with immune checkpoint blockade.Previously, we have shown that PLGA-MP mediated co-delivery of tumor antigens or lysates along with several experimental TLR ligands yielded potent and long-lasting anti-cancer immune responses in vivo affording suppression of tumor growth+ CTL induction with PLGA-NP or MP using different administration routes. PLGA particles containing ovalbumin (OVA) and GMP-certified pyrogen-free Riboxxim were injected either intraperitoneally (i.p.), intramuscularly (i.m.), intranodally (i.nd.), or subcutaneously (s.c.) into C57BL/6J mice. The maximal cytokine response on day 6 after vaccination was achieved after s.c. application  images show uniform spherical PLGA-NPs with a smooth surface of an average size of 250 nm in diameter. MPs produced by spray-drying possess a relatively broad size distribution ranging from nanosized up to 1.5 µm sized particles, notoriously suitable for cellular uptake and internalization by different APCs Fig. . Since tion Fig. . We deci+ bone marrow-derived DCs (BMDCs) and peritoneal macrophages (pMØ), as well as by human CD14+ monocytes, that have been in vitro differentiated into monocyte-derived DCs (MoDCs) or macrophages (MoMØ). Efficient particle engulfment of about 35% could also be demonstrated with CD1c+ and CD141+ primary human myeloid dendritic cells isolated from peripheral blood (mDCs). Of note, no in vitro cytotoxicity was detected after engulfment of PLGA-MP by BMDCs over several days  analysis showed unimodal size distribution with a mean particle size of ~1 µm in diameter and negative surface charge  and BDCA3+ (CD141+) cells, found in low numbers in human peripheral blood21.A proper activation of DCs is crucial for T-cell targeted cancer vaccines. Whether Riboxxim promotes upregulation of maturation markers and the release of cytokines by TLR3-expressing murine and human DCs was analyzed in comparison to conventional polyI:C (pIC) or a pyrogen-free polyI:C variant (pIC pyf). Endotoxin levels of ≤0.5 EU/ml further demonstrated safety of Riboxxim, while the most widely used conventional polyI:C exhibited a high level of endotoxin and pyrogen contaminations, which leads to a potential contribution of TLR4 signaling after addition of polyI:C , stimulation with Riboxxim achieved efficient elevation of co-stimulatory molecules, maturation markers, and MHC-I equivalent to the stimulation by polyI:C Fig. . This re+ and CD141+ DCs . TLR3-dependent upregulation of these markers was comparable to Riboxxim stimulation in WT cells. BMDCs deficient for the adapter molecule MAVS failed to release type I IFN with Riboxxim  into PLGA microparticles enabled the induction of peptide-specific CTL activation and cytotoxic effector function in chimeric human HLA-A*0201 transgenic AAD mice18. Encapsulation of STEAP1262–270 into PLGA-MP together with Riboxxim enabled even more potent CTL responses  epitope STEAP1ses Fig. . Notably+ T-cell responses by MP-OVA/R848 to MP-OVA/Riboxxim, a therapeutic administration induced only a mediocre antitumor response against established E.G7 tumors, which was similar to the antitumor efficacy of MP-OVA/polyI:C . Animals that had received a single vaccination of MP-OVA/Riboxxim or MP-OVA/polyI:C remained tumor-free throughout the whole experiment of 60 days Fig. . We then24 in order to direct the antitumor response towards the lung. A PLGA-MP booster application into the tumor microenvironment seems to recruit and amplify the antitumor response by systemic and local clonal expansion of tumor-specific T cells  was added to the therapeutic regimen 31 while mismatched uracil and guanosine residues were added to the polyI:C structure to decrease toxicity issues in Ampligen™ 32. The adjuvant effect of polyIC analogs includes dose-dependent stimulation and activation of DCs expressing pro-inflammatory cytokines and type I IFNs to promote Th1 polarization and activation of antigen-specific cytotoxic T cells, with too high doses leading to inhibition of the immune activity. The additional activation of MDA5 might also lead to overshooting immune responses. Over 60 clinical studies involve the administration of the dsRNA analog Hiltonol® in solid tumors in combination with peptide vaccines, fusion proteins, or immune checkpoint blockade. However, the therapeutic effect largely depends on multiple injections using high doses of polyICLC. Repeated systemic administration of TLR agonists may induce immune hypo-responsiveness, which is commonly known as TLR tolerance33. Only a few clinical studies encompassed Ampligen for cancer therapy by inducing DC maturation and Th1-type T-cell immunity34. The multimodal immunomodulatory properties of Ampligen are not extensively characterized, which marks Ampligen as a bona fide TLR3 agonist35.The importance of dsRNA agonists in cancer vaccines has been demonstrated in several studies as it facilitates potent DC activation and subsequent priming of tumor-specific CTLs with induction of a tumor-suppressive microenvironment36. The induction of a type I IFN response and apoptotic tumor cell death was enabled by targeted delivery of Riboxxol coupled to an anti-prostate stem cell antigen (PSCA) antibody derivative36. Matsumoto and colleagues created the chimeric TLR agonist ARNAX, which exclusively triggers TLR3 by the addition of a 5′phosphorothioate oligodeoxynucleotide (ODN)-cap of GpC DNA to the 140 bp dsRNA that specifically targets ARNAX to the endosome. ARNAX therapy-induced robust antitumor immune responses without the induction of systemic inflammation37. This non-inflammatory TLR3 agonist was shown to synergize with PD-L1 blockade in tumor eradication in immunocompetent hosts to overcome PD-1/PD-L1 resistance and provide long-term protection from tumor relapse. The authors highlight the necessity of both proper DC priming by a TLR3 ligand and checkpoint inhibitor blockade for efficient cancer immunotherapy43.In contrast to multiple prime-boost injections, PLGA particles carrying Riboxxim show antitumor responses after a single treatment. Compared to other dsRNA mimetics, Riboxxim displays superior properties as immune adjuvant due to the absence of systemic cytokinemia or adjuvant toxicity. The 50 bp derivative of Riboxxim, designated Riboxxol (RGIC®50), potently improves murine and human cDC1 activation to enhance T-cell proliferation+ CTLs and antitumor immunity in mouse models of melanoma or colorectal carcinoma45 and in human ovarian cancer46. The addition of 5′pppRNA to a cancer-peptide containing NP was shown to specifically boost CD8+ T-cell population while not inducing suppressive immune cell types45.Therapeutic targeting of RIG-I has recently emerged as another aim in antitumor immunotherapy and is currently explored in clinical trials. A combinatory vaccination scheme using 5′-triphosphorylated RNA-driven RIG-I stimulation with CTLA-4 or PD-1 blockade already has been demonstrated to induce potent cross-priming of CD8+ and CD141+ DCs as well as the induction of IFNα/β and IL-6/TNF , which has been most widely used in preclinical studies, probably originated from pyrogen contaminations, which was markedly reduced in pyrogen-free polyI:C (Invivogen) or in GMP-grade Riboxxim  2Enge/J (AAD) mice and B6;129-Mavstm1Zjc/J (Mavs−/−) mice were obtained from The Jackson Laboratory . B6;129S1-Tlr3tm1Flv/J (Tlr3−/−) mice were obtained from the Laboratory Animal Services Center (LASC)-Vivaria, University of Zurich, Switzerland. Transgenic animals are verified by genotyping of ear biopsies. Primers are listed in Supplementary Table + T cells in blood after vena facialis puncture. Animals were further bred in the accredited animal facility of the University of Konstanz under specific pathogen-free conditions on a 12-h light/dark cycle with lights on at 7 a.m. Mice were kept in air-conditioned rooms with controlled temperature (22 °C), 55% relative