Oncolytic adenovirus (oAd) elicits antitumor activity by preferential viral replication in cancer cells. However, poor systemic administrability or suboptimal intratumoral retainment of the virus remains a major challenge toward maximizing the antitumor activity of oAd in a clinical environment. To surmount these issues, a variety of non-immunogenic polymers has been used to modify the surface of oAds chemically or physically. Complexation of oAd with polymers can effectively evade the host immune response and reduces nonspecific liver sequestration. The tumor-specific delivery of these complexes can be further improved upon by inclusion of tumor-targeting moieties on the surface. Therefore, modification of the Ad surface using polymers is viewed as a potential strategy to enhance the delivery of Ad via systemic administration. This review aims to provide a comprehensive overview of polymer-complexed Ads, their progress, and future challenges in cancer treatment. Cancer is the second leading cause of death, responsible for loss of 10 million lives globally in 2020. Despite advancements in treatment options, including surgery, chemotherapy, and radiotherapy, the prognosis of patients with cancer remains poor because of the clinically elusive nature of the disease . As convMany oncolytic viruses (OV), including adenovirus (Ad), alphavirus, herpes simplex virus (HSV), poxvirus, Newcastle disease virus, poliovirus, measles virus, rhabdovirus, coxsackie virus, and vaccinia virus, are being actively investigated . A genetIn recent years, various strategies have been investigated to prevent rapid recognition and clearance of systemically administered Ad by the host immune system and its nonspecific sequestration into liver tissues . One of In this review, we highlight the advancements in polymer-complexed Ad delivery systems, aiming to explore how different types of polymers and targeting strategies are being used to overcome the limitations of conventional Ad. In addition, the review attempts to provide future directions in the design of efficient polymer-Ad vectors, briefly discussing the challenges toward the clinical development of polymer-Ad formulations, including biosafety and regulatory considerations, and how these challenges can be addressed.N-(2-hydroxypropyl methacrylamide) (pHPMA), and cationic polymers, have been used to mask the surface properties of Ads -L-glutamate) (PEG-PNLG) copolymers with different PEG molecular weights  and numbers of amine groups (2 or 5) [To further examine the role of cationic polymers in promoting Ad internalization in cancer cells, Choi et al. developed a series of six biocompatible polymers based on PEG-poly((2 or 5) . PEG-PNL(2 or 5) .Lee et al. developed Ad-coated with a cationic polymer, deoxycholic acid-conjugated poly(ethyleneimine) (DA3), to enhance Ad delivery into cancer cells with low CAR expression . The Ad/Numerous cationic polymers such as PEI, poly(lysine), and chitosan and liposomes have been used to complex with various gene therapeutics to enhance their gene delivery efficiency into a wide range of cell lines . Among tg-Arg-S-S-Arg-g-PEI-mPEG copolymer (PPSA) to coat Ad (Ad/PPSA) [For instance, Choi et al. developed a multi-degradable, bioreducible, and core-cross-linked PEI (rPEI) copolymer of varying molecular weights (16 and 32 kDa) as non-toxic and non-immunogenic material to coat the surface of oAd . In bothAd/PPSA) . PhysicoAd/PPSA) . The Ad/Ad/PPSA) . An oAd Ad/PPSA) .Similarly, biodegradable poly(disulfide amine) polymers with defined structures were synthesized using a Michael addition reaction between cystamine bisacrylamide and diamines . To imprRegardless of the bioreducible polymer types, all bioreducible polymers showed reduced toxicity in comparison with conventional 25 kDa PEI. The introduction of poly(disulfides) into the polymers allows rapid degradation of the polymer in the intracellular environment, which contains high concentration of glutathiones that reduce disulfide bonds and release of oAd from the polymer coating  (PEG-b-PHF) was developed to targeting acidic TME and inhibit tumor growth in a U87 tumor xenograft-bearing mice models [b-PHF complex was incubated at pH 6.4 compared to the complex incubated at pH 7.4, demonstrating the pH-responsiveness of the complex. Moreover, KOX/PEG-b-PHF (pH 6.4) elicited an enhanced cancer cell killing effect compared to both naked KOX and KOX/PEG-b-PHF (pH 7.4). Consequently, the systemically administered KOX/PEG-b-PHF complex induced more potent antitumor activity than naked KOX in the U87 tumor xenografts established in mice [b-poly(L-histidine) (PEG-b-pHis) copolymer [b-pHis copolymer was protonated in the acidic pH conditions of tumor tissues and effectively covered the Ad to generate a PEG-b-pHis/Ad complex. In comparison with naked Ad, the transduction efficiency of the PEG-b-pHis/Ad complex at pH 6.4 was increased by 3-, 9-, and 339-fold in U343, A549, and MCF7 cells, respectively. The therapeutic efficacy of the mPEG-b-pHis/Ad complex was significantly enhanced in both CAR-positive (C33A) and CAR-negative (MCF7) tumor models, showing 57 and 49% tumor growth inhibition in comparison with naked Ad, respectively. Collectively, these results demonstrate that PEG-b-pHis-mediated delivery of Ad is greatly augmented by acidic TME.A pH-responsive moiety often is used to enhance the tumor-targeting ability of the polymer because TME is mildly acidic where normal physiological pH is close to neutral . A pH-seopolymer . The Ad The introduction of bioreducible linkers provides greater pH sensitivity and improves the release of coated Ad in the intracellular compartment of cancer cells. For instance, Moon et al. synthesized a pH-responsive and bioreducible copolymer (PPCBA) to coat the surface of oAd . Owing tOther than passive targeting, introducing tumor-targeting moieties such as ligands and antibodies can actively target various receptors overexpressed on tumor tissues. For instance, Park et al. developed an folic acid (FA) anchored Ad/chitosan-PEG-FA nanocomplex to enable active tumor-targeting and tumor-specific transduction . Increaset al. used an EGFR-specific antibody, Erbitux-conjugated and PEGylated PAMAM dendrimer (PPE), to complex with Ad [9-fold greater than that of naked oAd/DCN-shMet, implying increased tumor accumulation and decreased liver sequestration of oAd/DCN-shMet/PPE. Consequently, systemically delivered oAd/DCN-shMet/PPE showed a 14.9-fold higher level of tumor growth inhibition in an EGFR-positive orthotopic lung tumor model in comparison with naked Ad, ultimately resulting in complete tumor regression in 66% of the treated mice. The pharmacokinetic profile of the oAd/DCN-shMet/PPE complex demonstrated a 290-fold higher viral genome in the blood circulation after 24 h, which implied that effective masking of Ad reduces immunogenicity during circulation and enhances Ad circulation time for tumor targeting. This was the first report demonstrating that the oAd nanocomplex can effectively treat an orthotopic xenograft tumor that closely emulates the disease progression and properties of a clinical tumor [In addition to natural cationic polymers, dendritic polymers with a well-defined structure, size, and surface charge have been explored in gene delivery. Poly(amidoamine) (PAMAM) dendrimers are highly water-soluble and non-immunogenic, and the presence of surface functional groups allows easy modification with drugs and targeting ligands. Yoon  with Ad . EGFR-taal tumor . TogetheCoating of Ad using cationic polymers provides numerous advantages including attenuation of the innate immune response, protection against blood proteins, prolonged circulation in the bloodstream, and increased targeting ability. The safety of cationic polymers can be further improved by the introduction of degradable linkers that can facilitate biodegradation and clearance of the polymers upon cellular internalization. For instance, to redirect cationic polymers containing multiple degradation linkers to tumor tissues, poly(cystaminebisacrylamide-diaminohexane) (CD) copolymer has been modified with a cyclic RGD (cRGDfC) to selectively target the tumor vasculature . Ad compIn summary, Ad complexed with cationic polymers conjugated with active tumor targeting moieties can enhance the antitumor activity of Ad via systemic administration Figs.  and 2. FTo maximize the therapeutic potential of oAds in a clinical setting, major limitations, such as poor systemic administrability and intratumoral retainment of the virus must be addressed. The development of a polymer-based carrier system for oAd is one of the promising approaches to address these inherent limitations of oAd, as these materials can endow novel properties such as low immunogenicity and improve the pharmacokinetics and tumor-targeting ability that are either impossible or difficult to achieve via conventional genetic engineering methods. Masking the oAd capsid using non-immunogenic polymers either by chemical conjugation or physical coating can prolong the circulation time of oAd in the bloodstream, attenuate induction of an antiviral immune response, and curtail nonspecific liver sequestration. Furthermore, inclusion of active tumor-targeting moieties can lead to tumor-specific accumulation of systemically administered oAd complex, simultaneously improving both the therapeutic efficacy and safety profile.Despite remarkable advancement within the field of polymer-based delivery of oAds in the last decades, many aspects that must be carefully investigated prior to its transition into a clinical environment. For example, scale-up studies, improvement of the homogeneity of the complex, optimization of Ad complex generation procedures in a good manufacturing practice (GMP) environment, and more in-depth toxicology studies must be conducted. To this end, new polymerization techniques that can synthesize polymers with defined structures will be useful for bulk synthesis. Therefore, the development of tailored polymers could collectively overcome the limitations oAds and improve tumor-specific accumulation via passive and active targeting to achieve enhanced gene therapy for cancer.


