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Derivatives of the potassium-sparing diuretic amiloride are preferentially cytotoxic toward tumor cells relative to normal cells, and have the capacity to target tumor cell populations resistant to currently employed therapeutic agents. However, a major barrier to clinical translation of the amilorides is their modest cytotoxic potency, with estimated IC50 values in the high micromolar range. Here we report the synthesis of ten novel amiloride derivatives and the characterization of their cytotoxic potency toward MCF7 (ER/PR-positive), SKBR3 (HER2-positive) and MDA-MB-231 (triple negative) cell line models of breast cancer. Comparisons of derivative structure with cytotoxic potency toward these cell lines underscore the importance of an intact guanidine group, and uncover a strong link between drug-induced cytotoxicity and drug lipophilicity. We demonstrate that our most potent derivative called LLC1 is preferentially cytotoxic toward mouse mammary tumor over normal epithelial organoids, acts in the single digit micromolar range on breast cancer cell line models representing all major subtypes, acts on cell lines that exhibit both transient and sustained resistance to chemotherapeutic agents, but exhibits limited anti-tumor effects in a mouse model of metastatic breast cancer. Nonetheless, our observations offer a roadmap for the future optimization of amiloride-based compounds with preferential cytotoxicity toward breast tumor cells.

Psoriasis, an immune-mediated inflammatory skin disorder characterized by a chronically relapsing-remitting course, continues to be primarily managed through topical therapy. While oral administration of tyrosine kinase 2 inhibitors (TYK2i) stands as an effective approach for psoriasis treatment, the potential efficacy of topical application of TYK2i remains unexplored. Herein, the carbomer/alginic acid hydrogel is embedded with borneol (BO) as a new topical carrier of TYK2i for achieving enhanced transdermal permeation and anti-psoriasis efficacy. The hydrogel system, i.e., TYK2i-BO-gel, exhibits significantly improved preventative and therapeutic effects in mice models of psoriasiform dermatitis, as evidenced by phenotypical images, psoriasis severity score index (PSI), histology, immunohistochemical staining, and PCR analysis. Remarkably, TYK2i-BO-gel outperforms conventional topical corticosteroid therapy by significantly preventing psoriatic lesion recurrence as measured by a nearly 50 % reduction in ear thickness changes (p

Implantable vascular devices are widely used in clinical treatments for various vascular diseases. However, current approved clinical implantable vascular devices generally have high failure rates primarily due to their surface lacking inherent functional endothelium. Here, inspired by the pathological mechanisms of vascular device failure and physiological functions of native endothelium, we developed a new generation of bioactive parylene (poly(p-xylylene))-based conformal coating to address these challenges of the vascular devices. This coating used a polyethylene glycol (PEG) linker to introduce an endothelial progenitor cell (EPC) specific binding ligand LXW7 (cGRGDdvc) onto the vascular devices for preventing platelet adhesion and selectively capturing endogenous EPCs. Also, we confirmed the long-term stability and function of this coating in human serum. Using two vascular disease-related large animal models, a porcine carotid artery interposition model and a porcine carotid artery-jugular vein arteriovenous graft model, we demonstrated that this coating enabled rapid generation of self-renewable "living" endothelium on the blood contacting surface of the expanded polytetrafluoroethylene (ePTFE) grafts after implantation. We expect this easy-to-apply conformal coating will present a promising avenue to engineer surface properties of "off-the-shelf" implantable vascular devices for long-lasting performance in the clinical settings.