humidity, and constant ventilation (17 air changes/h). Animals were provided ad libitum access to standard, autoclaved laboratory animal diet, and tap water. Male and female mice were used at 8–12 weeks of age. The number of animals in each group was determined according to statistical verification and previous studies.C57BL/6J mice and BALB/cAnNCrl (BALB/c) mice were originally purchased from Charles River . C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-1) mice and B6.Cg-Animal experiments have been conducted in compliance with ethical standards of German and EU guidelines after approval by the animal experimentation ethics committee of the Review Board of Governmental Presidium Freiburg, Germany . Human specimens were used in accordance with the requirements of the institutional ethics committee and national policies. Blood donations were approved by the Ethics Committee of the University of Konstanz, Germany. Written informed consent was obtained from each of the randomly enrolled healthy volunteers before blood donation following guidelines of the Review Board protocol of the University of Konstanz. Blood donations were performed by the transfusion unit at the Klinikum Konstanz in accordance with the precepts of the transfusion unit at the hospital and of the DRK (German Red Cross) with all applicable regulations, guidance, and local medical policies.+/GFP+ was kindly provided by Prof. Olaf van Tellingen . To maintain Luciferase expression, respective growth media were supplemented with 0.4 mg/ml G418 Sulfate . Positive expression of luciferase was verified before every injection into animals by in vitro bioluminescence imaging on the IVIS® Spectrum 200 platform (Perkin Elmer). Expression of ovalbumin was authenticated via immunoblotting using anti-chicken egg albumin antibody . The immature dendritic cell line DC2.4  was provided by Kenneth Rock  and maintained in RPMI 1640. The CD8+ splenic DC cell line MutuDC211459 was a kind gift of Prof. Hans Acha-Orbea  and cultured in IMDM with 10% (v/v) FCS, 100 IU/ml Penicillin, and 100 µg/ml Streptomycin. The B3Z T-cell hybridoma60 was a kind gift from Prof. Nilabh Shastri  and cultured in IMDM supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin, and 100 µg/ml Streptomycin. The CD4+ T-cell hybridoma DOBW61 was kindly contributed by Prof. Clifford V. Harding  and maintained in DMEM supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin and 100 µg/ml Streptomycin, 1 mM sodium pyruvate, 10 mM HEPES and 0.5 mM β-mercaptoethanol. The human monocytic cell line THP-1 (#TIB-202™) was purchased by ATCC®  and maintained in RPMI 1640. All culture media were supplemented with GlutaMAX™. Media and standard additives were purchased from Gibco® (ThermoFisher Scientific). Cell lines were regularly tested negative for Mycoplasma by PCR analysis of cell culture supernatants by Microsynth, Switzerland. Cell cultures were maintained at 37 °C with 5% CO2 in a humidified atmosphere. Adherent cells were detached by incubation with 0.05% trypsin-EDTA for 5 min at 37 °C. Primary DCs were detached by 5 mM EDTA/PBS for 15 min at 37 °C.The murine lymphoma cell line stably expressing OVA and Luciferase, E.G7-OVA-luc+ cells were kindly provided by Prof. Thomas Blankenstein  and maintained in RPMI 1640 plus 10% (v/v) FCS, 100 IU/ml Penicillin and 100 µg/ml Streptomycin. MO5 cells, murine melanoma cells stably expressing OVA, were provided by Dr. Antje Heit  and cultured in DMEM supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin, and 100 µg/ml Streptomycin. Luciferase-positive, OVA-expressing B16BL6 cells were generated by lentiviral transduction of cytosolic ovalbumin (amino acids 51-386) and cultured in DMEM and 10% (v/v) FCS, 100 IU/ml Penicillin, and 100 µg/ml Streptomycin. The original cell line B16BL6-luc62. 5 mg ovalbumin  and 0.05 mg adjuvant in 100 µl 0.1 M NaHCO3 were dissolved in the primary emulsion (100 mg PLGA in 3 ml dichloromethane (DCM)) and mixed with 25 ml of 2% poly . The mixture was stirred overnight at 4 °C to evaporate DCM. NPs were collected by ultra-centrifugation and lyophilized at −80 °C for 48 h. PLGA microparticles (MP) were prepared by spray-drying. For antigen/TLR3L co-encapsulation, 50 mg OVA or 10 mg of peptide antigens and 0.5 mg adjuvants  (HMW), InvivoGen; Riboxxim™, Riboxx Pharmaceuticals; MPLA-SM VacciGrade™, InvivoGen; R848 VacciGrade™, InvivoGen; α-Galactosylceramide ) were dissolved in 1 ml of 0.1 M NaHCO3 (aqueous phase) and emulsified with 1 g PLGA in 20 ml dichloromethane (organic phase) using a digital microtip sonicator. Riboxxim is provided at a 1 mg/ml solution in water for injection and is certified as GMP material sterile and endotoxin-free (European guidelines ICH-6). The HLA-A*0201 restricted tumor-associated peptides STEAP1262–270 (LLLGTIHAL); STEAP1292–300 (MIAVFLPIV); PAP135–143 (ILLWQPIPV); PSMA469–477 (LMYSLVHNL); Her2689–697 (RLLQETELV); TRP-2180–188 (SVYDFFVWL) and NY-ESO1157–165 (SLLMWITQV) were synthesized by the Institute for Biochemistry, Charité Berlin or were purchased by peptides&elephants, Germany. The obtained dispersion was immediately spray-dried with the Mini Spray-Dryer 290 (Büchi Labortechnik AG) at a flow rate of 1 ml/min and inlet/outlet temperatures of 25 °C/23 °C. Spray-dried microparticles were washed out of the spray-dryer’s cyclone with 0.05% Poloxamer 188  and collected on a cellulose acetate membrane filter. PLGA-MP were dried under vacuum at room temperature and subsequently stored under desiccation at 4 °C.PLGA particles were prepared from Resomer RG502H (Evonik Industries). Nanoparticles (NP) were produced by the double emulsification solvent evaporation method as previously describedThe amounts of encapsulated antigens and TLR ligands were determined after dissolving the PLGA particles in DMSO for 10 min. Phase separation of precipitated polymer and soluble compounds was achieved by the addition of 0.1 M NaOH. Antigen release was quantified using Pierce™ MicroBCA Protein Assay Kit (Thermo Fisher Scientific) after incubating particles in PBS pH 7.4 at 37 °C under continuous orbital agitation.The particle size and polydispersity index (PDI) were analyzed by cumulative analysis of dynamic light scattering (DLS) using the Zetasizer® Nano ZSP particle size analyzer and Zetasizer software version 7.12 . The ζ (zeta) potential of particles was measured by laser Doppler velocimetry in combination with phase analysis light scattering using the same device. Particles were dispersed in ultrapure water prior to measurement at 25 °C with the Helmholtz–Smoluchowski model.To characterize surface morphology using scanning electron microscopy (SEM) particles were mounted on aluminum stubs and sputter-coated with a 6 nm thick layer of platinum by an argon beam sputter coater . Micrographs were produced on a Zeiss Auriga 40 at 1, 3, or 5 kV regarding the sample with SmartSEM v6.0 .Endotoxin levels of PLGA particles were detected by ToxinSensor™ Chromogenic LAL Endotoxin Assay according to the manufacturer’s instructions .6 cells/well. Particle uptake was performed for 6 h at 37 °C or at 4 °C as a control (no uptake). Phase-contrast microscopy was performed for semi-quantitative estimation of particle uptake. After incubation, cells were harvested and incubated for 1–3 s in pure DMSO to remove unbound particles. After that, cells were washed three times with 1× DPBS. QD-positive fluorescent signals were assessed for each sample by flow cytometry.In vitro particle internalization by primary DCs and cell lines was determined using fluorescently labeled particles (PLGA-MP-OVA/Riboxxim/QD) . QD-loaded MPs were added to cells at a concentration of 10 µg/ml per 1 × 1063. Isolated bone marrow cells were cultured for 9 days in 10 cm dishes at 2 × 106 cells in RPMI 1640, supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin, 100 µg/ml Streptomycin, 1 mM