Despite the clinical success of the first bispecific antibody approved by the FDA against B cell malignancies (blinatumomab), many obstacles remain such as dosing, treatment resistance, and modest efficacy in solid tumors. To overcome these limitations, considerable efforts have been dedicated to the development of multispecific antibodies, opening up new avenues to address both the complex biology of cancer and the onset of anti-tumoral immune responses. Simultaneous targeting of two tumor-associated antigens is presumed to enhance cancer cell selectivity and reduce immune escape. Co-engagement of CD3, along with agonists of co-stimulatory molecules or antagonists of co-inhibitory immune checkpoint receptors in a single molecule, may revert T cell exhaustion. Similarly, targeting of two activating receptors in NK cells may improve their cytotoxic potency. And these are only examples of the potential of antibody-based molecular entities engaging three (or more) relevant targets. From the perspective of health care costs, multispecific antibodies are appealing, since a similar (or superior) therapeutic effect could be obtained with a single therapeutic agent as with a combination of different monoclonal antibodies. Despite challenges in production, multispecific antibodies are endowed with unprecedented properties, which may render them more potent biologics for cancer therapy. Finally, sdAb (12-15 kDa) consist of a unique variable domain derived from heavy-chain only antibodies present in camelids (named VHH) or cartilaginous fish (named VNAR). Obviously, the non-human nature of sdAb fragments makes them potentially immunogenic, although humanization approaches have been developed to mitigate this problem. Coding sequences of variable domains can be obtained from IgG with known specificities or by selection in vitro of large Ab libraries using phage, bacterial or yeast display. Of note, scFv and sdAb are easily produced as soluble, properly folded and stable recombinant proteins in bacteria, yeast and mammalian systems, with suitable ranges of affinities Fab fragments (50 kDa), comprise the full-length light (L) chain (VL and CL domains) and VH and CH1 domains of the heavy (H) chain from a conventional IgG, connected by an interchain disulfide bond. On the other hand, scFv are moieties of 25-30 kDa, which contain only VH and VL domains joined by a flexible polypeptide linker, usually (GGGGS)2 BsAb format, consists of two scFv fused in a single polypeptide chain by a five-amino acid linker Multimerization of these building blocks enables the modification of antibody size, shape, and valence to optimize half-life and tumor penetration. For instance, the tandem scFv or (scFv)On the other hand, IgG-like formats comprise molecules that partially or completely resemble natural antibodies, including a Fc region . Designing Fc-based TsAb facilities dimerization, purification and promotes stability and extended half-life. Most of IgG-like formats consist of asymmetric chains, which include modifications in CH3 domains (such as the knob into hole -KIH- technology) in order to enforce the heterodimerization ot two different heavy chains. The correct pairing of light chains is a challenge that is addressed with different strategies. However, a percentage of non-functional molecules or undesirable homodimers can still cause impurities. BsAb in IgG format may gain a third specificity by appending an antibody fragment to the N- or C-terminal ends of heavy or light chains. However, the large molecular weight of IgG-like TsAb formats hampers tumor penetration, although guarantee long serum half-lifes.The main application field of TsAb remains oncology, with at least one of the three specificities intended to bind T or NK cells, and at least one targeting a tumor-associated antigen (TAA) in most designs Immune cell engagers are molecules able to redirect immune effector cells (regardless of their antigen specificity) against cancer cells with the aim of triggering efficient tumor cell killing, acting as a bridge between immune cells and target cells Beyond increasing the avidity and size to overcome fast clearance in small antibody fragment-based constructs, the incorporation of a second in-cis binding antibody moiety targeting a different TAA expressed on the same tumor cells offers additional advantages. For example, dual TAA-targeting may contribute to prevent tumor escape by antigen loss caused by selective pressure in comparison to conventional single TAA-targeting TCE and may help to overcome antigenic heterogeneity., Schoonjans et al. described in 2000 a TsAb format, which they refer to as tribody, constituted by the fusion of two scFv against different TAA (BCL1 and hPLAP) to an anti-CD3 Fab, rendering an intermediate sized construct with a molecular weight around 100 kDa In a pioneering studyin vitro was inferior to that of the BsAb in vitro against CD19+ and/or CD22+ tumor cells and demonstrated significantly enhanced antitumor efficacy in a patient-derived xenograft (PDX) model of B-ALL, compared with the corresponding BsAb alone or in combination. Remarkably, leukemia relapse was observed in all animals, independently of the initial response, except in the group treated with the TsAb.Subsequently, dual TAA-targeting TsAb have been designed for leukemic cell killing, as an evolution of the CD19/CD3 BiTE (Bispecific T-cell Engager) blinatumomab, a CD3-based tandem scFv. This BsAb has shown remarkable efficacy in patients with B-ALL, although the durability of the response is limited, and the relapse rate is ~50% after 12-18 months. Interestingly, around 30% of blinatumomab-relapsed cases are characterized by CD19 negative leukemic cells Leukemia cells with mixed B/myeloid origin are found in a small percentage and usually have poor prognosis. To deal with this condition, a CD33/CD3/CD19 TsAb, termed triplebody, was designed. Triplebodies consist of three scFv in tandem fused in a single polypeptide chain, with a molecular weight around 80-90 kDa. The two distal binding domains recognize different TAA, while the central domain recruits effector T cells. The CD33/CD3/CD19 triplebody showed efficient lysis of different ALL and AML cell lines, with enhanced selectivity for CD19+ CD33+ double-positive cells over CD19+ with comparable antigen density in vitro, whereas PD-L1+ non-AML cells were spared. Moreover, the CiTE construct achieved complete AML eradication in a PDX model without immune-related adverse effects.Fine-tuning affinity in TAA-binding domains can help to further increase tumor selectivity. Another TsAb designed for AML treatment, named CiTE (Checkpoint Inhibitory T cell-Engaging) Ab, combined T cell redirection to CD33 on AML cells with PD-L1 blockade on the same cells in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo compared to control BsAb. To avoid systemic toxicity, it has been proposed that a dual-TAA targeting BsAb should preferentially bind to malignant cells rather than normal cells if the affinity of the individual binding domains are sufficiently low as to require the presence of both target antigens (through the avidity effect) for efficient binding In another work, a TsAb for dual targeting in solid tumors, termed trispecific T-cell engager (TriTE), was designed. This construct consists of a CD3-specific scFv flanked by an anti-epidermal growth factor receptor (EGFR) sdAb and an anti-epithelial cell (EpCAM) sdAb Recently, Liu and coworkers reported a thorough strategy to determine the most suitable fusion site of two VHH (anti-HER2 and anti-VEGFR2) to an anti-CD3 Fab for optimal antigen recognition. The resulting TsAb induced not only a more durable growth inhibition of double-positive tumors compared with the combination of corresponding BsAb, but also overcame tumor heterogeneity and elicited tumor regression in mice coimplanted with HER2+ or VEGFR2+ tumors in each flank. Moreover, the TsAb-based treatment inhibited tumor growth in a PC3 tumor model resistant to the approved mAb trastuzumab (anti-HER2) and ramucirumab (anti-VEGFR2). These results further supported the rationale that TAA dual targeting can improve the therapeutic index of TsAb in vitro for the killing of ovarian Antigen-independent stimulation of T cells with canonical bivalent anti-CD3 mAb was demonstrated decades ago. On the other hand, monovalent CD3 binding, as it occurs with TCE comprising a single CD3-targeting domain, can induce T cell activation only upon crosslinking to TAA on targets cells. However, this must be accompanied by co-stimulatory signals for optimal activation and T cell proliferation. Furthermore, activated T cells, in turn, express inhibitory checkpoint receptors to control the immune response. Hence, the design of trispecific TCE including agonists of co-stimulatory molecules or antagonists of checkpoint receptors, along with the anti-CD3 and anti-TAA moieties, constitutes an appealing strategy. Such molecules, comprising an agonist of the co-stimulatory receptor CD28 (TAA/CD3/CD28), were expressed as single-chain polypeptides (in scFv-sdAb-sdAb or scFv-scFv-sdAb formats) and characterized et al.in vitro, far above the potency of daratumumab (the anti-CD38 mAb approved for the treatment of MM). Similarly, the TsAb was superior in a humanized mouse model of MM, even at the lowest dose tested.However, it was the study by Wu in vitro was observed at CD38/CD3/CD28 TsAb concentrations far below serum levels well tolerated in non-human primates (NHP). The authors argue that monovalent CD28 binding by the TsAb is less prone to induce cytokine release than the bivalent anti-CD28 IgG used in the above-mentioned trial. This TsAb, coined SAR442257, is currently in a phase I clinical trial with MM patients which eventually will dilucidate if safety concerns are justified The use of CD28 agonists is associated to the risk of severe side effects due to cytokine release since the 2006 TeGenero clinical trial A second TsAb HER2/CD3/CD28, based on the same platform, has recently joined it in clinical trials. In a humanized breast cancer model, this TsAb mediated effective tumor regression, even in low HER2-expressing tumors, suggesting a potential benefit to breast cancer patients who are not candidates for current anti-HER2 therapies A third subtype of trispecific TCE comprises binding domains against a single TAA and a single T-cell activating receptor, along with a moiety for increased serum half-life. TsAb based on small Ab fragments, such as scFv and sdAb, usually exhibit molecular weights below the renal filtration cut-off, despite their multimeric nature. In fact, the 55-kDa BiTE blinatumomab has a serum half-life of only 2 hours and, therefore, continuous intravenous infusion is required The inclusion of albumin-binding sdAb is a widely used half-life extension strategy for small biotherapeutics. Albumin has a serum half-life of 3 weeks because of its size and FcRn-mediated recycling, properties shared with canonical IgG antibodies For example, the single-chain TriTAC (Trispecific T-cell Activating Construct) format, with a molecular weight of approximately 53 kDa, comprises a N-terminal humanized anti-TAA sdAb and a C-terminal humanized anti-CD3 scFv, separated by an anti-albumin sdAb which provides TriTAC with extended serum half-life. The serum levels of the anti-PSMA TriTAC HPN424 revealed a half-life of about 3.4 days in NHP The MATCH3 (Multispecific Antibody-based Therapeutics by Cognate Heterodimerization) technology allows the assembly of three binding domains by fusion of a single-chain diabody to a scFv (scDb-scFv). NM21-1480 (PD-L1/CD137/HSA) combines in such a construct the ability to target CD137-mediated T cell costimulation to PD-L1 overexpressed in the tumor microenvironment (avoiding liver toxicity which hampers anti-CD137 IgG) and simultaneously blocking the PD-1/PD-L1 axis. Combination of HSA binding with a molecular weight around 80 kD endows these molecules with a suggested plasma half-life in humans of up to 2 weeks ex vivo proliferation of T cells from NSCLC patients and to suppress tumor growth in vivo. In addition, NHP pharmacokinetics suggested a potential weekly clinical administration, which is a favorable regimen considering the small size of these molecules A similar strategy is exploited by the Humabody CB307, with three fully human sdAb targeting PSMA, CD137 and HSA (molecular weight <50 kDa), currently in phase I trial for the treatment of advanced PSMA+ tumors. CB307 functions as a CD137 agonist selectively in the presence of PSMA+ cancer cells, thereby enabling tumor-specific T cell activation and decreasing the risk of systemic side effects. Based on the same technology, CB213 is a tetravalent trispecific Humabody engaging simultaneously PD1 and LAG3 on T cells and HSA. CB213 showed superior activity compared to an anti-PD1 mAb to induce in vivo tumor inhibition However, non-covalent HSA binding by sdAb is susceptible to competition and displacement by endogenous ligands and other albumin binding drugs. In contrast, the genetic fusion of drugs to recombinant HSA offers the opportunity to control half-life by the inclusion of albumin sequences with different FcRn binding affinities Multispecific molecules targeting one (or two) activating NK cell receptors and a TAA constitute another type of ICE, developed to enhance tumor cell recognition and killing by NK cells As in TCE, concurrent binding to two different TAA has also been implemented in NKCE antibodies to increase their therapeutic window. Addition of an extra