Abstract Derivatives of the potassium-sparing diuretic amiloride are preferentially cytotoxic toward tumor cells relative to normal cells, and have the capacity to target tumor cell populations resistant to currently employed therapeutic agents. However, a major barrier to clinical translation of the amilorides is their modest cytotoxic potency, with estimated IC50 values in the high micromolar range. Here we report the synthesis of ten novel amiloride derivatives and the characterization of their cytotoxic potency toward MCF7 (ER/PR-positive), SKBR3 (HER2-positive) and MDA-MB-231 (triple negative) cell line models of breast cancer. Comparisons of derivative structure with cytotoxic potency toward these cell lines underscore the importance of an intact guanidine group, and uncover a strong link between drug-induced cytotoxicity and drug lipophilicity. We demonstrate that our most potent derivative called LLC1 is preferentially cytotoxic toward mouse mammary tumor over normal epithelial organoids, acts in the single digit micromolar range on breast cancer cell line models representing all major subtypes, acts on cell lines that exhibit both transient and sustained resistance to chemotherapeutic agents, but exhibits limited anti-tumor effects in a mouse model of metastatic breast cancer. Nonetheless, our observations offer a roadmap for the future optimization of amiloride-based compounds with preferential cytotoxicity toward breast tumor cells.

In addition to efficacious vaccines and antiviral therapeutics, reliable and flexible in-home personal use diagnostics for the detection of viral antigens are needed for effective control of the COVID-19 pandemic. Despite the approval of several PCR-based and affinity-based in-home COVID-19 testing kits, many of them suffer from problems such as a high false-negative rate, long waiting time, and short storage period. Using the enabling one-bead-one-compound (OBOC) combinatorial technology, several peptidic ligands with a nanomolar binding affinity toward the SARS-CoV-2 spike protein (S-protein) were successfully discovered. Taking advantage of the high surface area of porous nanofibers, immobilization of these ligands on nanofibrous membranes allows the development of personal use sensors that can achieve low nanomolar sensitivity in the detection of the S-protein in saliva. This simple biosensor employing naked-eye reading exhibits detection sensitivity comparable to some of the current FDA-approved home detection kits. Furthermore, the ligand used in the biosensor was found to detect the S-protein derived from both the original strain and the Delta variant. The workflow reported here may enable us to rapidly respond to the development of home-based biosensors against future viral outbreaks.

Conventional approaches for screening anticancer drugs rely on chemical reactions, which are time consuming, labor intensive, and costly. Here, we present a protocol for label-free and high-throughput assessment of drug efficacy using a vision transformer and a Conv2D. We describe the steps for cell culture, drug treatment, data collection, and preprocessing. We then detail the building of deep learning models and their use to predict drug potency. This protocol can be adapted for screening chemicals that affect the density or morphological features of cells. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Abstract Determining one’s confidence in a decision is a vital part of decision-making. Traditionally, psychological experiments have assessed a person’s confidence by eliciting confidence judgments. The notion that such judgments can be elicited without impacting the accuracy of the decision has recently been challenged by several studies which have shown reactivity effects—either an increase or decrease in decision accuracy when confidence judgments are elicited. Evidence for the direction of reactivity effects has, however, been decidedly mixed. Here, we report three studies designed to specifically make reactivity effects more prominent by eliciting confidence judgment contemporaneously with perceptual decisions. We show that confidence judgments elicited contemporaneously produce an impairment in decision accuracy, this suggests that confidence judgments may rely on a partially distinct set of cues/evidence than the primary perceptual decision and, additionally, challenges the continued use of confidence ratings as an unobtrusive measure of metacognition.

As a measure of cytotoxic potency, half-maximal inhibitory concentration (IC50) is the concentration at which a drug exerts half of its maximal inhibitory effect against target cells. It can be determined by various methods that require applying additional reagents or lysing the cells. Here, we describe a label-free Sobel-edge-based method, which we name SIC50, for the evaluation of IC50. SIC50 classifies preprocessed phase-contrast images with a state-of-the-art vision transformer and allows for the continuous assessment of IC50 in a faster and more cost-efficient manner. We have validated this method using four drugs and 1,536-well plates and also built a web application. We anticipate that this method will assist in the high-throughput screening of chemical libraries (e.g., small-molecule drugs, small interfering RNA [siRNA], and microRNA and drug discovery).