sodium pyruvate, 55 µM β-mercaptoethanol and 20 ng/ml recombinant murine GM-CSF . Peritoneal macrophages were elicited by i.p. injection of 0.5 ml of 3% Brewer thioglycolate medium (w/v)64. Cells were harvested on day 3 after injection by washing cells from the peritoneal cavity by lavage with 5 ml of ice-cold, sterile PBS/3%FCS. After a centrifugation step at 300 × g for 5 min, cells were seeded at 1 × 106 cells/well in RPMI 1640, supplemented with 10% (v/v) FCS, 100 IU/ml Penicillin, 100 µg/ml Streptomycin, 55 µM β-mercaptoethanol. Non-adherent cells were discarded after 6 h. Adherent macrophages were used after overnight cultivation.DCs from proliferating mouse bone marrow progenitors were isolated from femur and tibia of 8–10-weeks-old wild-type C57BL/6J mice and differentiated according to Lutz et al.6 cells. If required, cells were stained after RBC‐lysis with 1.66% NH4Cl w/v. Live/Dead cell discrimination was performed by the addition of Zombie Green™ Fixable Viability Dye . Flow cytometry was performed using BD LSRFortessa™ or BD FACSLyric™ instruments. Data were acquired using BD FACSDiva™ Version 8.0.1 and BD FACSuite™ 1.2.1 (BD Biosciences). Data were analyzed using FlowJo™ v10.7.1 software (BD Biosciences).Antibodies and dilutions used in this study are listed in Supplementary Table 5 THP-1/ml in the presence of rhGM-CSF (20 ng/ml) and rhIL-4 (20 ng/ml) for 5 days65. THP-1 cells were differentiated into monocyte-derived macrophages (MoMØ) using 100 nM of phorbol 12-myristate 13-acetate  for 72h66. Blood samples from healthy donors were collected as whole blood donation in 500 ml blood bags supplemented with CPDA-1 stabilizer. Human peripheral blood mononuclear cells (PBMC) were isolated within the first hour of acquisition by Ficoll®Paque™ PLUS  centrifugation of 35 ml fresh blood in 50 ml LeucoSep (Greiner) tubes at 800×g for 15 min at RT without brake. Mononuclear cells at the interphase were purified by multiple washing steps with 1× DPBS. Human primary cells were cultured in AIM-V medium  supplemented with 2% heat-inactivated human AB serum  and 50 μM β-mercaptoethanol (complete AIM-V). CD14+ monocyte-derived DCs (MoDCs) were enriched by positive magnetic selection using anti-CD14 microbeads according to the manufacturer’s instructions . Purified CD14+ monocytes were differentiated into MoDCs by culturing 2 × 106 cells/ml in complete AIM-V medium supplemented with 500 U/ml recombinant human GM-CSF  and 250 U/ml recombinant human IL-4  for 5 days. CD14+ monocytes were differentiated into CD68 + MoMØ by culturing 2 × 106 cells/ml in a complete AIM-V medium supplemented with 50 ng/ml rhGM-CSF for 5 days67. CD1c+ (BDCA-1+) and CD141+ (BDCA-3+) myeloid DCs were purified from CD14-negative PBMCs using MACS isolation kits  or magnetic MicroBeads , respectively. CD1c+ and CD141+ cells were used directly.The human monocytic leukemia cell line THP-1 was differentiated into immature DC with similar characteristics to human monocyte-derived DCs (MoDCs) by culturing 2 × 10−6 M SIINFEKL peptide for 5 h in the presence of 10 µM/ml Brefeldin A . For surface staining, cells were incubated with anti-CD8α APC  for 30 min at 4 °C; then cells were fixed and permeabilized using a Cytofix/Cytoperm Kit  according to the manufacturer’s instructions as to preparation for intracellular staining using anti-IFNγ BV421 antibody  in BD Perm/Wash™ buffer . CD8+IFNγ+ cells were analyzed 16 h later by flow cytometry.Intracellular cytokine staining (ICS) was performed to detect intracellular IFNγ as a measure of cytotoxic T lymphocyte activation. Splenocytes or tumor-infiltrating lymphocytes (TILs) were cultured with or without 105 cells per well onto pre-coated MultiScreen®HTS Filter Plates (Merck Millipore) and re-stimulated with 10 μM of the respective peptide for 20 h. After incubation with the biotinylated secondary antibody specific for IFNγ, a streptavidin-alkaline phosphatase enzyme conjugate was added. After the addition of the BCIP®/NBT substrate solution , a purple precipitate is formed as spots at the sites of captured IFNγ. Automated spot analysis and quantification were performed using the ImmunoScan® analyzer and Immunospot software v.6.0.0.2 .IFNγ production of OVA-specific cells was analyzed using a commercially available mouse ELISPOT antibody pair according to the manufacturer’s protocol . Briefly, splenocytes of immunized mice were seeded at 5 × 10Equal amounts of supernatants of PLGA-MP release assays were resolved in reducing conditions on 10% SDS-polyacrylamide gels for electrophoresis. Silver Staining was performed according to the manufacturer’s instructions .Supernatants of DC cultures and BMDC-DC co-cultures were analyzed for different cytokines by ELISA according to the manufacturers’ instructions. Commercially available ELISA Kits were purchased from Thermo Fisher Scientific (Invitrogen™ eBioscience™ ELISA Ready-SET-Go!™) except for mIFNγ  and mIFNβ . Mouse IFNα, as well as human IFNα and IFNβ were purchased from PBL .+ T-cell hybridoma, specific for the SIINFEKL peptide of ovalbumin (OVA257–264) and the CD4+ T-cell hybridoma DOBW specific for the ovalbumin class II epitope ISQAVHAAHAEINEAGR (OVA323–339). 1 × 105 hybridoma cells were co-cultured overnight with 2 × 105 DCs loaded with PLGA particles. To determine the relative maximum of T-cell activation, DCs were pulsed with SIINFEKL peptide . 100 μl of lacZ buffer ) was added for an additional 4-h incubation step68. The absorbance of the chromogenic reaction was measured at 570 nm with background subtraction at 620 nm using a spectrofluorometer . Presentation on MHC class II was similarly assessed as described above, except for using the OVA323–339-specific hybridoma cell line DOBW. IL-2 secretion was measured from supernatants of DOBW-DC co-cultures using a mouse IL-2 ELISA kit  according to the manufacturer’s protocol.Antigen (cross-) presentation of SIINFEKL peptide was assessed using the B3Z CD8+ T cells were isolated from OT-I spleens and LN using mouse CD8a (Ly-2) MicroBeads , and then labeled with 1 μM of the proliferation and cell tracking dye CFSE . 1 × 105 labeled CD8+ cells were co-cultured with an equal number of PLGA particle loaded DCs or splenocytes of PLGA-MP immunized mice in complete medium. After 72 h, cells were recovered, and OT-I proliferation was measured by CFSE dye dilution of CD8+ T cells using flow cytometry. Additionally, IFNγ secretion of culture supernatants was measured by mouse IFNγ ELISA kit  according to the manufacturer’s protocol.CD869. On day 5 post-vaccination, splenocytes from congenic C57BL/6J mice were pulsed with 1 µM SIINFEKL peptide (vaccine-specific target cells) or left unpulsed (non-specific control cells). Immunized mice were i.v. injected with a 1:1 mixture of 107 cells labeled with either 5 µM CFSE  (peptide-pulsed) or 0.5 µM CFSE (unpulsed). The following day, ratios of the CFSE bright to dim fluorescent peaks of CD45+ splenocytes were analyzed. OVA-specific cytotoxicity was calculated using the following equation: {1−(PLGA-MP immunized mice) [CFSEhigh(%)/CFSElow(%)]/naive mice [CFSEhigh(%)/CFSElow(%)]} × 100Cytotoxic activity of OVA-specific CTLs in PLGA-MP immunized mice were analyzed in vivo4 cells per well for 48 h at 37 °C. The number of live  cells was evaluated by the addition of 10% of Deep Blue Cell Viability™ Kit  according to the manufacturer’s protocol. The reduction of resazurin into resorufin was photometrically detected using a spectrofluorometer  at 550 nm. To demonstrate cytotoxicity, cells were treated with 10% (w/v) Triton®X-100 (Merck) for the last 4 h as a positive control.The viability of murine BMDCs was determined by incubation of DCs with varying concentrations of PLGA-MP in 96-well plates at 5 × 10Mavs−/−, Tlr3−/− or AAD mice were immunized either subcutaneously at the base of the tail or via the intraperitoneal route using 5 mg PLGA particles loaded with antigen  and dsRNA analogs (2.5 µg/mouse) in PBS. The following applications were performed under isoflurane anesthesia. For intranasal immunizations, mice were vaccinated with a suspension of 2.5 mg PLGA particles  in 50 μl PBS (25 μl per nostril). 