anti-CD19 scFv to a BsAb CD19/CD16 rendered a construct with a molecular weight around 90 kDa termed single-chain Fv triplebody Another CD16-directed trispecific, but tetravalent antibody format, termed aTriFlex, has been generated for the redirection of NK cell cytotoxicity to two multiple myeloma antigens. This format consists of an anti-CD16 monospecific diabody with two scFv in both ends, one anti-BCMA and another anti-CD200. Dual-targeting of multiple myeloma cells resulted in enhanced selectivity and 17-fold increase in potency in vitro compared to the parental BsAb IgG-like TsAb in this section include a sdAb-based SEEDbody, generated using the Strand-Exchanged Engineered Domains (SEED) heterodimerization technology, by replacement of the VH and VL regions of a conventional antibody by two different sdAb domains and grafting a third sdAb onto the hinge region of the second heavy chain Full activation of NK cells to induce anti-tumor immunity has been shown to require the co-engagement of different cell-surface receptors. Based on this consideration, a series of molecules which bind a TAA and trigger two NK cell activation receptors have been designed.et al. reported the characterization of a trifunctional NKCE consisting of two Fab antibody fragments targeting NKp46 and a TAA , separated by an Fc domain to promote ADCC via CD16 in vitro, with no off-target cytotoxicity. In Raji B lymphoma models, mice treated with Fc-silenced NKCE or trifunctional NKCE controlled disease better than the group treated with the mAb rituximab, but the trifunctional construct was significantly more potent and outperformed a mixture of reagents activating NKp46 and CD16 separately. In a related work, trifunctional NKCE were designed targeting CD19 or CD20 and triggering either NKp46 or NKp30, aimed to the treatment of pediatric B-ALL In vitro experiments demonstrated efficient NK cell-mediated killing by all NKCE of leukemia cell lines and primary blasts, including the NK cell-resistant MHH-CALL-4. Moreover, a clinical study using an anti-NKp46-based ANKET targeting CD123 (IPH6101/SAR443579) is ongoing in patients with AML (NCT05086315). Another trifunctional ANKET (IPH6401/SAR'514), directed against BCMA, has recently been selected for clinical development.The Antibody-based NK cell Engager Technology (ANKET) platform has generated a range of promising NKCE targeting different tumor antigens. In a pioneering work, Gauthier A different platform for the development of multifunctional NKCE has been named TriNKET (Trispecific NKCE Therapies). DF1001 is one of several agents belonging to this platform, which targets HER2 and coengages CD16 and NKG2D et al.TriKEs (Trispecific Killer cell Engagers) are not strictly speaking TsAb, since they bind a TAA with one arm and CD16 with the other, while their third moiety is IL-15, which activates the recruited NK cells via interaction with the corresponding receptor. Nevertheless, since they are antibody-based, trifunctional molecules, they will be considered here, as proposed by Elsiathy in vitro. In a HL-60 mouse tumor model, the TriKE also showed enhanced antitumor activity relative to BiKE. Interestingly, TriKE-treated animals had substantial numbers of circulating human NK cells three weeks after, when they were hardly detectable in untreated or BiKE-treated animals. A phase 1 clinical trial (NCT03214666) with this TriKE, named GTB-3550, demonstrated absence of severe side effects, expansion of NK cells and clinical effect in patients with AML, with significant reduction in the numbers of leukemic blasts in the bone marrow The first IL-15-based TriKE reported in the literature was an evolution of a previously characterized BiKE (Bispecific Killer cell Engager) based on a tandem scFv CD33/CD16 Second generation TriKE were based on a humanized anti-CD16 sdAb, since the camelid CD16-engaging moiety provided further potency than the corresponding scFv Very recently, it has been reported a second-generation TriKE with a novel mechanism of action, directed against the stroma-associated antigen TEM8  In this section, we will review TsAb which do not bridge tumor and effector cells, as they recognize in-cis antigens expressed on the surface of the same cell.HH fused in the C-terminal end of TsAb has been previously described by different groups Small molecule EGFR tyrosine kinase inhibitors are used for the treatment of NSCLC driven by EGFR mutations, but resistance frequently appears due to different mechanisms such as cMET amplification. The BsAb amivantamab, which recognizes EGFR and cMet and blocks their respective signalling pathways, was approved in 2021 for the treatment of NSCLC with EGFR exon 20 insertion mutations. The incorporation of a third binding domain to an in-cis targeting TsAb could potentially increase its efficacy. Indeed, a HER2/EGFR/cMet TsAb has been obtained using Fab interface designs to induce correct chain pairing, denoted OrthoTsAb. TsAb were designed with varied geometries, and overall, every construct demonstrated strong trispecific binding although their therapeutic potential was not addressed Another example of multiple tumor targeting is the IgG-like TriMAb directed against EGFR, IGF1R and cMet or HER3 In vitro, the TsAb blocked phosphorylation of both receptors and inhibited the proliferation of NSLC cells with different EGFR mutations. Significant in vivo anti-tumoral efficacy of GB263T was shown in several tumor models of EGFR exon 20 insertion, EGFR C797S mutation, cMET amplification, and cMET exon 14 skipping mutation. Currently, GB263T is under clinical testing in a phase 1 study (NCT05332574) A different approach is under the design of GB263T, an IgG-like TsAb recognizing EGFR and two different cMET epitopes, with enhanced ADCC function. On the other hand, ephrin (Eph) receptors constitute another family of tyrosine kinase receptors less explored as therapeutic targets, with EphA2, EphA4, and EphB4 found overexpressed in different types of carcinoma An interesting approach could be the use of TsAb-based immunoconjugates for tumor delivery of drugs, toxins or radioisotopes, potentially increasing selectivity and therefore their therapeutic window.et al. the generation of a TsAb combining antagonists of PD-1 and CTLA-4 with a CD137 agonist was reported, although it was not functionally characterized Immunomodulatory mAb include immune checkpoint inhibitors and agonists of costimulatory receptors. Given the nonredundant signaling of the respective pathways, it is conceivable that combinations of agonist and antagonist antibodies could synergize to obtain higher response rates Combinations of three different binding moieties in a single molecule offer a wide variety of new therapeutic options. A good example is the TsAb HER2/FAP/mPEG (scFv/scFv/Fab format) for conversion of mPEG (methoxypolyethylene glycol)-coated liposomes containing doxorubicin to immunoliposomes directed towards HER2+ breast cancer cells and tumor-associated fibroblasts (TAF) expressing FAP (fibroblast activation protein) If antibody engineering allows the assembly in a single functional molecule of three different binding domains, adding a fourth one would not be a cumbersome task. Indeed, tetraspecific Ab (TtsAb) are not a future perspective any longer, but a reality. So real that some of them have reached clinical testing.in vivo relative to combinations of BsAb, and they did so at lower antibody concentrations enabled by higher-avidity binding of tumor cells.One of the most interesting platforms for this purpose is named CrossMAb. This technology is based on the crossover of VH and VL or CH1 and CL domains in one Fab arm of a Bs IgG to promote the correct assembling of light chains, along with the KIH technology to enforce the heterodimerization of heavy chains Another format amenable to TtsAb is coined MATCH4, which can be applied to produce homogeneous and stable antibody-derived molecules The Tetramab concept is based on the combination of single-chain Fab and Fv fragments in an IgG antibody format enhanced by KIH technology in vitro, although not significant differences were observed among them.Tetraspecific NKCEs also come in different formats. The DuoBody (DB)-VHH incorporates VHH with different specificities attached to the heavy chain C-terminal ends of a bispecific, Fc-silent IgG An evolution of TriKE format is TetraKE, which has been described to simultaneously engage EpCAM and CD133 for targeting carcinoma cells and cancer stem cells, along with a CD16-engaging moiety and IL-15 for NK cell expansion Furthermore, a new type of multifunctional molecule based on the ANKET technology (named ANKET4) incorporates an IL-2 variant, binding IL-2 receptor β and γ chains, to NKp46 and CD16 co-engagement and targeting of a single TAA. ANKET4 induced NK cell proliferation and accumulation at the TME and had a higher anti-tumor efficacy than approved therapeutic antibodies targeting the same TAA. In NHP, a CD20-directed ANKET4 resulted in sustained CD20+ B-cell depletion with minimal systemic cytokine release and no clinical sign of toxicity On the other hand, the TriTECM platform enables the fusion of trispecific TCE to an anti-IL6R scFv antibody in order to mitigate the CRS mainly promoted by IL6. It is well known that on-target T cell activation is associated with release of cytokines that can potentially result in CRS, being this one of the major safety concerns intrinsic to the mode of action of TCE. Several EGFR/PD-L1/CD3/IL6R TriTECM candidates were generated by fusing one or two anti-IL6R scFv to the TsAb and ensuring the heterodimerization using KiH technology. In both cases, TriTECM antibodies inhibited simultaneously and dose-dependently EGFR and IL6R signaling pathways and triggered efficiently T cell activation and cytotoxicity against EGFR+ cells. In addition, TriTECM-mediated antitumor T cell activity was attenuated in comparison with EGFR/PD-L1/CD3 TsAb variants, indicating TriTECM potential as TCE with cytokine release modulator activity A search in clinicaltrials.gov as of November 2022 renders at least three tetraspecific TCE: GNC-038 (CD3/41BB/PD-L1/CD19), being tested in phase 1 trials with patients with NHL (NCT04606433) and DLBCL (NCT05192486); GNC-035 (CD3/41BB/PD-L1/ROR1) in patients with breast cancer; and GNC-039 (CD3/41BB/PD-L1/EGFRvIII) in glioma (NCT05160545).An alternative to Ab-based constructs is the use of non-immunoglobulin scaffolds as building blocks Another interesting approach to widen the therapeutic window of TsAb is the use of conditionally active TCE, administered as inactive prodrugs, and designed for prolonged release (to avoid CRS) or tumor-restricted activation (to avoid on target-off tumor side effects) Other strategies include proteolytic cleavage sites in the TCE design to take advantage of the enhanced protease activity in the TME, as the ProTriTAC platform. An alternative to the masking peptide has been named COBRA  where anti-CD3 VH and VL domains are forced to pair with inactive VL and VH domains in a diabody-like structure. Anti-EGFR and anti-HSA sdAb were added to the N- and C-terminal ends in a two-chain or single chain configuration Another EGFR/CD3/HSA trispecific construct named tumor-activated T cell engager (TRACTr) Finally, TwoGATE (Two component Guided Antibody Tumor Engager) platform consists of two HLE BsAb each targeting a different TAA and forming an active anti-CD3 moiety only when both bind to a cancer cell. Each of the CD3-targeting split paratopes are associated with an inactivating domain cleaved by tumor-specific proteases in the TME.In all approaches described above, the HLE domain  is lost upon proteolytic activation, reducing the risk of systemic side effects if the active form of the TCE is released from the tumor.The development of complex therapeutics such as TsAb and TtsAb offers important challenges in terms of manufacturability. Some constructs may suffer from compromised stability, resulting in unfolding and aggregation. In addition, production of multichain multispecifics may be hampered by non-desired by-products arising due to incorrect assembly. Good manufacturing practice-compliant processes should be optimized to solve these problems, although in some cases production yields for TsAb were found to be better than for BsAb in vivo data, and overall, they reveal the growing interest in the TsAb field that has blossomed in recent years. Indeed, multispecific antibodies are expanding the toolbox of antitumor reagents given their improved ability to target the multifactorial nature of cancer.Many of the studies commented in this review are only proof-of-concept, focusing mainly on the design, expression, and preliminary functional characterization of TsAb, but a portion of them offer compelling Specially interesting will be the demonstration of the TsAb therapeutic effect in non-hematological cancers compared to leukemias or lymphomas, currently a challenge for more advanced clinical trial-stage strategies such as BsAb and CAR-T cells, hampered by the lack of suitable single target antigens, intratumoral heterogeneity and the hostile tumor microenvironment promoting exclusion and/or exhaustion of recruited effector cells. It can be expected that TsAb, in their variety of flavors, will be better equipped to deal with these hurdles.Ongoing early-stage clinical trials, most of them in patients with solid tumors, will provide relevant information in the next future on the added value of TsAb compared to the plethora of immunotherapies in development.