Significant progress has been made in designing bone materials capable of directing endogenous cells to promote vascularized bone regeneration. However, current strategies lack regulation of the specific endogenous cell populations for vascularized bone regeneration, thus leading to adverse tissue formation and decreased regenerative efficiency. Here, we engineered a biomaterial to regulate endogenous cell adhesion and promote vascularized bone regeneration. The biomaterial works by presenting two synthetic ligands, LLP2A and LXW7, explicitly targeting integrins α4β1 and αvβ3, respectively, expressed on the surfaces of the cells related to bone formation and vascularization, such as mesenchymal stem cells (MSCs), osteoblasts, endothelial progenitor cells (EPCs), and endothelial cells (ECs). In vitro, the LLP2A/LXW7 modified biomaterial improved the adhesion of MSCs, osteoblasts, EPCs, and ECs via integrin α4β1 and αvβ3, respectively. In an adult rat calvarial bone defect model, the LLP2A/LXW7 modified biomaterial enhanced bone formation and vascularization by synergistically regulating endogenous cells with osteogenic and angiogenic potentials, such as DLX5+ cells, osteocalcin+ cells, CD34+/CD45- cells and CD31+ cells. In a fetal sheep spinal bone defect model, the LLP2A/LXW7 modified biomaterial augmented bone formation and vascularization without any adverse effects. This innovative biomaterial offers an off-the-shelf, easy-to-use, and biologically safe product suitable for vascularized bone regeneration in both fetal and adult disease environments.

Previous research has shown that eliciting retrospective confidence ratings during problem-solving enhances performance in high self-efficacy individuals but impairs performance in low self-efficacy individuals. In the current study, we examined whether judgments of solvability, a metacognitive rating made prior to participants’ first-order responses are similarly reactive and whether, like retrospective confidence ratings, this effect is moderated by pre-existing self-efficacy. In two experiments, we showed that judgments of solvability are reactive, but this effect is not moderated by pre-existing self-efficacy. We argue that eliciting metacognitive ratings prior to a participant’s response changes metacognitive control, without necessarily activating self-evaluative thoughts. These results are encouraging for the use of judgments of solvability as an education intervention.

Cognitive flexibility is the ability to adapt to changing contingencies and demands over time. It is often assessed by complex problem solving tasks (e.g., microworld simulations), which require individuals to maintain a goal state over trials. Yet typically analyses summarises performance by mean accuracy per person, which assumes performance is a constant and ignores the trial-by-trial trajectory of individuals that reveal strategy . We tested n = 83 psychology students aged 16 to 45 in a microworld task under different conditions of stochasticity and delay. We modelled trial-by-trial performance according to a prospective model of the true task contingencies versus a reactive strategy adapting to feedback from the last trial,. Most individuals (~66%) appeared to adopt a reactive strategy, however when the task contingencies were immediate, people were more likely to act prospectively. Performance in the microworld task depends on the strategy which varies by person and with the task contingencies.

The lack of vascularization associated with deep burns delays the construction of wound beds, increases the risks of infection, and leads to the formation of hypertrophic scars or disfigurement. To address this challenge, we have fabricated a multi-functional pro-angiogenic molecule by grafting integrin αvβ3 ligand LXW7 and collagen-binding peptide (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY (LDS), and further employed this to functionalize collagen-based Integra scaffolds. Using a large deep burn wound model in C57/BLK6 mice (8-10 weeks old, 26-32g, n = 39), we demonstrated that LDS-modified collagen-based Integra scaffolds loaded with endothelial cells (ECs) accelerate wound healing rate, re-epithelialization, vascularization, and collagen deposition. Specifically, a 2 cm × 3 cm full-thickness skin burn wound was created 48 h after the burn, and then wounds were treated with four groups of different dressing scaffolds, including Integra + ECs, Integra + LDS, and Integra + LDS + ECs with Integra-only as the control. Digital photos were taken for wound healing measurement on post-treatment days 1, 7, 14, 21, 28, and 35. Post-treatment photos revealed that treatment with the Intgera + LDS + ECs scaffold exhibited a higher wound healing rate in the proliferation phase. Histology results showed significantly increased re-epithelialization, increased collagen deposition, increased thin and mixed collagen fiber content, increased angiogenesis, and shorter wound length within the Integra + LDS + ECs group at Day 35. On Day 14, the Integra + LDS + ECs group showed the same trend. The relative proportions of collagen changed from Day 14 to Day 35 in the Integra + LDS + ECs and Integra + ECs groups demonstrated decreased thick collagen fiber deposition and greater thin and mixed collagen fiber deposition. LDS-modified Integra scaffolds represent a promising novel treatment to accelerate deep burn wound healing, thereby potentially reducing the morbidity associated with open burn wounds. These scaffolds can also potentially reduce the need for autografting and morbidity in patients with already limited areas of harvestable skin.