50 µl of a 1 mg/ml PLGA particle solution in PBS  were administered intramuscularly into the hind limb thigh muscle. The intranodal injection was also performed by administering 10 µl of a 1 mg/ml PLGA particle suspension in PBS  into the inguinal lymph node. Control mice were treated with respective amounts of empty PLGA particles or particles containing only dsRNA analogs. On day 6 after immunization, target organs were resected and homogenized into single-cell suspension by smashing through 70 µm cell strainers  for further analysis. Lung tissue was dissociated using the mouse Lung Dissociation Kit  with a gentleMACS™ octo Dissociater according to the manufacturer’s protocol.For immunization studies, 8-to-12-week-old female and male C57BL/6J, Female BALB/c mice were s.c. injected with 5 mg of QuantumDot containing PLGA-MP-OVA/Riboxxim into the left flank. The left non-injected flank served as a negative control. Fluorescence signals were acquired at indicated time points with the IVIS® Spectrum 200 In Vivo Imaging System (Perkin Elmer) using the Far-red filter set. Popliteal and inguinal LNs were dissected at different time points post-immunization and incubated in 10% neutral formalin for subsequent paraffin-embedding and histology.Organs were excised and immediately fixed in 10% neutral-buffered formalin for 24 h followed by paraffin-embedding and sectioning to 5-µm-thick slides. H&E staining was performed according to standard protocols. For IHC staining, tumor-bearing tissues were collected and processed as described before. Paraffin-embedded tissue sections of 3 µm thickness were rehydrated and subjected to heat-induced antigen retrieval (HIER) in 10 mM citrate buffer (pH 6). Blocking was performed with 10% goat serum. Sections were incubated with rat primary antibodies specific for mouse CD8a (1:200)  at 4 °C overnight and then stained with an avidin-biotin-based peroxidase system . Positive immunoreactions were visualized using peroxidase substrate kits . Sections were counterstained with hematoxylin. Results of three random fields per section were recorded with AxioObserver or AxioImager microscopes and ZEN 3.2 (blue edition) imaging software (Carl Zeiss Imaging Inc.) and analyzed in a blinded manner.n = 5). The establishment of lung metastases was performed by i.v. injection of 105 MO5 melanoma cells. To direct antitumor responses to the site of tumor formation, mice were immunized with PLGA particles in a prime-boost setting as previously described24. To this aim, mice received a subcutaneous prime immunization followed by an intranasal boost 14 d later. Therapeutic PLGA-MP treatment was performed by simultaneous s.c. and i.n. double route-immunization. Solid tumors were established by s.c. injection of 5 × 105 syngeneic E.G7-OVA-luc+ cells into the left flank of each mouse. Tumor volumes were measured at regular intervals by using bioluminescence and caliper measurements of two perpendicular tumor dimensions. The tumor volume was calculated using the following formula: Tumor volume (mm3) = [(long diameter) × (short diameter)2] × 0.5. A single dose of PLGA-MP treatment was administered either in a protective manner 6 days before tumor challenge or therapeutically when tumor nodules became palpable. To deplete CD8+ T cells, mice were i.p. injected with 100 µg of InVivoMAb anti-mouse CD8β/Lyt 2.1 mAb (clone 116-13.1 (HB129), #BE0118, BioXCell) 3 days before and on the day of tumor cell inoculation followed by weekly injections until the end of the experiment. Depletion of CD8+ T cells in peripheral blood was validated by flow cytometric analysis with 100% loss of CD8+ T cell at the peak of PLGA-MP-mediated antitumor CTL response (day 6 post-vaccination). Immune checkpoint blockade was achieved by multiple i.p. injections of GoInVivo™ purified anti-mouse CD152  with a cumulative dose of 250 µg anti-CTLA-4 per mouse or a total of 1500 µg purified anti-PD-1 mAb per mouse ). Detailed treatment regimens of immune checkpoint blockade and PLGA-MP vaccinations are outlined in schemes and figure legends. The tumor size was measured every 3 days using a caliper and bioluminescent signals were acquired at indicated time points using the IVIS® 200 imaging system (Perkin Elmer). Mice were anesthetized by isoflurane inhalation and D-Luciferin*K  was administered intraperitoneally at 150 mg/kg prior to luminescence imaging. Luminescent signals were acquired using a 20 cm field of view, small binning, and 120 s exposure time. Regions of interest (ROI) were pre-set around the signal on pseudo-color luminescent images using Living Image® v4.1 Software (Perkin Elmer). The emitted signal intensity (photon flux) was integrated over these ROIs and it is expressed as relative light units  with the lower signal threshold set to 5% of the maximum signal value. Body weights were monitored three times a week. Tumor-bearing mice were euthanized in case s.c. tumors exceeded 1500 mm3 or exhibit necrotic or ulcerative pathologies and when animals showed signs of distress or rapid weight loss of 20% of the initial weight.C57BL/6J mice were randomized into different groups  followed by Tukey’s or Sidak’s post-hoc tests was used for comparison of multiple groups. The Kaplan–Meier survival analysis was used to estimate statistical significance in overall survival distribution between the groups and Log-rank (Mantel–Cox) tests were applied to compare survival rates. Unless otherwise stated in the figure legends, all data were derived from at least three independent experiments and are presented as means ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8.4.1 . Statistical significance was achieved when P < 0.05.Statistical significance was determined by applying a two-tailed Student’s Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationPeer Review FileReporting summary


Background: Cardiovascular disease, and more recently, subclinical cardiac dysfunction have both been implicated as important risk factors for cognitive decline. Several measures have been used to detect subclinical cardiac dysfunction, with global longitudinal strain (GLS) emerging as an important and more sensitive indicator than traditional measures. Yet, the association of GLS with cognitive function remains relatively unexplored. Objective: The aim of this review is to systematically summarize the literature exploring the association between GLS and cognitive function. Methods: We conducted a systematic review of the literature following PRISMA guidelines using the following databases: PubMed, OVID Medline, Embase, Web of Science, and CINAHL. Inclusion criteria were observational studies published in English, measuring GLS and assessing cognitive function through neuropsychiatric tests or brain imaging. Quality assessment was done using the Newcastle Ottawa Scale. Results: The initial search revealed 394 studies, of which three met inclusion criteria and were included for final review. The three studies included were cross-sectional and of high quality. They all reported that lower GLS scores were associated? with worse cognitive function and more brain abnormalities in both bivariate and multivariable analysis. Conclusion: Subclinical cardiac dysfunction, identified by GLS, was associated with worse cognitive function and presence of cerebral abnormality on brain imaging. The underlying mechanism could be attributed to dysfunctional autoregulatory and microvascular processes occurring in the brain vasculature. Further longitudinal studies are needed to better delineate the relationship between GLS and cognitive function.