The rapid rollout of COVID-19 vaccines in 2021 sparked general optimism toward controlling the severe form of the disease, preventing hospitalizations and COVID-19-associated mortality, and the transmissibility of SARS-CoV-2 infection . HoweverSince December 2021, the Omicron variant, whose spike protein highly diverges from previous viral strains, spread aggressively worldwide, also among vaccinated individuals, rapidly becoming the dominant variant by January 2022 ,7. AlthoThe mild clinical presentation of Omicron progressively shifted public health attention from the containment of morbidity to the prevention and control of SARS-CoV-2 infection. With a rapidly mutating virus heading toward become endemic, herd immunity by mass vaccination proved ineffective yet costly for providing long-standing protection against community transmission of SARS-CoV-2. However, harmless treatments that are easy to administer in outpatient settings were immediately indicated to be critically important since the early phase of the pandemic for controlling the transmissibility of SARS-CoV-2 from patients affected by mild–moderate disease ,15,16.Off-label therapies have been recommended or tested before and even after COVID-19 vaccines became available to tackle the saturation of hospital beds and shortage of healthcare force. Several studies, conducted predominantly in vitro, tested the efficacy of different active compounds in the early phase of infection, as post-exposure prophylaxis to reduce viral shedding time (VST) and mitigate the disease progression ,17,18. SARS-CoV-2 enters the human body predominantly through the nasal cavity, where the virus first infects the multi-ciliated cells of the nasopharynx or the sustentacular cells of the nasal olfactory mucosa . Aerosolp = 0.0005) in vitro, followed by cetylpyridinium chloride . Mouthwashes with 0.07% cetylpyridinium chloride completely inactivated different SARS-CoV-2 variants  up to the limit of detection in suspension assays [A systematic review and meta-analysis of 33 published studies (11 in vivo and 22 in vitro) investigated the virucidal efficacy of various compounds as mouth rinses and nasal sprays to reduce the salivary load of SARS-CoV-2 [Following povidone–iodine, chlorhexidine was the most efficacious intervention used to reduce SARS-CoV-2 salivary viral load in vivo, with a mean difference in viral load of 72% for the former versus 86% for the latter . HoweverAnother compound tested against SAR-CoV-2, both in vitro and in vivo, is hydrogen peroxide, an antiseptic solution yielding hydroxyl free radicals reacting against membrane lipids and other essential cell components of micro-organisms ,24. It wFurther inhaling agents proposed against SARS-CoV-2 during the course of the pandemic included alcohol-based preparations and acetic acid ,25. EthaWhilst emerging evidence from in vivo studies using hydrogen peroxide, cetylpyridinium chloride, and various other active agents remains inconclusive, povidone–iodine and chlorhexidine mouth rinses are confirmed to be the most efficacious clinical interventions to reduce the oral load of SARS-CoV-2, regardless of their concentration. Routine use of mouth rinses of povidone–iodine and chlorhexidine in asymptomatic or uninfected individuals may therefore greatly contribute to the containment of VST in patients infected by SARS-CoV-2, especially in health care settings . However, all compounds mentioned above, including povidone–iodine and chlorhexidine, are not physiological substances, and thus tolerability in real life may be an issue, especially in routinely administered nasal formulations. For instance, hypothyroidism has been linked to exposure to povidone–iodine antiseptics in neonates, and transient hyper-thyrotropinemia was reported in infants whose mothers were exposed to povidone–iodine as a skin disinfectant ,28,29,30Furthermore, despite several antiseptics reducing SARS-CoV-2 load by 3–4 log10 in 15–30 s in vitro , all labTherefore, supporting the aspecific physiological defenses of human airways against the spread of a highly mutating virus such as SARS-CoV-2 compels us to look at natural agents already part of innate defences of human airways’ mucosae.Edinburgh and Lothians Viral Intervention Study (ELVIS) tested hypertonic saline nasal irrigation and gargling against other types of coronaviruses in a randomized controlled clinical trial, reporting reduction of VST by 2.6 days in patients treated with hypertonic saline lavages [Regressed Nasal Infectivity and Shedding of SARS-CoV-2 by Achieving Negativization for COVID-19 Earlier (RE.NA.I.S.S.A.N.C.E.) clinical trial recently tested in vivo the virucidal activity of an existing formulation of sweater plus additives (xylitol and panthenol and lactic acid) sprayed in the nasal cavity of patients with mild–moderate COVID-19 infected by Omicron, to reduce the respective VST. In the latter study COVID-19 patients treated with a nasal spray of seawater turned negative an average of two days earlier compared to controls if treatment was administered within the first 5 days following COVID-19 diagnosis [One of these candidates proposed and tested for nasal disinfection against SARS-CoV-2 due to its intrinsic health safety is hypertonic saline solution . Hyperto lavages . HoweverAlthough saline solutions are known to be harmless, the over-production of HOCl in the nasal cavity may generate some irritation to the local epithelium in real life application. −), produced in the human airways from three components [Another candidate considered for nasal administration against SARS-CoV-2 infection is hypothiocyanite (OSCNmponents :H2O2 + SLactoperoxidase (LPO), secreted by goblet cells and serous cells of the submucosal glands;−), released by duct cells of submucosal gland;Thiocyanate anion (SCN2O2), produced by epithelial cells of the airways.Hydrogen peroxide , fungi and viruses [− showed an aspecific strain-independent virucidal activity likely to be effective against any SARS-CoV-2 variants [A recent study tested the virucidal activity of enzyme-free OSCNtoferrin . The exaplicated . In partcated [− . At micr viruses ,39. Sincvariants ,41.− could eradicate an early nasal settlement of SARS-CoV-2, preventing also the downward progression of infection to the lungs [Whilst highly present in the airways epithelium, the LPO system is almost absent in the pulmonary parenchyma . Aerosolhe lungs .− in vivo, since also the above experiment in vitro employed Vero cells [− against SARS-CoV-2 infection should not encounter ethical issues, since the reagent is part of the physiological defences of human airways against the threat of pathogens; it already overcame a phase 1 clinical trial and did not show any cytotoxicity in vitro [− was extemporaneously produced via a two-step bio-catalytic pathway, removing enzymes from the solution by ultrafiltration with a single-use dialysis micromodule. Enzyme-free OSCN− is featured by a high intrinsic reactivity, thus it persists for a limited amount of time (15 minutes) in an environment, implying some limitations in real-life aerosol nasal applications [However, clinical trials on humans are needed to confirm the effect of OSCNro cells . A cliniin vitro ,18,38,43ications .N-chlorotaurine (NCT) is another natural oxidant belonging to the aspecific physiological defences of human airways, yielded from HOCl and taurine amino acid :HOCl + T−, NCT has a recognized broad-spectrum activity against bacteria, fungi, parasites, and viruses. The ciliary beat frequency of epithelial cells of the nasal mucosa, a very sensitive parameter for cytotoxicity, only moderately and reversibly decreased following exposure to 1% NCT, hence NCT is eligible to be applied in sensitive body districts as endogenous disinfectant [Similar to OSCNnfectant .− or NCT or both, with the view of supporting the innate aspecific defences of human airways against SARS-CoV-2 and any future respiratory pathogens, responding to criteria of wide-spectrum virucidal efficacy, health safety, tolerability and cost-effectiveness.Taken together, the above evidence narrows down the attention on a nasal formulation including hypertonic saline solution combined with either SCN− into OSCN− and, separately, taurine into NCT, two natural oxidants less reactive though less toxic than HOCl [The highly reactive HOCl, overproduced by nasal administration of hypertonic saline solution in fact oxidizes SCNhan HOCl ,18,46. T


Therefore, identifying novel diagnostic and prognostic biomarkers for these patients is urgently needed. Predictive markers and therapeutic strategies for tamoxifen-resistant ER+ breast cancer are not clear, and micro (mi)RNAs have recently become a focal research point in cancer studies owing to their regulation of gene expressions, metabolism, and many other physiological processes. Therefore, systematic investigation is required to understand the modulation of gene expression in tamoxifen-resistant patients. High-throughput technology uses a holistic approach to observe differences among expression profiles of thousands of genes, which provides a comprehensive level to extensively investigate functional genomics and biological processes. Through a bioinformatics analysis, we revealed that glutamine synthetase/glutamate-ammonia ligase (GLUL) might play essential roles in the recurrence of tamoxifen-resistant ER+ patients. GLUL increases intracellular glutamine usage via glutaminolysis, and further active metabolism-related downstream molecules in cancer cell. However, how GLUL regulates the tumor microenvironment for tamoxifen-resistant ER+ breast cancer remains unexplored. Analysis of MetaCore pathway database demonstrated that GLUL is involved in the cell cycle, immune response, interleukin (IL)-4-induced regulators of cell growth, differentiation, and metabolism-related pathways. Experimental data also confirmed that the knockdown of GLUL in breast cancer cell lines decreased cell proliferation and influenced expressions of specific downstream molecules. Through a Connectivity Map (CMap) analysis, we revealed that certain drugs/molecules, including omeprazole, methacholine chloride, ioversol, fulvestrant, difenidol, cycloserine, and MK-801, may serve as potential treatments for tamoxifen-resistant breast cancer patients. These drugs may be tested in combination with current therapies in tamoxifen-resistant breast cancer patients. Collectively, our study demonstrated the crucial roles of GLUL, which provide new targets for the treatment of tamoxifen-resistant breast cancer patients.Although adjuvant tamoxifen therapy is beneficial to estrogen receptor-positive (ER National Center for Health Statistics, the incidence and mortality of cancer increases with an estimated 1,9 million newly diagnosed cases and 600,000 deaths nationwide in 2022 GLUL), also named glutamine synthetase (GLNS), is the enzyme responsible for the de novo biosynthesis of glutamine from glutamate and ammonia in an ATP-dependent reaction in vitroGLUL regulates the immune microenvironment and cancer development through the inflammatory pathways are largely lacking.Glutamate-ammonia ligase (Micro (mi)RNAs, a class of small single-stranded RNAs, belong to the noncoding (nc)RNA family which functions in inhibiting protein translation or degrading transcripts by binding to the 3'-untranslated region (UTR) of messenger (m)RNAs. In particular, recent studies report that miRNAs contributed to the progression of breast cancer after tamoxifen treatment. The mechanisms include the activation of estrogen receptor alpha (ERα), progression of the cell cycle, regulation of apoptosis, and controlling epithelial-to-mesenchymal transition + breast cancer using both high-throughput technology and wet-lab approaches. Microarray-based mRNA and miRNA expression profiles of tamoxifen-resistant (TR) and tamoxifen-sensitive (TS) ER+ breast cancer patients were extracted from relevant datasets available at the Gene Expression Omnibus (GEO), followed by DEMs analyses and DEGs analyses, prediction of downstream target genes, and subsequently gene regulatory network to determine potential biomarkers associated with tamoxifen resistance. Finally, wet lab experiments were performed to validate the preceding analytical results.Therefore, our study aimed to provide a comprehensive understanding of the mechanisms involved in tamoxifen-resistant ER2). GLUL gene silencing was generated using a small hairpin (sh)RNA knockdown vector system. All of the shRNA vectors harboring puromycin