Human serum albumin (HSA) is the most abundant protein in human blood plasma. It plays a critical role in the native transportation of numerous drugs, metabolites, nutrients, and small molecules. HSA has been successfully used clinically as a noncovalent carrier for insulin (e.g., Levemir), GLP-1 (e.g., Liraglutide), and paclitaxel (e.g., Abraxane). Site-specific bioconjugation strategies for HSA only would greatly expand its role as the biocompatible, non-toxic platform for theranostics purposes. Using the enabling one-bead one-compound (OBOC) technology, we generated combinatorial peptide libraries containing myristic acid, a well-known binder to HSA at Sudlow I and II binding pockets, and an acrylamide. We then used HSA as a probe to screen the OBOC myristylated peptide libraries for reactive affinity elements (RAEs) that can specifically and covalently ligate to the lysine residue at the proximity of these pockets. Several RAEs have been identified and confirmed to be able to conjugate to HSA covalently. The conjugation can occur at physiological pH and proceed with a high yield within 1 h at room temperature. Tryptic peptide profiling of derivatized HSA has revealed two lysine residues (K225 and K414) as the conjugation sites, which is much more specific than the conventional lysine labeling strategy with N-hydroxysuccinimide ester. The RAE-driven site-specific ligation to HSA was found to occur even in the presence of other prevalent blood proteins such as immunoglobulin or whole serum. Furthermore, these RAEs are orthogonal to the maleimide-based conjugation strategy for Cys34 of HSA. Together, these attributes make the RAEs the promising leads to further develop in vitro and in vivo HSA bioconjugation strategies for numerous biomedical applications.

PurposeImmune checkpoint inhibitors (ICI) in general have shown poor efficacy in bladder cancer. The purpose of this project was to determine whether photodynamic therapy (PDT) with bladder cancer-specific porphyrin-based PLZ4-nanoparticles (PNP) potentiated ICI.Experimental designSV40 T/Ras double-transgenic mice bearing spontaneous bladder cancer and C57BL/6 mice carrying syngeneic bladder cancer models were used to determine the efficacy and conduct molecular correlative studies.ResultsPDT with PNP generated reactive oxygen species, and induced protein carbonylation and dendritic cell maturation. In SV40 T/Ras double-transgenic mice carrying spontaneous bladder cancer, the median survival was 33.7 days in the control, compared with 44.8 (P = 0.0123), 52.6 (P = 0.0054), and over 75 (P = 0.0001) days in the anti-programmed cell death-1 antibody (anti-PD-1), PNP PDT, and combination groups, respectively. At Day 75 when all mice in other groups died, only 1 in 7 mice in the combination group died. For the direct anti-tumor activity, compared with the control, the anti-PD-1, PNP PDT, and combination groups induced a 40.25% (P = 0.0003), 80.72% (P < 0.0001), and 93.03% (P < 0.0001) tumor reduction, respectively. For the abscopal anticancer immunity, the anti-PD-1, PNP PDT, and combination groups induced tumor reduction of 45.73% (P = 0.0001), 54.92% (P < 0.0001), and 75.96% (P < 0.0001), respectively. The combination treatment also diminished spontaneous and induced lung metastasis. Potential of immunotherapy by PNP PDT is multifactorial.ConclusionsIn addition to its potential for photodynamic diagnosis and therapy, PNP PDT can synergize immunotherapy in treating locally advanced and metastatic bladder cancer. Clinical trials are warranted to determine the efficacy and toxicity of this combination.