The obtained results show that the energy levels can be modulated by changing the strength of the acceptor unit. Among the three investigated end-groups, 1,1-dicyanomethylene-3-indanone exhibited the largest bathochromic shift and the lowest band gap suggesting the strongest electron-withdrawing character. Moreover, the emissive properties of the investigated systems vary greatly with the nature of the terminal group and are generally lower compared to their precursor aldehyde derivatives.The synthesis of some novel donor-acceptor and acceptor-donor-acceptor systems containing a 2,2′-bi[3,2- I–III, while compounds with a core structure IV showing a S(IV) atom are not stable and could not be isolated.Among the aromatic-fused 5,5 heterocyclic systems with one heteroatom in each ring, fused bithiophenes stand out due to their versatile properties and vast applications ,4,5,6,7.b]thienothiophene  is the most investigated -TT derivatives are also connected to converting solar energy into electricity. Thus, they were employed for the fabrication of efficient dye-sensitized solar cells (DSSCs) -TT derivatives, we considered it of interest to expand our research in the field of molecules with exciting optoelectronic properties [b]thienothiophene derivatives belonging to D-A (V) or A-D-A (VI) systems  with the hexyloxy (compound 2) groups was mandatory to ensure good processability for the structure and properties investigations of the targeted new derivatives with 2,2′-bithienothiophene. The homocoupling of 2, leading to 3,3′6,6′-tetrakis(hexyloxy)-2,2′-bithienothiophenes 4  or diformyl (6) derivatives  molar ratio.The first step to accessing the target compounds of types reported . We starorted -TT central unit.Concerning the low-energy absorption bands, their theoretical absorption maxima ranges from 546 nm to 645 nm within the O) Egap,  trend foO) Egap, . More pr(A-)D-A motifs, with a lower coefficient on the acceptor moiety in the case of the DCV- and InOCN-based compounds. Similarly, LUMO is delocalized over both the donor and acceptor units within the A-D-A molecules, but with a considerably higher coefficient on the acceptor part in the case of the D-A counterparts  and internal charge transfer (ICT), while in the case of the A-D-A counterparts, only local transitions take place. To further prove these statements, we plotted the electron density difference between first excited state and ground state for each molecule  the absence of a π-spacer between D and A that could facilitate the localization of the HOMO on the donor side and the LUMO on the acceptor moiety ; (ii) the presence of two acceptor moieties, which pull the electron density in opposite direction and do not allow its polarization toward one EWG; and (iii) the imbalance between the donation ability of the -TT platform and accepting ability of EWGs.As HOMO and LUMO have the strongest impact on the optoelectronic properties of our studied compounds, it is useful to depict their spatial distribution in order to obtain more information about the nature of the electronic transitions. On this note, careful inspection of the wave functions shows a distribution of HOMO over the entire molecule in all terparts . This ormolecule . Upon ex1H NMR (400 or 600 MHz) and 13C NMR (100 or 150 MHz) spectra were recorded in CDCl3 or CD2Cl2 at room temperature using the solvent line as a reference.254 TLC plates. All plates were visualized by UV irradiation at 254 and 365 nm.Thin layer chromatography (TLC) was conducted on silica gel 60 FSolvents were dried and distilled under argon using standard procedures. Chemicals were purchased from TCI Chemicals, and Alfa Aesar and Sigma Aldrich and were used without further purification.Melting points were determined with a Kleinfeld apparatus and are uncorrected.HRMS were recorded using an LTQ XL OBITRAP mass spectrometer equipped with ESI/APCI sources. UV-vis optical data in solution and in films were recorded with an UV-vis 1900 Shimadzu spectrometer. Fluorescence spectra were recorded on a JASCO FP-8300 spectrofluorometer using glass cuvettes (1 cm). Solutions for UV-vis, CV, HRMS and fluorescence measurements were prepared in HPLC grade solvents .Films were obtained from chloroform solutions using a classical spin coater.Fluorescence quantum yields were calculated using fluoresceine  or rhodamine B  as standards.5 and 6; 520 nm for 7, 8, 10, 11; and 640 nm for 9) and at a 40 MHz repetition rate. The signal was collected from the compounds in a CHCl3 solution with a UPlanSApo 60 ×/NA = 1.2 water immersion objective and filtered thereafter with a 50 μm pinhole and adapted long-pass emission filters.Fluorescence lifetime measurements were performed on a MicroTime200 time-resolved confocal fluorescence microscope system , described in detail elsewhere . The exc4NPF/CHCl3 at 100 mV/s, Pt electrode references, SCE.Cyclic voltammetry (CV) measurements were carried out with a Biologic SP-150 potentiostat using a three-electrode cell equipped with a platinum electrode, a calomel reference electrode (SCE) and a platinum wire counter electrode. All experiments were performed in 0.10 M Bun-butyllithium (1.5 M in hexane)  was added dropwise over 10 min at −78 °C to a solution of 3,6-bis(hexyloxy)thienothiophene  in dry THF (65 mL) and the stirring was continued for another 2 h at the same temperature. Then, CuCl2  was added in one portion and the reaction mixture was gradually warmed up to room temperature and then stirred overnight. After adding water (30 mL) and triethylamine (10 mL), the mixture was extracted with dichloromethane (3 × 30 mL) and the combined organic layers were dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure. The crude product was purified with silica gel gradient column chromatography (elution system started with pentane/CH2Cl2 = 2/0.2 and ended with pentane/CH2Cl2 = 2/0.4) to afford a yellow solid .Under stirring and in an argon atmosphere, 4  in dry 1,2-dichloroethane (10 mL) was purged with argon and cooled to 0 °C. Then, DMF  and phosphoryl chloride  were slowly added and the reaction mixture was additionally stirred for 1 h at 0 °C. Then, the reaction mixture was heated under stirring at 60 °C for 18 h. Afterwards, the mixture was poured into a NaOAc solution  and vigorously stirred for 2 h at rt. The final solution was extracted with dichloromethane (3 × 30 mL) and the combined organic layers were dried over magnesium sulfate. The solvents were removed under reduced pressure and the crude product was purified by silica gel gradient column chromatography (elution system started with pentane/CH2Cl2/Et3N = 2/1/0.02 and ended with pentane/CH2Cl2 = 2/1.5) to afford an orange solid for 5  or a red solid for 6 .A solution of 7: 47 mg, 0.71 mmol; 10: 107 mg, 1.62 mmol) and triethylamine (0.1 mL) were subsequently added to an argon purged solution of 5  or 6  in toluene (10 mL) and the reaction mixture was stirred at room temperature for 18 h. Then, the solvent was removed under reduced pressure, the crude product was triturated with ethanol, and the precipitate was filtered off to afford a metallic green solid .Malononitrile  and piperidine  were subsequently added to an argon-purged solution of 5  or 6  in toluene  and the reaction mixture was stirred at 70 °C for 20 h. Then, the solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography . Pure samples were obtained with further purification by crystallization through slow evaporation from a 2/1 mixture of dichloromethane/ethanol for 8, and in the case of 11, by precipitation with ethanol from its solution in chloroform. The products were obtained as metallic green solids .3-Ethyl-2-thioxothiazolidin-4-one (H-inden-1-ylidene)malononitrile  and triethylamine (0.4 mL) were added subsequently to an argon-purged solution of 5  in toluene (30 mL) and the reaction mixture was stirred at 60 °C for 3 h. The solvent was removed afterwards under reduced pressure and the crude product was purified by silica gel column chromatography (elution system: pentane/CH2Cl2/Et3N = 4/5/0.04). Further purification by crystallization from a 2/1 mixture chloroform/ethanol afforded a metallic green solid .2-. 