and enhanced green fluorescent proteins, including two respective recombinant encoding human GLUL shRNA, and one non-target control (pLKO.1) shRNA against luciferase (shLuc), were constructed by the National RNAi Core Facility . The MCF-7 cells were seeded into six-well plates 24h prior to being incubated with three respective lipofectamine and shRNA vectors for 24h, in RPMI-1640 medium containing polybrene (8µg/mL). During an additional 48 hours of lipofectamine transfection, cells were subsequently maintained in a regular medium containing 10% FBS, and stable clones expressing shRNA were selected by constant treatment with puromycin (2µg/mL) from 72 hours onward. A non-target control (pLKO.1) shRNA against luciferase (shLuc) was employed as an expression control. At day 28 post-transfection with lipofectamine, the efficacy of gene silencing was further confirmed by a quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and Western blotting in two populations of shGLUL-transfected MCF-7 cells relative to vector control group.The MCF-7 human epithelial breast cancer cell line, characterized by the presence of the ER, PR, and glucocorticoid receptors, was a kind gift from Prof. Chun Hei Antonio Cheung of National Cheng Kung University . MCF-7 cells were maintained in RPMI-1640 complete medium  supplemented with 10% fetal bovine serum  plus 1% penicillin/streptomycin , and were kept inside a humidified incubator under typical condition  prior to being subjected to complementary synthesis using a PrimeScript Synthesis Kit , with triplicate determinations. The RT-qPCR was performed using GoalBio SYBR green master mix  on a Roche Light Cycler 96 platform. Primer pair sequences targeting GLUL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were provided by Origene  and were constructed by MDBio, Inc. The relative fold changes in expression of the GLUL gene were calculated by the delta-delta Ct (2-∆∆Ct) method after being normalized against the mRNA level of GAPDH as the housekeeping gene.Total RNA extracted from stable clones of shGLUL-transfected MCF-7 and shGLUL-transfected MCF-7 cells were subjected to Western blotting following the manufacturer's protocol . For subsequent analyses, an anti-GLUL (GTX109121) rabbit polyclonal antibody (polyAb)  and anti-GAPDH (GTX124502) rabbit polyAb (GeneTex) were employed as primary antibodies.Total protein extractions derived from cell lysates of stable clones of shFor the colony-formation assay, MCF-7 cells of the three experimental groups were respectively seeded at the low density (1000 cells/well) into six-well plates for 2~3 weeks until macroscopic colonies had formed. The medium was discarded, and cells were fixed with absolute methanol fixation (20 min at room temperature), followed by an incubation with 2% methylene blue staining. The number of proper colonies formed was counted by a stereomicroscope (×100), and experiments were performed in triplicate data.+ and/or PR+ breast cancer patients treated with tamoxifen, regardless of their clinical outcomes (with or without recurrence), were collected from the GEO database. Differential mRNA and miRNA expression profiles of primary and relapsed ER+ breast tumors following tamoxifen treatment were extracted from two groups of datasets, GSE9893 combined with GSE7378, and GSE46823 combined with GSE83292 p-value less than 0.05. A list of significant DEGs was imported into the Ingenuity Pathway Analysis (IPA) and MetaCore platform to construct related biological networks, biological processes (BPs), and diseases, with significant cutoff points for the enrichment of a pathway or an annotated gene group set to p<0.05 as mentioned above + breast cancer patients compared to TS ones. Finally, four down-regulated miRNAs and five common up-regulated between these two datasets, and then subjected to evaluation of the repressive strength of miRNA binding to its target mRNA, allowing producing target gene prediction. A miRmap score of > 95 was applied as the selection threshold for this analysis In this study, datasets containing molecular profiles of either ERp-value of <0.05 and an NES of >1.5 were set as thresholds The Search Tool for the Retrieval of Interacting proteins (STRING) is a huge database consolidating more than twenty billion interactions among approximately sixty-seven million proteins of nearly fourteen thousand organisms +) T cells, and CD8+ T cells) along with myeloid lineages ) in the tumor microenvironment (TME) compared to adjacent normal tissues, using the DiffExp module with default parameters. Expression scatterplots between a pair of given genes in specific cancer types were constructed according to the Spearman correlation and estimated statistical significance, adjusted for tumor purity if necessary Tumor Immune Estimation Resource (TIMER) provides comprehensive and systematic analyses of tumor-infiltrating immune cells across various cancer types, specifically six immune cell populations across 31 tumors from The Cancer Genome Atlas (TCGA). In other words, this web server helps estimate differences in infiltration levels of lymphoid lineages , using a one-way analysis of variance (ANOVA) and Tukey's multiple-comparison test. A We identified the differential expressions of mRNAs and miRNAs between TR and TS patients using miRtest, and evaluated individual miRNAs and their predicted target genes in the same analysis for verifying consistency. The up and down-regulated mRNAs and miRNAs were analyzed using numerous bioinformatics tools. The IPA was used to find upstream and downstream networks of miRNAs and mRNAs, STRING to build up PPI networks, and DAVID to investigate miRNA- and mRNA- associated functions and pathways. The miRWalk2.0, miRmap, and TargetScan were used to predict targets of the upregulated and downregulated miRNAs and to screen potential miRNA-mRNA interactions Figure .+ TR breast cancer. Multiple oncogenes and tumor suppressor genes are contained in this canonical pathway, including G-protein-coupled receptors (GPCR) signaling, RAS/integrin signaling, AKT signaling, transforming growth factor (TGF)-β/bone morphogenetic protein (BMP) signaling, WNT signaling, Notch and Hedgehog (Hh) signaling, and death receptor signaling . The resulting three clusters with a core cluster containing all genes were related to cancer progression and metastasis.The potential genes for miRNA-mRNA interactions in TR patients were identified in the previous section, and the potential pathways and networks were explored in this section. The potential genes targeted by miRNA in GSE46823 and GSE83292 datasets were uploaded to the IPA. The canonical pathway was shown in Figure For comprehensive exploration, we utilized a public database, the Gene Set Enrichment Analysis (GSEA), to verify the importance of targeted pathways in tamoxifen-treated primary breast cancer patients compared to controls. Upregulated genes were analyzed to retrieve the Gene Ontology enrichment results and Kyoto Encyclopedia of Genes and Genomes (KEGG), including cellular components (CCs), biological process (BPs), and molecular functions (MFs). The potentially regulated networks identified included peptide metabolic process, reactome cell cycle, fatty acid metabolism, adipogenesis, stem cells, epithelial-mesenchymal transition, estradiol response, oxidative phosphorylation, estrogen response late, G2 M cell cycle, glycolysis, IL-4 signaling, TNFA signaling via nuclear factor (NF)-κB, mitotic metaphase, and anaphase  was selected for further analyses , which promote the cell cycle G1/S phase transition. The increasing amount of information regarding the IL-4 pathway proved the importance of IL-4 in regulating metabolic processes. Meanwhile, an evolving tumor microenvironment is a convoluted and continuously growing entity. The configuration of the tumor microenvironment varies among tumor types. Several research reveal the important role of tumor microenvironment in cancer progression. We further evaluated the correlation coefficient between GLUL expression in breast cancer cells and activation of tumor-infiltrating immune cells, including B cells, M1 macrophages, M2 tumor-associated macrophages (M2 TAMs), neutrophils, and dendritic cells. A higher level of correlation coefficient was shown in red color, and these data suggested that GLUL expression was positively correlated with the function of immune cells  staining provided by the Human Protein Atlas database. Staining levels of GLUL were increased in cancer specimens compared to normal breast , and it is currently a recommended drug for metastatic ER+ breast cancer patients. Methotrexate ranked number 25 (from -1 to 1) and is currently one of the cytotoxic agents for adjuvant chemotherapy. Other compounds were potential drugs for ER+ TR patients  and the EGFR  gene DGCR5  gene, along with resistance to apoptosis by activating the nuclear factor-κB (NF-κB) signaling pathway BRCA-related breast cancer In our study, expression levels of four down-regulated miRNAs: hsa-miR-1323, hsa-miR-711, hsa-miR-4287, hsa-miR-650, as well as the five miRNAs, including hsa-miR-532-5p, hsa-miR-1180, hsa-miR-152, hsa-miR-578, hsa-miR-128, were upregulated in combined analyses of GSE46823 and GSE83292 datasets. The prediction software of miRNA-targeting genes showed potential genes in interest. In previous literature, hsa-miR-1323 widely regulate cancer progression and radiotherapy effects + breast cancer patients obtained from the GSE9893 dataset were matched with those from the GSE7378 dataset. We found that multiple oncogenic pathways were linked with tamoxifen resistance in the present study, including WNT, GPCR, RAS, AKT, TGF-β/BMP, Notch, Hedgehog, and death receptor signaling. In particular, mutation of ESR1 gene is a well-known mechanism of tamoxifen resistance and upregulation of WNT signaling is detected in an in vitro model of tamoxifen-resistant breast cancer On the other hand, upregulated mRNAs in TR compared to TS ERGLUL plays an important role in cancer progression. GLUL protein promotes cell proliferation in breast cancer GLUL results in drug resistance in cancer cells GLUL in tamoxifen resistance or recurrence of breast cancer is still absent. Meanwhile, through MetaCore and TIMER analysis revealed that “Immune response_IL-4-induced regulators of cell growth, survival, differentiation and metabolism”, and immune-related pathways were correlated with GLUL expression in breast cancer patients from TCGA and METABRIC datasets. IL-4 is a pleiotropic cytokine regulating cell differentiation, survival, and proliferation. IL-4 stimulates cell population proliferation through the activation of several Cyclin-dependent kinases (CDKs), which promote the transition of the cell cycle G1/S phase. IL-4 is also important in the regulation of metabolic processes. Specifically, IL-4/JAK/STAT6 signaling enhances the anabolic actions of insulin and is involved in the metabolism process for different types of cells. These shreds of evidence were consistent with our data and GLUL may regulate tamoxifen resistance or the recurrence of breast cancer. Meanwhile, in addition to GLUL, we also found several potential candidate genes showing high expression in tamoxifen-treated recurrence breast cancer patients, which may serve as ideal targets for the treatment of breast cancer (Figure Activation of RAS/PI3K/AKT signaling blocks tamoxifen-induced anoikis + breast cancer in vitroin vitro study Through the CMap analysis, we revealed that particular compounds with highly negative correlation is served as potential therapeutic agents for TR breast cancer patients. Fulvestrant and methotrexate are listed as potential drugs for TR patients. Fulvestrant is one of the standard salvage drugs for metastatic ER+ breast cancer patients and further wet-lab validation, our study presented GLUL as a remarkable factor that associated with ER+ breast cancer. Furthermore, we also identified the immune signaling pathways by the top co-expressed genes with GLUL in ER+ breast cancer patients developing tamoxifen resistance. Our results provide a better understanding of the molecular mechanisms leading to tamoxifen resistance in patients undergoing treatment. Finally, computational connectivity map-based production of drugs that can serve as alternative treatments for tamoxifen-resistant ER+ breast cancer.To summarize, through comprehensive bioinformatics analyses of the transcriptome of four datasets of ERSupplementary figures and tables.Click here for additional data file.