Low dose non-toxic disulfide cross-linked micelle (DCM) encapsulated paclitaxel (PTX) was found to be highly efficacious as a radiosensitizer against oral cancer preclinical model. Intensity-modulated radiation therapy was locally administered for three consecutive days 24 h after intravascular injection of DCM-[PTX] at 5 mg/kg PTX. DCM-[PTX] NPs combined with conventional radiotherapy (2 Gy) resulted in a 1.7-fold improvement in therapeutic efficacy compared to conventional PTX plus radiotherapy. Interestingly, we found that radiotherapy can decrease tight junctions and increase the accumulation of DCM-[PTX] in tumor sites. Stereotactic body radiotherapy (SBRT) given at 6 Gy was used to further investigate the synergistic anti-tumor effect. Tumor tissues were collected to analyze the relationship between the time interval after SBRT and the biodistribution of the nanomaterials. Compared to combination DCM-[PTX] with conventional radiation dose, combination DCM-PTX with SBRT was found to be more efficacious in inhibiting tumor growth.

Abstract Prior research has established that people use reappraisal to regulate others’ emotions in higher-emotional intensities but use distraction in lower-emotional intensities. However, research has not compared different reappraisal subtypes, such as reconstrual versus minimizing. In three pre-registered studies, participants completed a novel advice-giving task where they selected regulation strategies (distraction, reconstrual, or minimizing) to help a ‘partner’ who was ostensibly experiencing stimuli of differing emotional intensities and types (the partner was, in fact, non-existent). In Experiment 1, participants selected reconstrual over distraction significantly more for low versus high intensity stimuli. In Experiment 2, participants showed no significant preference for minimizing over distraction on low versus high intensity stimuli. In Experiment 3, participants selected reconstrual over minimizing significantly more on low versus high intensity stimuli. Results indicate that previous findings regarding the effect of emotional intensity on ‘reappraisal’ preference are limited to reconstrual and may not generalize to other reappraisal subtypes (i.e., minimizing) which require lower cognitive costs and emotional engagement with the stimuli.

Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. However, the efficacy of ICB therapy is limited by the ineffective infiltration of T effector (Teff) cells to tumors and the immunosuppressive tumor microenvironment (TME). Here, we report a programmable tumor cells/Teff cells bispecific nano-immunoengager (NIE) that can circumvent these limitations to improve ICB therapy. The peptidic nanoparticles (NIE-NPs) bind tumor cell surface α3β1 integrin and undergo in situ transformation into nanofibrillar network nanofibers (NIE-NFs). The prolonged retained nanofibrillar network at the TME captures Teff cells via the activatable α4β1 integrin ligand and allows sustained release of resiquimod for immunomodulation. This bispecific NIE eliminates syngeneic 4T1 breast cancer and Lewis lung cancer models in mice, when given together with anti-PD-1 antibody. The in vivo structural transformation-based supramolecular bispecific NIE represents an innovative class of programmable receptor-mediated targeted immunotherapeutics to greatly enhance ICB therapy against cancers.

Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor-like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on human oolemma and played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.

Tumor heterogeneity poses one of the greatest challenges to a successful treatment of cancer. Tumor cell populations consist of different subpopulations that have distinct phenotypic and genotypic profiles. Such variability poses a challenge in successfully targeting all tumor subpopulations at the same time. Relapse after treatment has been previously explained using the cancer stem cell model and the clonal evolution model. Cancer stem cells are an important subpopulation of tumor cells that regulate tumor plasticity and determine therapeutic resistance. Tumor plasticity is controlled by genetic and epigenetic changes of crucial genes involved in cancer cell survival, growth and metastasis. Targeting epigenetic modulators associated with cancer stem cell survival can unlock a promising therapeutic approach in completely eradicating cancer. Here, we review various factors governing epigenetic dysregulation of cancer stem cells ranging from the role of epigenetic mediators such as histone and DNA methyltransferases, histone deacetylases, histone methyltransferases to various signaling pathways associated with cancer stem cell regulation. We also discuss current treatment regimens targeting these factors and other promising inhibitors in clinical trials.