1H-NMR  δ (ppm): 0.89–0.93 , 1.33–1.38 , 1.45–1.55 , 1.80–1.92 , 4.07 , 4.32 , 6.19 . 13C-NMR  δ (ppm): 14.20, 14.21, 22.75, 25.75, 25.82, 29.22, 30.13, 31.69, 70.76, 72.60, 97.46, 118.22, 127.60, 128.01, 144.86, 150.54. HRMS (ESI): m/z calcd for [C36H54O4S4 + H]+: 679.2978; found: 679.2966.3,3’,6,6’-tetrakis(hexyloxy)-2,2’-bithienothiophene-5-carbaldehyde 5: orange solid; mp: 105–106 °C; 58 mg, 0.08 mmol, 66%; Rf = 0.7 (pentane/CH2Cl2/Et3N = 2/1/0.02). 1H-NMR  δ (ppm): 0.90–0.93 , 1.33–1.37 , 1.45–1.56 , 1.81–1.94 , 4.08 , 4.32 , 4.42 , 4.54 , 6.26 , 10.00 . 13C-NMR  δ (ppm): 14.15, 14.20, 14.22, 22.70, 22.73, 22.79, 25.61, 25.68, 25.80, 25.86, 29.17, 29.96, 30.13, 30.15, 31.56, 31.65, 31.68, 31.72, 70.91, 72.70, 72.82, 73.07, 98.82, 115.96, 122.61, 124.29, 126.52, 126.59, 130.05, 135.90, 144.48, 146.61, 150.48, 157.96, 181.29. HRMS (ESI): m/z calcd for [C37H54O5S4 + H]+: 707.2927; found: 707.2957.3,3’,6,6’-tetrakis(hexyloxy)-2,2’-bithienothiophene-5,5’-dicarbaldehyde 6: red solid; mp: 192–193 °C; 140 mg, 0.19 mmol, 86%; Rf = 0.5 (pentane/CH2Cl2/Et3N = 2/1/0.02). 1H-NMR  δ (ppm): 0.90–0.94 , 1.35–1.39 , 1.48–1.56 , 1.84–1.93 , 4.42 , 4.55 , 10.03 . 13C-NMR  δ (ppm): 14.15, 14.21, 22.70, 22.77, 25.60, 25.78, 29.96, 30.16, 31.56, 31.68, 72.93, 73.27, 123.56, 123.73, 126.29, 134.39, 146.16, 157.62, 181.53. HRMS (ESI): m/z calcd for [C38H54O6S4 + H]+: 735.2876; found: 735.2906.3,3’,6,6’-tetrakis(hexyloxy)-2,2’-bithienothiophen-5-yl)methylene)malononitrile 7: metallic green solid; mp: 189–190 °C; 44 mg, 0.06 mmol, 82%; 1H-NMR  δ (ppm): 0.90–0.95 , 1.35–1.40 , 1.47–1.56 , 1.81–1.94 , 4.09 , 4.32 , 4.48 , 4.58 , 6.31 , 7.90 . 13C-NMR  δ (ppm): 14.17, 14.20, 14.23, 22.70, 22.71, 22.74, 22.80, 25.65, 25.68, 25.80, 25.88, 29.18, 29.91, 30.09, 30.15, 31.57, 31.68, 31.72, 68.51, 70.98, 72.81, 73.20, 73.34, 99.70, 115.11, 115.19, 116.36, 116.48, 122.64, 126.12, 129.86, 131.15, 137.91, 144.18, 146.41, 147.67, 150.46, 157.94. HRMS (APCI): m/z calcd for [C40H54N2O4S4 + H]+: 755.3039; found: 755.3036.2--2,2’-bithienothiophen-5-yl)methylene)-2-thioxothiazolidin-4-one 8: metallic green solid; mp: 138–139 °C; 40 mg, 0.05 mmol, 55%; Rf = 0.65 (pentane/CH2Cl2/Et3N = 2/1/0.02). 1H-NMR  δ (ppm): 0.91–0.96 , 1.25 , 1.36–1.41 , 1.47–1.52 , 1.55–1.62 , 1.81–1.95 , 4.07 , 4.14 , 4.35 , 4.42 , 4.52 , 6.28 , 7.99 . 13C-NMR  δ (ppm): 12.56, 14.36, 14.38, 14.42, 14.45, 23.15, 23.17, 23.19, 23.21, 26.06, 26.17, 26.21, 26.30, 29.65, 30.46, 30.57, 30.60, 32.07, 32.14, 32.16, 32.17, 40.34, 71.42, 73.24, 73.43, 73.56, 99.37, 116.00, 116.29, 119.12, 123.90, 125.28, 125.55, 127.00, 130.24, 134.54, 144.71, 146.97, 150.81, 154.44, 167.49, 192.57. HRMS (APCI): m/z calcd for [C42H59NO5S6 + H]+: 850.2790; found: 850.2819.(Z)-2--2,2’-bithienothiophen-5-yl)methylene)-2,3-dihydro-1H-inden-1-ylidene)malononitrile 9: metallic green solid; mp: >360 °C; 25 mg, 0.03 mmol, 57%; Rf = 0.7 (pentane/CH2Cl2/Et3N = 4/5/0.04). 1H-NMR  δ (ppm): 0.92–0.97 , 1.38–1.43 , 1.47–1.54 , 1.59–1.64 , 1.85 , 1.91 , 1.93–1.99 , 4.04 , 4.43 , 4.48 , 4.66 , 6.21 , 7.45 , 7.50 , 7.73 , 8.37 , 9.00 . 13C-NMR  δ (ppm): 14.22, 14.25, 14.27, 14.28, 22.77, 22.82, 25.72, 25.85, 25.86, 25.93, 29.94, 29.86, 30.14, 30.26, 31.69, 31.73, 31.81, 31.85, 65.62, 70.88, 72.89, 73.07, 73.77, 99.80, 115.43, 115.63, 116.19, 117.51, 120.53, 122.55, 122.85, 124.29, 125.78, 131.17, 131.75, 133.04, 133.17, 133.85, 136.64, 140.01, 142.63, 144.75, 148.02, 150.37, 160.49, 161.33, 188.87. Elemental analysis: calcd. for C49H58N2O5S4: C 66.63, H 6.62, N 3.17, S 14.52; found: C 66.87, H 6.48, N 3.11, S 14.36.bis(methan-1-yl-1-ylidene)dimalononitrile 10: metallic green solid; mp: 311–312 °C; 65 mg, 0.08 mmol, 96%; 1H-NMR  δ (ppm): 0.90–0.95 , 1.33–1.40 , 1.48–1.52 , 1.56–1.59 , 1.87–1.93 , 4.47 , 4.59 , 8.00 . 13C-NMR  δ (ppm): 14.15, 14.19, 22.67, 22.75, 25.62, 25.82, 29.93, 30.13, 31.54, 31.70, 71.35, 73.47, 73.58, 114.56, 115.58, 117.87, 125.33, 125.36, 135.55, 146.65, 146.77, 157.53. HRMS (APCI): m/z calcd for [C44H54N4O4S4 + H]+: 831.3101; found: 831.3090.2,2’--2,2’-bithienothiophene-5,5’-diyl)bis(methan-1-yl-1-ylidene)bis(3-ethyl-2-thioxothiazolidin-4-one) 11: metallic green solid; mp: 249–250 °C; 135 mg, 0.132 mmol, 75%; Rf = 0.75 (toluene/CH2Cl2 = 2/0.2). 1H-NMR  δ (ppm): 0.92–0.96 , 1.28 , 1.37–1.42 , 1.52 , 1.61 , 1.87 , 1.94 , 4.17 , 4.42 , 4.49 , 8.03 . 13C-NMR  δ (ppm): 12.46, 14.19, 14.27, 22.72, 22.81, 25.66, 25.89, 30.08, 30.22, 31.64, 31.79, 40.05, 73.06, 73.15, 116.90, 120.09, 122.20, 123.42, 126.80, 132.70, 145.88, 153.61, 167.27, 191.89. HRMS (ESI): m/z calcd for C48H64N2O6S8: 1020.2525; found: 1020.2581.(5b]thienothiophene donor block and various electron-withdrawing groups as acceptors was synthesized and characterized. An in-depth study of the terminal electron-acceptor unit’s impact on the optical and electrochemical properties was carried out. It was shown that the introduction of electron-withdrawing groups in parent 2,2′-bithienothiophene led to a red shift of the main absorption band due to a decrease in the HOMO-LUMO band gap. All investigated systems display emission maxima in the green region, with the A-D-A compounds exhibiting poorer photoluminescence properties compared to their D-A counterparts. Finally, theoretical studies shed light on the observed optoelectronic behaviours by revealing the type and nature of the transitions that can take place and also by showing how various electron-accepting groups tune the optical bandgaps.In conclusion, a series of symmetrical and unsymmetrical conjugated systems based on a 2,2′-bi[3,2-


Older adults overwhelmingly prefer to age at home and in their community, yet research shows uneven access to quality home and community-based services (HCBS), especially among diverse racial and ethnic groups. Our research in Georgia and New York employs participatory research methods, including key informant interviews and focus groups conducted in 2021 and 2022, to identify root causes of disparities in access to, quality of, and experience with HCBS for older racial and ethnic and LGBTQI+ communities. Some root causes identified include: complexity in and lack of funding, tension between equal versus equitable service provision, and meaningful community outreach, particularly in Latino communities. We then identify scalable opportunities policy and programmatic interventions to improve care and service equity for older adults in those communities.