Lobectomy was performed and histopathologic examination showed an extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue.With the widespread use of computed tomography (CT), subsolid nodules are more frequently encountered in daily practice. We present the case of a 74-year-old man with a large persistent well-defined subsolid nodule on CT. Although the lesion had a predominant ground-glass appearance on CT, Teaching Point: When large persistent subsolid nodule with a predominant ground-glass aspect shows moderate uptake on 18F-FDG-PET, other possible diagnoses than adenocarcinoma should be kept in mind, including primary pulmonary lymphoma. Primary pulmonary lymphoma is a rare, malignant monoclonal lymphoid proliferation within the lung parenchyma. On imaging it can have different imaging appearances, including single or multiple lesions, nodules and masses, and/or areas of consolidation or ground glass. Whereas subsolid pulmonary nodules most often correlate on histopathology to adenocarcinoma with lepidic growth, in rare cases these lesions may represent primary pulmonary lymphoma.18F-FDG-PET was performed  subsolid nodules are more frequently encountered in daily practice. Subsolid nodules comprise both pure ground-glass and part-solid nodules. The most common diagnosis of persistent subsolid nodules is adenocarcinoma and its precursors : atypicaPrimary pulmonary lymphoma is very rare, accounting only for up to 0.4% of all lymphoma. Most common are non-Hodgkin lymphoma subtypes including extranodal marginal zone lymphoma (EMZL), mucosa-associated lymphoid tissue (MALT) and diffuse large B-cell lymphoma (DLBCL). An association with autoimmune disorders such as Sjögren syndrome, multiple sclerosis, rheumatoid arthritis or systemic lupus erythematosis is known . Clinica18F-FDG-avid tumors [Lymphoma is one of the great mimickers with numerous different imaging features depending on the type of lymphoproliferative disorder. EMZL can present in different ways, both with solitary and multiple lesions. In the majority of cases >70%) lesions are multiple and bilateral . More co4560% lesion


Aim: 2D speckle-tracking echocardiography (2D-STE) has been used to assess cardiac recovery during the COVID-19 patient follow-ups within the pandemic. The novel role of STE in predicting adverse outcomes of COVID-19 has received attention due to its high sensitivity in identifying subclinical myocardial dysfunction. We reviewed the studies on using 2D-STE to assess COVID-19 prognosis. Methods: a literature search was conducted on PubMed and Scopus for eligible articles, 24 of which discussed using prognostic 2D-STE for COVID-19 patients. Results: 2D-STE predicts cardiovascular impairments more rapidly and precisely than conventional echocardiography. The 2D-STE technique presents an independent prognostic factor in COVID-19 infection. Conclusion: 2D-STE could be considered a time-efficient and accurate risk predictor of all-cause mortality in COVID-19 patients. In this review, we have gathered every article that discusses the association between COVID-19 prognosis and speckle-tracking echocardiography, which is a novel, fast and accurate method and does not need expert operators to perform. We have shown that according to the current literature, we can use this imaging technique on the right and left heart ventricles to estimate the prognosis of the patients infected with COVID-19. Initially known as a respiratory tract infection, coronavirus disease 2019 (COVID-19) is a multi-organ inflammatory disease that has led to high mortality and morbidity rates worldwide . MyocardCardiovascular involvement in COVID-19 infection may be due to direct myocardial injury, increased systemic inflammatory response, hypoxia or micro-thrombogenesis resulting from a hypercoagulable state ,5. SeverWe herein aim to examine the role of 2D-STE as novel method in predicting adverse outcomes in COVID-19 patients. We have reported the prognostic value of 2D-STE imaging for each cardiac chamber individually.A review of the scientific literature was performed to investigate available articles focusing on the application of STE in COVID-19 patients as an imaging predicting parameter. A comprehensive literature search was performed on PubMed and Scopus to identify suitable studies that were published until March 2022. Our search strategy is as follows: Initially, a manual search was applied in PubMed. The resulting articles were used to obtain the list of keywords and Medical Subject Headings (mesh) terms. The search strategy was repeated multiple times in PubMed and Scopus search engines until the keywords list was completed and no further studies were found. Subsequently, a manual search of the bibliography was performed to find other potentially eligible studies. Search results were screened by title and abstract, and potentially eligible studies were further investigated based on full text. Throughout the review, no language restriction was applied. Data extraction was performed using the double data extraction method by two independent reviewers , followed by a scrutinous review of both included and excluded articles by two other members of the team .We included 24 articles in our study, of which 20 were original, and the remaining 4 were either editorial, letters to the editor, or short communication.  The sampSeveral strains were measured in the left ventricle, namely the global longitudinal strain or left ventricular longitudinal strain (GLS/LS), free wall longitudinal strain (FWLS), global circumferential strain (GCS), and basal longitudinal strain (BLS).The most common strain measured for the left ventricle was GLS/LS, which appeared across 15 studies. Nine articles reported a significant association among the 13 studies focused on GLS/LS and mortality rate. Any associations other than mortality rate would be unconcluded since current literature lacks sufficient data for other adverse effects .In the right ventricle, the measured strains were as follows: (1) right ventricular longitudinal strain, or global strain, or longitudinal strain (RV-LS/GS/LS); (2) total wall global longitudinal strain (TWGLS); (3) free wall strain, free wall longitudinal strain, or free wall global longitudinal strain (FWS/ FWLS/ FWGLS); The most common of which being RV-LS/GS/LS (12-times) and FWS/FWLS/FWGLS (nine-times).A significant correlation was observed between mortality rates and RV-LS measurements in 5 studies, whereas the other four did not show any associations. In the case of FWS, 6 of 8 articles reported a significant association with mortality rates.LS was measured in both left and right atrium , resulting in a correlation between left atrial longitudinal strain and atrial fibrillation. However, neither left nor right global longitudinal strains were associated with mortality.Multiple factors could exacerbate cardiovascular function in critically ill COVID-19 patients, including systemic inflammatory response, hypoxemia, direct myocardial injury, myocarditis, arterial dysfunction, and pulmonary embolism . EffectiPatients evaluated with 2D-STE benefit from a highly sensitive quantitative approach for both global and regional myocardial function. For instance, GLS has a -20% cut-off value in identifying subjects at risk of developing cardiac events (18). 2D-STE would also allow us to detect subclinical cardiac dysfunction, as previously observed in patients with diabetes mellitus, hypertension, and nonobstructive coronary artery disease. 2D STE may ease the diagnosis of acute myocarditis when cardiac magnetic resonance (CMR) or endomyocardial biopsy (EMB) are unavailable . AlthougThis review highlights the potentially favorable role of 2D-STE in assessing the cardiovascular system in all COVID-19 patients. The current literature implies that left ventricular global longitudinal strain (LV GLS) and free wall global longitudinal strain (RV FWGLS) seem more reliable independent predictors of mortality rate, providing additional prognostic implications over conventional echocardiographic parameters of COVID-19 patients. Several limitations are applied in this new field of imaging in COVID-19 patients. First, there is a limited number of studies on pediatric patients with COVID-19 infection. In two of the literature, the only association examined was between global and regional strain measurements with the severity of multisystem inflammatory syndrome in children (MIS-C) .Moreover, studies have demonstrated the link between co-morbidities such as heart failure, chronic kidney disease, and chronic obstructive pulmonary disease with decreasing strain values . BecauseIt is worth mentioning that since STE detects subclinical myocardial dysfunction and impairment sensitively, it may also be a useful imaging modality for predicting outcomes in other multi-system inflammatory infectious diseases.We have stated that 2D-STE could be considered as an additional risk predictor of all-cause mortality in COVID-19 patients. The significant advantage of 2D-STE is being time-efficient and easy to measure by non-experts. However, the association between 2D-STE results and ICU requirements, intubation rate, hospitalization, and COVID-19 severity has been controversial, according to the present literature.Future research should consider the potential of 2D-STE in pediatric patients more carefully. Moreover, the role of atrial strains _especially left atrium_ in determining COVID-19 outcomes need further research which may lead to promising results. Third, the impact of 2D-STE on prognosing other multi-systemic inflammatory disorders and viral infections with a similar mechanism as COVID-19 may further help us to manage patients other than COVID-19 infection.2D-STE is a novel, highly sensitive, fast, and objective modality that may benefit the management of COVID-19 patients.Twenty-four articles that studied 2d-STE on each heart chamber were included in this review.Thirteen studies focused on the association between measurement of the strain GLS/LS of left ventricle and Mortality rate, nine of which found significant association.In case of the right ventricle, five studies found significant association between RV-LS measurements and mortality.