Diabetic ischemic wound treatment remains a critical clinical challenge. Neovascularization plays a significant role in wound healing during all stages of the tissue repair process. Strategies that enhance angiogenesis and neovascularization and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic conditions. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates vascular endothelial growth factor (VEGF) receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a multi-functional pro-angiogenic molecule by grafting LXW7 and collagen-binding peptides (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY, and further employed this multi-functional molecule to functionalize collagen-based extracellular matrix (ECM) scaffolds. We confirmed that LXW7-DS-SILY modification significantly promoted EPC attachment and growth on the ECM scaffolds in vitro and supported EPC survival in vivo in the ischemic environment. When applied in an established Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model, LXW7-DS-SILY-functionalized ECM scaffolds loaded with EPCs significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

Implantable vascular devices are widely used in clinical treatments for various vascular diseases. However, current approved clinical implantable vascular devices generally have high failure rates primarily due to their surface lacking inherent functional endothelium. Here, inspired by the pathological mechanisms of vascular device failure and physiological functions of native endothelium, we developed a new generation of bioactive parylene (poly(p-xylylene))-based conformal coating to address these challenges of the vascular devices. This coating used a polyethylene glycol (PEG) linker to introduce an endothelial progenitor cell (EPC) specific binding ligand LXW7 (cGRGDdvc) onto the vascular devices for preventing platelet adhesion and selectively capturing endogenous EPCs. Also, we confirmed the long-term stability and function of this coating in human serum. Using two vascular disease-related large animal models, a porcine carotid artery interposition model and a porcine carotid artery-jugular vein arteriovenous graft model, we demonstrated that this coating enabled rapid generation of self-renewable “living” endothelium on the blood contacting surface of the expanded polytetrafluoroethylene (ePTFE) grafts after implantation. We expect this easy-to-apply conformal coating will present a promising avenue to engineer surface properties of “off-the-shelf” implantable vascular devices for long-lasting performance in the clinical settings.

In the tumor micro-environment, tumor associated macrophages (TAMs) represent a predominant component of the total tumor mass, and TAMs play a complex and diverse role in cancer pathogenesis with potential for either tumor suppressive, or tumor promoting biology. Thus, understanding macrophage localization and function are essential for cancer diagnosis and treatment. Typically, tissue biopsy is used to evaluate the density and polarization of TAMs, but provides a limited "snapshot" in time of a dynamic and potentially heterogeneous tumor immune microenvironment. Imaging has the potential for three-dimensional mapping; however, there is a paucity of macrophage-targeted contrast agents to specifically detect TAM subtypes. We have previously found that sulfated-dextran coated iron oxide nanoparticles (SDIO) can target macrophage scavenger receptor A (SR-A, also known as CD204). Since CD204 (SR-A) is considered a biomarker for the M2 macrophage polarization, these SDIO might provide M2-specific imaging probes for MRI. In this work, we investigate whether SDIO can label M2-polarized cells in vitro. We evaluate the effect of degree of sulfation on uptake by primary cultured bone marrow derived macrophages (BMDM) and found that a higher degree of sulfation led to higher uptake, but there were no differences across the subtypes. Further analysis of the BMDM showed similar SR-A expression across stimulation conditions, suggesting that this classic model for macrophage subtypes may not be ideal for definitive M2 subtype marker expression, especially SR-A. We further examine the localization of SDIO in TAMs in vivo, in the mammary fat pad mouse model of breast cancer. We demonstrate that uptake by TAMs expressing SR-A scales with degree of sulfation, consistent with the in vitro studies. The TAMs demonstrate M2-like function and secrete Arg-1 but not iNOS. Uptake by these M2-like TAMs is validated by immunohistochemistry. SDIO show promise as a valuable addition to the toolkit of imaging probes targeted to different biomarkers for TAMs.