Logistic regression analyses assessed the association between their perceived risk of having COVID when smoking LCCs and pandemic-related behavioral changes in CAI and CAB use . Findings showed that users with a higher perceived risk of getting COVID-19 when smoking LCCs were more likely to endorse trying to quit CAI and CAB during the pandemic. Compared to the non-Hispanic White population, the non-Hispanic Black population were less likely to endorse smoking less CAI and trying to quit CAB during the pandemic. Dual users of CAI and CAB and females were more likely to endorse smoking more CAB compared to CAB-only users and males, respectively. Tailored cessation strategies are needed for dual users, non-Hispanic Black young adults, and young women. Raising awareness about the risks of LCC use can be an effective strategy for LCC smoking cessation.We examined the smoking behaviors of U.S. young adults ages 18–36 regarding little cigars and cigarillos (LCCs) during the COVID-19 pandemic. Survey data were collected from a nationally representative sample of young adults between October and November 2020. Respondents who reported using LCCs with tobacco (CAI) and/or with marijuana (CAB) within the past 6 months prior to the survey ( Respondents were included in the study only if they reported having used CAI or CAB within the past 6 months or less.Based on the findings from a previous focus group study in which LCC users indicated that they most often smoke a part of LCC product occasionally,  respondeRespondents were asked individual questions about changes in their CAI and/or CAB smoking behaviors because of the COVID-19 pandemic. This was measured based on the questions, “Have any of the following happened to your little cigar or cigarillo smoking [as sold/as blunt] behavior because of COVID-19?” Respondents rated their level of agreement to the following items: smoked CAI/CAB more than usual, smoked CAI/CAB less than usual, tried to stop smoking CAI/CAB, and worried about smoking CAI/CAB because of the risk associated with COVID-19. Dual users responded to both CAI and CAB-specific questions. The responses were measured on a 7 point scale ranging from 1 (strongly disagree) to 7 (strongly agree).Respondents were asked about using other tobacco products, including cigarette, large cigars, hookah, and e-cigarette (with and without marijuana) in the past 6 months prior to the survey. This was measured based on the questions, “Have you ever used a [cigarette/large cigars/hookah/e-cigarette (with and/or without marijuana)] at least once, in your lifetime? If yes, when was the most recent time you used a [cigarette/large cigars/hookah/e-cigarette (with and/or without marijuana)] even one or two puffs?” Pictures and examples of brand names of these tobacco products were shown in the survey to help respondents accurately answer the survey questions.All analyses were weighted to the general U.S. young adult population and were performed using SAS version 9.4 . Chi-square tests were used to examine the differences in demographic characteristics among the user groups. Logistic regression was used to examine COVID-19 risk perception and subsequent behavioral changes specific to CAI and CAB use, controlling for demographic-level factors  and user type . The responses for the behavioral changes in LCC use during the COVID-19 pandemic were categorized as: those who “agreed” , “disagreed” , and “neither agree nor disagree”  to experiencing changes in LCC use behaviors. Those who neither agreed nor disagreed were excluded from the regression analysis.p < 0.01).Among 399 past 6 month LCC users in our sample, 72 were CAI-only users (14.5%), 160 were CAB-only users (38.0%), and 167 were dual users (47.4%) . The meaPast 6 month CAI-only users were 68.4% male, 86.8% heterosexual, 42.4% Hispanic, and 73.9% had some college experience. Past 6 month CAB-only users were 56.4% female, 70.4% heterosexual, 43.7% non-Hispanic White, and 63.4% had some college experience. Past 6 month dual users were 63.6% male, 79.9% heterosexual, 35.7% non-Hispanic White, and 57.6% had high school or less education.p < 0.01).Consistent with previous studies ,25, polyp < 0.01) between CAB-only and dual users regarding worrying about smoking CAB, trying to quit CAB, and smoking less CAB during the pandemic.Overall, respondents did not report experiencing substantial changes in CAI and CAB use during the first several months of the COVID-19 pandemic . Among CCompared to the non-Hispanic White population, the non-Hispanic Black population were more likely to endorse worrying about CAI use because of the risk associated with COVID-19 , controlling for other sociodemographic characteristics and risk perceptions . Those wCompared to CAB-only users, dual users were less likely to endorse worrying about CAB use because of the risk related to COVID-19  and smoking less CAB during the pandemic  after controlling for other characteristics. They were also more likely to endorse smoking more CAB  than CAB-only users. Those with greater perceived risk of having COVID-19 if using LCCs were more likely to endorse worrying about CAB  and trying to quit CAB  during the pandemic. The non-Hispanic Black population were less likely to endorse trying to quit CAB  compared to non-Hispanic White population. Moreover, compared to males, females were more likely to endorse smoking more CAB  during the pandemic.The current study examined factors that are associated with the reported behavioral changes in LCC use during the first several months of the COVID-19 pandemic among young adult users, specified by the modality of use , LCC use with marijuana as blunts (CAB) and user group status . Descriptive analysis of our sample indicated that dual use of CAI and CAB was the most prevalent (47.4%), compared to exclusive CAB (38.0%) and CAI use (14.5%). Such a finding is in line with emerging studies that reported increased sales of cannabis in some U.S. states  and incrRespondents in our sample did not report drastic changes in LCC use during the pandemic. Still, we observed racial and ethnic differences in CAI and CAB use behaviors. Compared to the non-Hispanic White population, the non-Hispanic Black population were less likely to endorse trying to quit CAB and smoking less CAI during the pandemic, although they were more likely to endorse worrying about smoking CAI because of risk related to COVID-19. The heightened concern about smoking CAI may be due to the disproportionate impact of COVID-19 on communities of color . Yet, thIn addition, females were less likely to endorse smoking less CAI and more likely to endorse smoking more CAB than males during the pandemic. One likely explanation for the observed racial/ethical and gender differences is that pandemic-induced stressors, such as financial concerns, death of loved ones, feeling isolated ,32, and We also found that user type  had an impact on COVID-related behavioral changes in LCC use. Compared to CAB-only users, dual users were more likely to endorse smoking more CAB and less likely to endorse smoking less CAB during the pandemic. Because dual users use multiple tobacco products and consume both tobacco and marijuana, they may experience greater nicotine dependence ,37,38 thOur findings showed that users with a higher perceived risk of having COVID-19 when smoking LCCs were more likely to endorse trying to quit both CAI and CAB during the pandemic. Nevertheless, the perceived risk of having COVID-19 when smoking LCCs was low in our sample. This finding builds upon previous research that reported cigar smokers, including LCC users, tend to underestimate its health risks compared to cigarettes ,39,40. MPreliminary research shows that tobacco use is associated with more severe symptoms and progression of COVID-19 ,42. In aThis study has a few limitations. The behavioral changes in LCC use during the COVID-19 pandemic were self-reported and were not directly measured using biological measures. There is also a potential recall bias in reporting past LCC use. Moreover, while this study is representative of the young adult population in the U.S., it is important to note the limited sample size included in the logistic regression analysis, particularly for the CAI-only users.We examined young adult LCC users’ perceived risk of COVID-19 and their behavioral changes in LCC use in response to the COVID-19 pandemic. Our findings showed that young adult LCC users, particularly the non-Hispanic Black population, dual users, and females, are at higher risk of increased LCC use during the pandemic. Smoking behaviors formed during the first few waves of the COVID-19 pandemic, as well as during the repeated outbreaks caused by new variants and subvariants of the virus, will continue to affect the health of LCC users. Furthermore, because the Food and Drug Administration (FDA) is considering stricter regulations on tobacco products, such as a JUUL e-cigarette ban and reducing allowable nicotine levels in cigarettes, it is possible that users of these tobacco products switch to using LCCs. Therefore, our findings may inform the development of tobacco prevention and cessation interventions. Programs seeking to reduce LCC use must have focused efforts to reduce all modalities of LCC use, including LCC use with tobacco (CAI) and with marijuana as blunts (CAB), among vulnerable sub-populations of young adult LCC users.