Redox-active organic materials have emerged as promisingalternativesto conventional inorganic electrode materials in electrochemical devicesfor energy storage. However, the deployment of redox-active organicmaterials in practical lithium-ion battery devices is hindered bytheir undesired solubility in electrolyte solvents, sluggish chargetransfer and mass transport, as well as processing complexity. Here,we report a new molecular engineering approach to prepare redox-activepolymers of intrinsic microporosity (PIMs) that possess an open networkof subnanometer pores and abundant accessible carbonyl-based redoxsites for fast lithium-ion transport and storage. Redox-active PIMscan be solution-processed into thin films and polymer–carboncomposites with a homogeneously dispersed microstructure while remaininginsoluble in electrolyte solvents. Solution-processed redox-activePIM electrodes demonstrate improved cycling performance in lithium-ionbatteries with no apparent capacity decay. Redox-active PIMs withcombined properties of intrinsic microporosity, reversible redox activity,and solution processability may have broad utility in a variety ofelectrochemical devices for energy storage, sensors, and electronicapplications. However, thereare growing concerns about these inorganic electrode materials regardingtheir high cost, heavy metal toxicity, environmental pollution, andsophisticated end-of-life recycling. In contrast, redox-active organicmaterials that can reversibly store and release charges have emergedas promising alternatives to inorganic metal oxides owing to theirvarious inherent merits such as chemical diversity, structural tunability,processability, environmental sustainability, and potential low cost.2 Inspired by the ubiquitous redox cofactors  in biological systems, the discovery and development of redox-activeorganic materials have attracted considerable interest for use inbatteries and supercapacitors, photo-/electrocatalysis, and sensors.Redox-active organic molecules often have a high density of redoxsites per mass, which provides high capacity when used as electrodematerials in batteries, such as carbonyl-based small molecules, lithiumrhodizoate3 (580 mAh g–1) and cyclohexanehexone4 (902 mAh g–1). Nevertheless, these molecular species tend to dissolvein electrolyte solvents, such as carbonates and ethers, leading torapid performance degradation and a short lifetime.1 To avoid this dissolution problem, redox-active segmentsare directly polymerized or incorporated as pendant groups into polymerchains/networks to afford insoluble solids.8 However, these redox-active polymersoften pack efficiently in a dense amorphous solid state, so that iontransport and electron transfer are slower relative to their molecularanalogues, resulting in ineffective utilization of redox sites withinthe materials and poor electrochemical performance at the device level.With the rapid developmentof renewable energy and electric vehicles,there are increasing demands for efficient and cost-effective batteriesfor grid storage and transport applications. Lithium-ion batteries(LIBs) dominate the market of portable electronics and electric vehicleswith electrode materials mainly produced from inorganic metal oxides,such as LiCoO10 For example, covalent organic frameworks (COFs) have been extensivelystudied as electrode materials in supercapacitors and metal-ion batteries,13 and show rapid redox processes owing to their ordered ion-transportchannels, enabling high-power batteries.15 However, complicated processing is often required to fabricate thesecrystalline materials into battery electrodes to provide good dispersionand contact between active materials and carbon additives, which arecrucial for mixed ionic-electronic conduction.16 Several fabrication methods have been reported, such asexfoliation of COF solids into nanosheets,17 growth of conductive polymers within COF pores,18 and in situ growth of COF nanosheets on carbon nanotubes15 and graphene.20 These possessing steps add additional complexity and cost to thefabrication process and can be difficult to reproduce or scale up.Hence, it is highly desired to develop redox-active microporous polymersthat can be dissolved in certain organic solvents for ease of processingbut remain insoluble in electrolytes.The recent development of porous organic materials provides extendedchemical space for designing redox-active organic materials with improvedelectrochemical performance. The presence of molecular-sized channelsconstitutes effective pathways for efficient ion diffusion and masstransport, making redox sites more accessible to metal ions and analytes.22 PIMs have shown great promise as next-generation membrane materialsfor molecular separations,25 batteries,29 and fuel cells.30 We anticipate that if incorporated with redox-active structuralunits, PIMs with fast ion-transport channels could be used as high-performanceelectrode materials in electrochemical devices. However, the electrochemicalproperties of PIMs have been rarely investigated, with the exceptionof using PIM-7 as a membrane separator in Li-S batteries.31 We surveyed the library of reported PIMs andnoticed four examples that were initially designed for gas separationapplications but contain a redox-active structural unit, namely thephenazine in PIM-732 and TPIM-133 as well as the conjugated diimide in PMDA-TMDATand NTDA-TMDAT34 . However, a large portion of redox-inactive structuralunits is essential in these PIMs to enforce a rigid, contorted chainstructure, resulting in a low theoretical charge capacity with valuesin the range of 90–130 mAh g–1. The relativelylow density of redox sites represents an obstacle to the immediateuse of these PIMs in electrochemical devices. Although an alternativeapproach toward electroactive PIMs can be achieved via vacuum thermolysisthat produces microporous carbon electrodes for supercapacitor energystorage,35 the intrinsic merits of PIMs are sacrificed during the postsynthetictreatment. Therefore, a molecular redesign is required to maximizethe mass ratio of redox-active units within the rigid, contorted polymerchains to provide a new generation of solution-processable redox-activePIMs.Polymers of intrinsicmicroporosity (PIMs) are solution-processablemicroporous materials with subnanometer channels generated from theinefficient packing of the rigid, contorted polymer chains.2 g–1 and a high theoretical capacity ofup to 248 mAh g–1. Moreover, Li+ iondiffusion is enhanced by the highly interconnected subnanometer poresof redox-active PIMs was selected for polymerization, a rigid diamine monomer featuringa site of contortion is required to avoid an intractable linear polymer.Based on a previous battery electrode work in the literature, a triptycenequinone molecule was selected as the structural unit as it had previouslyshown multielectron redox reactions as high-capacity cathode material.37 Although its redox cycling performance was reportedto be poor due to its partial solubility in battery electrolytes,we anticipated that utilization of this structural unit in redox-activePIMs would simultaneously serve as sites of contortion while providingabundant redox-active functionality.Cycloimidization reaction was chosen asthe polymerization route to redox-active PIMs as the imide linkinggroup possesses robust redox chemistryS1 and S2). Additional experimental details, materials, andmethods are provided in the Supporting Information. To compare PIM-TMTrip-MQ with structurally similar polymers, whichcontain imide and quinone functionalities but with a less microporousor nonporous structure, PI-Trip-MQ and PI-AQ were prepared  owingto its rigid and contorted chain structure that reduces interchaincohesion and facilitates the absorption of solvent, enabling the fabricationof mechanically robust films by solution casting .The incorporation of redox-active sites in both the structural unitand the linking group provides high theoretical capacity values of189 mAh g–1 for PIM-TMTrip-MQ, 209 mAh g–1 for PI-Trip-MQ, and 248 mAh g–1 for PI-AQ basedon a reversible four-electron redox reaction.The target redox-activePIM polymer (PIM-TMTrip-MQ) was synthesized using a diamine monomerbased on triptycene monobenzoquinone that contains tetramethyl groupsto prohibit rotation around the C–N single bond . In contrast, PI-Trip-MQ displayed negligible nitrogen uptake andan apparent BET surface area of only 6.4 m2 g–1. NLDFT pore size distribution derived from the nitrogen sorptionisotherm confirms the absence of any micropores in PI-Trip-MQ thatare accessible to nitrogen. Microporosity is consistent for PIM-TMTrip-MQand PI-Trip-MQ with their hydroquinone precursors . Interestingly, substantialcarbon dioxide (CO2) adsorption at 273K . Importantly, pore size distributionsderived from molecular models agree well with those from the DFT analysisof CO2 sorption (S8). It can be concludedthat a continuous network of interconnected free volume elements  is formed in PIM-TMTrip-MQ owing to its chain rigidityand contortion. In contrast, the absence of any restriction for C–Nbond rotation in PI-Trip-MQ leads to more efficient packing of thepolymer chains, generating predominantly ultramicropores that areonly accessible to smaller carbon dioxide molecules at a higher temperature(273 K) but not to larger nitrogen molecules. In addition, the lowestcarbon dioxide adsorption in PI-AQ that is composed of planar structuralunits further verifies triptycene monobenzoquinone as a porosity-generatingstructural unit in PIM-TMTrip-MQ and PI-Trip-MQ (High uptake of nitrogen at low relative pressureat 77 K indicatesfreely accessible microporosity in PIM-TMTrip-MQ with a high apparentBrunauer–Emmett–Teller (BET) surface area value of 702m2 sorption and molecular simulation, with the most microporousPIM-TMTrip-MQ showing the highest sorption of 36.5 wt % and the nonporousPI-AQ showing the lowest of 19.0 wt % at 273 K and 116 mbar. In additionto the siphoning effect driven by the capillary forces from micropores,solvent percolation in these polyimides of varied microporosity isalso influenced by polymer swelling.Percolationof electrolyte solvents within redox-active materialsis crucial to the formation of ion-transport channels for fast diffusionof metal ions with a short response time during battery charging anddischarging. Methanol was used to probe the accessibility of microporesto organic solvents and assess the corresponding solvent uptake capacity2f. Metha2.2E1/2 values of 2.11, 2.37, 2.64,and 2.84 V vs Li/Li+, respectively . However,in previous work on other diimide polymers, the utilization of allfour carbonyls has been shown to result in structural damage to thepolymer,39 which does not appear to be occurring withinthe voltage range in this study. Similar CV curves as for PIM-TMTrip-MQwere observed in PI-Trip-MQ, but with broader peak widths and largercathodic peak-to-anodic peak separation . Even largerpeak separation was observed in PI-AQ, which showed broad and overlappingpeaks at a scanning rate greater than 0.2 mV s–1. The difference in ΔEp suggestslarger polarization and lower utilization of carbonyl groups in theless porous PI-Trip-MQ and PI-AQ than in PIM-TMTrip-MQ, while thestronger intramolecular electronic interactions for the less porouspolymers may also explain the broad and poorly resolved peaks.Conventionalcoin cells with lithium foil anodes were prepared with cathodes fabricatedfrom slurries of each polymer with carbon black and PVDF binder inanhydrous NMP. Solid-state cyclic voltammetry (CV) measurements showedthat PIM-TMTrip-MQ exhibited four pairs of well-resolved redox peakswith ectively 3a,b. TheSI). Solid-state CV measurementsof the model compounds suggest that the imide units were