AimsOne of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardioprotective effects of DS-IkL after acute myocardial infarction.Methods and resultsIn vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 min of left anterior descending coronary artery ligation-induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischaemia/reperfusion of the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischaemia/reperfusion and 24 h later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls.ConclusionsOur findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

Glioblastoma multiforme (GBM) remains the top challenge to radiotherapy with only 25% one-year survival after diagnosis. Here, we reveal that co-enhancement of mitochondrial fatty acid oxidation (FAO) enzymes (CPT1A, CPT2 and ACAD9) and immune checkpoint CD47 is dominant in recurrent GBM patients with poor prognosis. A glycolysis-to-FAO metabolic rewiring is associated with CD47 anti-phagocytosis in radioresistant GBM cells and regrown GBM after radiation in syngeneic mice. Inhibition of FAO by CPT1 inhibitor etomoxir or CRISPR-generated CPT1A-/-, CPT2-/-, ACAD9-/- cells demonstrate that FAO-derived acetyl-CoA upregulates CD47 transcription via NF-κB/RelA acetylation. Blocking FAO impairs tumor growth and reduces CD47 anti-phagocytosis. Etomoxir combined with anti-CD47 antibody synergizes radiation control of regrown tumors with boosted macrophage phagocytosis. These results demonstrate that enhanced fat acid metabolism promotes aggressive growth of GBM with CD47-mediated immune evasion. The FAO-CD47 axis may be targeted to improve GBM control by eliminating the radioresistant phagocytosis-proofing tumor cells in GBM radioimmunotherapy.

Although stem cell-derived extracellular vesicles (EVs) have remarkable therapeutic potential for various diseases, the therapeutic efficacy of EVs is limited due to their degradation and rapid diffusion after administration, hindering their translational applications. Here, we developed a new generation of collagen-binding EVs, by chemically conjugating a collagen-binding peptide SILY to EVs (SILY-EVs), which were designed to bind to collagen in the extracellular matrix (ECM) and form an EV-ECM complex to improve EVs' in situ retention and therapeutic efficacy after transplantation. Methods: SILY was conjugated to the surface of mesenchymal stem/stromal cell (MSC)-derived EVs by using click chemistry to construct SILY-EVs. Nanoparticle tracking analysis (NTA), ExoView analysis, cryogenic electron microscopy (cryo-EM) and western-blot analysis were used to characterize the SILY-EVs. Fluorescence imaging (FLI), MTS assay, ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to evaluate the collagen binding and biological functions of SILY-EVs in vitro. In a mouse hind limb ischemia model, the in vivo imaging system (IVIS), laser doppler perfusion imaging (LDPI), micro-CT, FLI and RT-qPCR were used to determine the SILY-EV retention, inflammatory response, blood perfusion, gene expression, and tissue regeneration. Results:In vitro, the SILY conjugation significantly enhanced EV adhesion to the collagen surface and did not alter the EVs' biological functions. In the mouse hind limb ischemia model, SILY-EVs presented longer in situ retention, suppressed inflammatory responses, and significantly augmented muscle regeneration and vascularization, compared to the unmodified EVs. Conclusion: With the broad distribution of collagen in various tissues and organs, SILY-EVs hold promise to improve the therapeutic efficacy of EV-mediated treatment in a wide range of diseases and disorders. Moreover, SILY-EVs possess the potential to functionalize collagen-based biomaterials and deliver therapeutic agents for regenerative medicine applications.

Non-small cell lung cancer (NSCLC) is the largest contributor to cancer mortality in the United States. Traditional chemotherapies are toxic and prone to the development of drug-resistance. Recently, several drug candidates were shown to induce lysosomal membrane permeabilization (LMP) in aggressive cancers. This has led to increased interest in lysosome dysregulation as a therapeutic target. However, approaches are needed to overcome two limitations of current lysosomal inhibitors: low specificity and potency. Here, we report the development of a transformable nanomaterial which is triggered to induce LMP of lysosomes in NSCLC. The nanomaterial consists of peptide amphiphiles, which self-assemble into nanoparticles, colocalize with the lysosome, and change conformation to nanofibrils due to lysosomal pH shift, which leads to the disruption of the lysosome, cell death, and cisplatin sensitization. We have found that this cell-penetrating transformable peptide nanoparticle (CPTNP) was cytotoxic to NSCLC cells in the low-micromolar range and it synergized cisplatin cytotoxicity four-fold. Moreover, we demonstrate CPTNP's promising antitumor effect in mouse xenograft models with limited toxicity when given in combination with low dose cisplatin chemotherapy. This is the first example of enhanced LMP via transformable peptide nanomaterial and offers a promising new strategy for cancer therapy.