Background: Myocardial infarction (MI), characterized by reduced blood flow to the heart, is a coronary artery disorder with the highest morbidity and mortality among cardiovascular diseases. Consequently, there is an urgent need to identify effective drugs to treat MI. Rhizoma Corydalis (RC) is the dry tuber of Corydalis yanhusuo W.T. Wang, and is extensively applied in treating MI clinically in China. Its underlying pharmacological mechanism remains unknown. This study aims to clarify the molecular mechanism of RC on MI by utilizing network pharmacology and experimental verification.Methods: Based on network pharmacology, the potential targets of the RC ingredients and MI-related targets were collected from the databases. Furthermore, core targets of RC on MI were identified by the protein-protein interaction (PPI) network and analyzed with Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Molecular docking was used to validate the binding affinity between the core targets and the bioactive components. Oxygen-glucose deprivation (OGD) was performed on H9c2 cells to mimic MI in vitro. A Cell Counting Kit-8 assay was used to assess the cardioprotective effect of the active ingredient against OGD. Western blot analysis and RT-qPCR were used to measure the cell apoptosis and inflammation level of H9c2 cells.Results: The network pharmacology obtained 60 bioactive components of RC, 431 potential targets, and 1131 MI-related targets. In total, 126 core targets were screened according to topological analysis. KEGG results showed that RC was closely related to the phosphatidylinositol 3-kinase (PI3K)/Protein kinase B  signaling pathway. The experimental validation data showed that tetrahydropalmatine (THP) pretreatment preserved cell viability after OGD exposure. THP suppressed cardiomyocyte apoptosis and inflammation induced by OGD, while LY294002 blocked the inhibition effect of THP on OGD-induced H9c2 cell injury. Moreover, the molecular docking results indicated that THP had the strongest binding affinity with Akt over berberine, coptisine, palmatine, and quercetin.Conclusion: THP, the active ingredient of RC, can suppress OGD-induced H9c2 cell injury by activating the PI3K/Akt pathway, which in turn provides a scientific basis for a novel strategy for MI therapy and RC application. Myocardial infarction (MI), characterized by myocardial necrosis, is a major cause of morbidity and mortality worldwide, resulting in an estimated 7.4 million deaths per year . The ackR. Corydalis (RC) is the dried tuber of C. yanhusuo W.T. Wang, which belongs to the Papaveraceae family. RC was first recorded by Shennong Herbal Classic and believed to be able to activate blood, move “Qi”  and alleviate painful conditions  Database , the EncHomo sapiens. The UniProtKB database was used to unify and standardize the targets  network of RC potential targets together with MI-related targets , and topp < 0.05, and the species was limited to H. sapiens.To explore the biological processes of core targets, Gene Ontology (GO) biological function and KEGG pathway enrichment analyses were performed with the online tool DAVID Bioinformatics Resources 6.8 . The scrThe crystal structure of core targets (macromolecules) was sought in the Protein Data Bank, and the structure (in SDF format) of the ligand (component) was retrieved from the PubChem database. We then changed the format of the molecules to pdb using OpenBabel software, and utilized AutoDockTools (version 1.5.6) for molecular docking. PyMOL software was then employed to display the docking model. All websites of the databases used in this study are listed in Tetrahydropalmatine   was purchased from Shanghai Yuanye Biotechnology Co., Ltd. . The THP powder was dissolved in dimethyl sulfoxide . Diazoxide (01131863) was provided by Adamas-beta , and LY294002 (S1105) was provided by Selleck .6 cells and then maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were grown at 37°C in humidified 5% CO2. Before the subsequent experiments, a serum-free conditioned medium was used for cell culture.H9c2 cells were plated into a dish (35 mm) at a density of 1 × 10A Cell Counting Kit-8 (CCK-8) assay kit  was used to examine cell activity. Briefly, H9c2 cells were seeded into 96-well plates. After treatment, the cells were incubated with 10 μl CCK-8 solution per well for 2 h at 37°C, and after incubation, the absorbance value at 450 nm was measured using a microplate reader .Western blot assays were performed as we previously reported . PrimaryAfter overnight incubation, appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were added and incubated at room temperature for 1 h. The protein level signals were visualized using ECL-enhanced chemiluminescence , and the band intensities were quantified by ImageJ software (Bio-Rad).−ΔΔCt method.The H9c2 cells were lysed with Accurate Biology RNAex Pro Reagent  to extract total RNA. Then 2 μg of RNA was reversely transcribed to cDNA with the Evo M-MLV RT Kit . The mRNA levels were determined with a SYBR Green qPCR kit  by QuantStudio 5 Real-Time PCR instrument . Rat-specific primers for BCL2 Associated X (Bax), B cell lymphoma-2 (Bcl-2), p53, interleukin-6 (IL-6), interleukin-1 beta (IL-1β), tumor necrosis factor-α (TNF-α), and β-actin were synthesized by Sangon Biotech (Shanghai) . All semA one Step TdT-Mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Assay Kit  was used to measure cell apoptosis. H9c2 cells were washed with phosphate buffered saline (PBS) three times and fixed with 4% paraformaldehyde for 10 min. After washing with PBS three times, H9c2 cells were incubated with 50 μl of TUNEL detection solution at 37°C for 1 h in the dark and then incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min to visualize nuclei. The green spectrum and blue spectrum were used to detect TUNEL-positive cells and nuclei, respectively. Finally, the cells were photographed by confocal scanning microscopy . TUNEL-positive cells were quantified and analyzed from four different views. Data were expressed as the percentage of the number of TUNEL-positive nuclei in the total number of nuclei detected by DAPI staining. ImageJ software was used to calculate the number of TUNEL positive cells.p < 0.05 was considered statistically significant.The data in this study are presented as mean ± SD from three independent replicates. One-way analysis of variance (ANOVA) with the Bonferroni post-hoc test was performed among the multiple groups. In all cases, To collect the components of RC, we searched three databases, and in all, 60 bioactive components were collected by filtering the TCMSP, ETCM, and TCMID databases by the limitations of OB ≥ 30% and DL ≥ 0.18. The active ingredients mainly included berberine, sitosterol, tetrahydropalmatine, corydaline, and quercetin. The information about the components is listed in p < 0.05 and absolute fold change of 1.5 or greater. After deleting duplicates, 1,131 targets were obtained in total.To facilitate statistics, we set screening conditions for MI targets derived from corresponding databases. After taking the third quartile value, a relevance score ≥ 3.66 was set as the threshold for targets from the GeneCards database, and a score ≥ 0.1 was set as the threshold for those from the DisGeNET database. While searching GEO datasets, we selected the reference series GSE48060 to obtain the differentially expressed genes between normal cardiac function controls and first-time MI patients . The filWe used the Cytoscape plugin BisoGenet to construct a PPI network of RC potential targets   and MI-rp = 0.000000000020993, 31 targets/981), negative regulation of transcription from RNA polymerase II promoter , transcription, DNA-templated , viral process , negative regulation of apoptotic process , negative regulation of transcription, DNA-templated , cell-cell adhesion , positive regulation of transcription, DNA-templated , negative regulation of gene expression, epigenetic  and cellular protein metabolic process .To identify the biological function of the core targets, GO and KEGG enrichment analyses of the 126 core targets were performed using the DAVID online tool. Ultimately, a total of 126 targets were analyzed by GO, while 105 targets were enriched in KEGG pathways. The top 10 results of biological process analysis, cellular component analysis, and molecular function analysis of GO function and the top 10 major KEGG enriched pathways are shown in p = 0.000000000000000, 94 targets/5,415), nucleoplasm , cytoplasm , cytosol , membrane , extracellular exosome , protein complex , nucleolus , cell-cell adherens junction  and focal adhesion .The top 10 cellular components were nucleus , poly(A) RNA binding , ubiquitin protein ligase binding , enzyme binding , ATP binding , DNA binding , cadherin binding involved in cell-cell adhesion , protein heterodimerization activity , identical protein binding  and transcription factor binding .The major molecular functions were protein binding . As As the KEGG analysis of the core target results showed, among the 10 pathways, we found that the 126 core targets were primarily involved in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B  signaling pathway  significantly inhibited the overproduction of these inflammatory biomarkers compared to the OGD-induced group . CollectThe results from KEGG enrichment showed that RC and THP were closely related to the PI3K/Akt signaling pathway against MI and that most components could regulate Akt. Therefore, to confirm whether THP exhibits a protective effect via the regulation of Akt, the phosphorylation level and protein expression of Akt were detected. As shown in The PI3K/Akt signaling pathway is significant in defending against myocardial infarction damage . AccordiMI is a severe disease with high motility that places a heavy burden on individuals and society. The current mechanisms of MI include cell death, mitochondrial dysfunction, inflammatory response, oxidative stress, and ATP depletion . In addiThe chemical components of RC can be classified as alkaloids  . In thisMoreover, we obtained a PPI network by merging the active component target PPI of RC and the potential target PPI of MI . Then, ain vivo  The internal absorption and utilization of RC is a complicated process, meaning the function of RC may not simply accumulate the effects of multiple compounds. 2) The predicted targets are greatly affected by the quality of the databases and the algorithm. 3) The anti-apoptotic effect and the exact mechanism of THP against MI should be further demonstrated with animal models and AKT knockout animals.In conclusion, the present study aimed to investigate the effect and mechanism of RC against MI via network pharmacology analysis, experimental validation and molecular docking studies. We identified 60 bioactive components with 431 potential targets and 1,131 MI-related targets through network pharmacology analysis. Then, 126 core targets were screened by PPI network topology analysis. The KEGG enrichment results and the experimental validation results suggested that RC achieves the protective effect against MI via the PI3K/Akt signaling pathway. The molecular mechanisms by which RC inhibits MI were associated with a high binding affinity between THP and AKT. In light of these findings, understanding the function and mechanism of RC can provide novel insight for therapeutic strategies to treat MI.