reduced atless positive potentials of 2.06 and 2.25 V vs Li/Li+ incomparison to the more positive potentials required to reduce triptycenemonobenzoquinone . The molecularelectrostatic potential (MESP) for the bare hydrogen-capped PIM-TMTrip-MQrepeat unit was first calculated to predict the possible Li bindingsites. The surface minima revealed the carbonyl oxygens to be themost preferable sites for lithiation, where areas of high electrondensity were most likely to attract a Li+ ion. Based onthe possible Li binding sites, a conformational search was then performedfor the hydrogen-capped PIM-TMTrip-MQ repeat unit with different numbersof Li . The lithiation pathway was predicted based on the minimumenergy principle, and MESP plots were calculated for each preferredintermediate (Table S1), while the 5th and 6th lithiation of the PIM-TMTrip-MQrepeat unit, theoretically forming the diimide tetra-anions, showedconsiderably less positive predicted reduction potential, providingfurther evidence to support the proposed four-electron redox mechanism.Model compounds 2,6-diphenyl-pyromellitic diimide(DPPMDI) andtetramethyl-triptycene monobenzoquinone (TMTrip-MQ) were preparedto confirm the assignment of CV peaks of PIM-Trip-MQ . FastLi+ ion diffusion in the most microporous PIM is also supportedby its high ionic conductivity as measured with a solution-cast filmusing electrochemical impedance spectroscopy (EIS) with values rangingfrom 1.7 to 2.7 mS cm–1 over the temperature rangeof 30–80 °C . Thedefect-free structure of the films used for the conductivity testwas confirmed by single gas permeation measurements (Table S2). However, we were not able to process PI-Trip-MQand PI-AQ into dense films due to their poor solubility in organicsolvents, precluding the direct comparison of their ionic conductivity.To understand the influence of polymer microporosity on Liurements 3b were aof 0.974 3c, much 2.343 (S15). Time-of-flight secondaryion mass spectrometry (ToF-SIMS) further revealed the heterogeneousspatial distribution of different components in the electrode composites,as visualized by the signals of lithium (Li+) and fluorine(Cs2F+) that correspond to redox-active polymersand PVDF binder, respectively. The spatial distributions of redox-activepolymers and PVDF were complementary to each other, exhibiting scatteredlarge polymer particles (1–10 μm in diameter) embeddedin the PVDF matrix , while the apparent size of the carbon blackparticles was noticeably larger than that in a control sample fabricatedusing only carbon blacks and PVDF binder . ToF-SIMS showed homogeneous spatial distribution ofthe redox-active polymer and PVDF binder across the electrode composite. The cell polarizationremained low throughout the cycling test , while removing carbonadditives from the composite electrode led to the “shut-down”of redox activity . In contrast,some carbonyl polymers are demonstrated with high capacity at highcharge and discharge rates, such as poly5 which delivers 69% of their low-rate capacity(181 mAh g–1) at 20C as well as a lithium salt polymerof dihydroxyanthraquinone46 that displaysthe capacity of 236 mAh g–1 at 10C. The high ratingperformance of these polymers is based on their high electronic conductivity,which is ensured by the π-conjugated structure. To improve electrontransfer within the composite electrode, we further utilized carbonnanotubes (CNT) as conductive additives to construct a more conductingnetwork. The discharge capacity of PIM-TMTip-MHQ/CNT composite electrodewas improved to the range of 80–90 mAh g–1 at 5C from none for the electrode without CNT . Importantly, the solution-processed electrode exhibited a stablecycling performance over 1400 cycles of charge and discharge, furtherconfirming the important role of solution processing in achievinghigh-capacity retention  based onthe differentiation (dQ/dV) of thecharging–discharging profiles, a method widely used as a diagnostictool to investigate battery state of health. The 2nd cycle of chargingshowed four well-resolved peaks in the incremental capacity curveat 2.47, 2.81, 3.07, 3.31 V, respectively, in good agreement withCV measurements. However, the two peaks at lower potentials  underwent a continuing increase in peak area duringcycling, accompanied by a shift of redox potential toward higher values and a decrease in peak area forthe other two peaks. The mechanism for the change of potential plateausduring cycling merits further study.In general, redox-active organic materials haverelatively poorelectronic conductivity aside from doped conductive polymersactivity S12. In cactivity S12. In c2.447), (2) greater electronic conjugationcould be introduced to these microporous polymers, or (3) the conceptof PIMs is extended to other redox chemistries where electronic conjugationis not a prerequisite for high performance, such as organic radicalpolymers.49Reaction kinetics in electrode materials is controlledby ionic transport, electron transfer as well as ionic–electroniccoupling. The influence of ionic transport has been extensively studiedfor porous network polymers, particularly covalent organic frameworks(COFs). As the electron transfer in COFs is usually efficient, superiorrating performance can be achieved through the enhancement of ionictransport. For conventional nonporous conjugated polymers, the highdegree of π-conjugation ensures efficient intrachain electronmovement through delocalized π-orbitals and interchain electronmovement where there is sufficient π–π overlapbetween polymer chain segments. Ion transport in these nonporous polymersmainly relies on electrolyte swelling and could be sluggish for somepolymer chemistries. For redox-active PIMs reported in this work,ionic transport is effectively enhanced by their intrinsic micropores;however, their nonconjugated structure leads to inferior electrontransfer. Consequently, the apparent redox kinetics and rate performanceof redox-PIMs are found poorer than some previously reported COFsand conventional conjugated polymers. Nevertheless, strategies toenhance ionic transport and improve battery lifetime, even when electrontransfer is limited, broaden the scope of the potential materialsthat can be used in energy storage, and may inspire the developmentof better materials that show optimal performance in all aspects ofthe evaluating matrix. We envision that optimal performance wouldbe achieved if (1) redox-active PIMs are employed in systems whereion uptake and transport are more critical . Furtherenhancement of specific capacity could be achieved based on recentprogress in making multielectron redox quinone materials includingpillarquinone macrocycles3+ diffusion and stable cycling performancein lithium-ion batteries. The strategy of designing redox-active PIMswith combined properties of intrinsic microporosity, reversible redoxactivity, and solution processability may inspire the developmentof redox-active polymers with applications in a variety of electrochemicaldevices for energy storage, electrochemical sensors, supercapacitors,and photonic devices.The design and synthesisof redox-active PIMs with accessible microporosityand high specific capacity involves, first, the use of a triptycenebenzoquinone structural unit to achieve a high density of redox-activesites while maintaining the highly rigid and contorted structure,and second, the introduction of tetramethyl groups to provide a sufficientsteric hindrance to restrict rotation about the C–N imide singlebond. Importantly, these redox-active PIMs can be readily solution-processedinto thin films and electrodes with a well-defined microstructure,enabling enhanced Li


Barrett’s esophagus is a major complication of gastro-esophageal reflux disease and an important precursor lesion for the development of Barrett’s metaplasia and esophageal adenocarcinoma. However, the cellular and molecular mechanisms of Barrett’s metaplasia remain unclear. Inflammation-associated oxidative DNA damage could contribute to Barrett’s esophagus. It has been demonstrated that poly(ADP-ribose) polymerases (PARPs)-associated with ADP-ribosylation plays an important role in DNA damage and inflammatory response. A previous study indicated that there is inflammatory infiltration and oxidative DNA damage in the lower esophagus due to acid/bile reflux, and gastric acid could induce DNA damage in culture esophageal cells. This review will discuss the mechanisms of Barrett’s metaplasia and adenocarcinoma underlying oxidative DNA damage in gastro-esophageal reflux disease patients based on recent clinical and basic findings. Barrett’s esophagus (BE) most commonly arises from gastro-esophageal reflux disease (GERD), which is defined as the retrograde flow of gastric and (or) duodenal contents into the esophagus, inducing discomfort symptoms and (or) esophageal mucosal pathological lesions. The incidence rate of GERD varies from 10 to 20% in the Eastern and Western countries . Indeed,GERD is a chronic inflammation of the esophagus stimulated by repeated acid/bile acids. Thus, esophagitis is the major pathological manifestation for patients with GERD and BE . FurtherThe reflux-induced epithelial injury could be repaired by squamous cell regeneration and differentiated columnar epithelium in the distal esophagus. It is reviewed that different types of cells have been proposed to develop intestinal metaplasia during GERD-induced BE by direct or indirect trans-differentiation. Many signaling pathways may also be involved in this process . Columna−/− mice, the stratified squamous epithelium fails to form, while the esophagus is lined by simple columnar epithelium  esophageal insufficiency, esophageal hiatal hernia, leading to gastric acid, pepsin, and bile reflux into the esophagus. Bile salts or hydrochloric acid treatment could increase the levels of ROS, inducing an increase in the levels of 8-hydroxydeoxyguanosine (8-OH-dG) and p-H2AX which are markers of oxidative DNA damage and DNA double-strand breaks, respectively . It is wdiseases . Recent diseases . Since Gdiseases . Both CDdiseases . Correspdiseases . In this+ as the substrate and the negatively charged ADP-ribose (ADP) group is covalently added to the target proteins. The most common target sites for PARylation are the side chains of arginine, aspartic acid, and glutamic acid residues. After catalyzing the addition of the first ADPr onto the target proteins, other ADPrs can be covalently polymerized onto the first ADPr leading to the formation of both linear and branched polymers, called poly(ADP-ribose) (PAR)  polymerase (PARPs) which catalyze poly(ADP-ribose)relation (PARylation) at the sites of damage . These ee) (PAR) . Among te) (PAR) . PARP-1 ll death . Thus, t2O2 or bile acid-induced oxidative damage and cell death. PARP1 also positively regulates the viability of esophageal epithelial cells, which reveals a potential candidate for a therapeutic strategy for BE  Adding extrinsic NF-κB or activating PARP-1/NF-κB signaling to detect the DNA damage repair and inflammatory response between PARP-1 knockout esophageal stem cell lines and wild-type cell lines with induced ROS. So what is the detailed mechanism among NF-κB/∆Np63/EMT in the development of BE and EAC. 3) What is the exact role of PARP-1 in esophageal stem cells, Barrett’s esophageal columnar cells, and adenocarcinoma cells.We speculate that PARP-1/NF-κB signaling and ADP-ribosylation-dependent DNA damage response may be involved in the occurrence of BE and incomplete DNA repair due to low levels of ADP-ribosylation possibly lead to Barrett’s adenocarcinoma. However, there are still many open questions existing in the field that require further studies. 1) The degree of oxidative DNA damage, the level of PARP-1/NF-κB signaling, and NAD