Self-assembling peptides have shown tremendous potential in the fields of material sciences, nanoscience, and medicine. Because of the vast combinatorial space of even short peptides, identification of self-assembling sequences remains a challenge. Herein, we develop an experimental method to rapidly screen a huge array of peptide sequences for self-assembling property, using the one-bead one-compound (OBOC) combinatorial library method. In this approach, peptides on beads are N-terminally capped with nitro-1,2,3-benzoxadiazole, a hydrophobicity-sensitive fluorescence molecule. Beads displaying self-assembling peptides would fluoresce under aqueous environment. Using this approach, we identify eight pentapeptides, all of which are able to self-assemble into nanoparticles or nanofibers. Some of them are able to interact with and are taken up efficiently by HeLa cells. Intracellular distribution varied among these non-toxic peptidic nanoparticles. This simple screening strategy has enabled rapid identification of self-assembling peptides suitable for the development of nanostructures for various biomedical and material applications.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Abstract Animal pollinators are vital for the reproduction of ~90% of flowering plants. However, many of these pollinating species are experiencing declines globally, making effective pollinator monitoring methods more important than ever before. Pollinators can leave DNA on the flowers they visit, and metabarcoding of these environmental DNA (eDNA) traces provides an opportunity to detect the presence of flower visitors. Our study, collecting flowers from seven plant species with diverse floral morphologies, for eDNA metabarcoding analysis, illustrated the value of this novel survey tool. eDNA metabarcoding using three assays, including one developed in this study to target common bush birds, recorded more animal species visiting flowers than visual surveys conducted concurrently, including birds, bees, and other species. We also recorded the presence of a flower visit from a western pygmy possum; to our knowledge this is the first eDNA metabarcoding study to simultaneously identify the interaction of insect, mammal, and bird species with flowers. The highest diversity of taxa was detected on large inflorescence flower types found on Banksia arborea and Grevillea georgeana. The study demonstrates that the ease of sample collection and the robustness of the metabarcoding methodology has profound implications for future management of biodiversity, allowing us to monitor both plants and their attendant cohort of potential pollinators. This opens avenues for rapid and efficient comparison of biodiversity and ecosystem health between different sites and may provide insights into surrogate pollinators in the event of pollinator declines.

Systemic chemotherapy is efficacious against triple-negative breast cancer (TNBC), but it is often associated with serious side effects. Here, a luteinizing hormone-releasing hormone (LHRH) receptor-targeted and tumor microenvironment-responsive nanoparticle system to selectively deliver chemotherapeutic drugs to TNBC cells, is reported. This delivery system (termed "LHRH-DCMs") contains poly(ethylene glycol) and dendritic cholic acid as a micellar carrier, reversible intra-micellar disulfide bond as a redox-responsive crosslink, and synthetic high-affinity (D-Lys)-LHRH peptide as a targeting moiety. LHRH-DCMs exhibit high drug loading efficiency, optimal particle size, good colloidal stability, and glutathione-responsive drug release. As expected, LHRH-DCMs are more efficiently internalized into human TNBC cells through receptor-mediated endocytosis, resulting in stronger cytotoxicity against these cancer cells than the non-targeted counterpart when encapsulated with paclitaxel (PTX). Furthermore, near-infrared fluorescence and magnetic resonance imaging demonstrate that LHRH-DCMs facilitate the tumor distribution and penetration of payloads in three different animal models of breast cancer, including cell line-derived xenograft (CDX), patient-derived xenograft (PDX), and transgenic mammary carcinoma. Finally, in vivo therapeutic studies show that PTX-LHRH-DCMs outperform both the corresponding nontargeted PTX-DCMs and the current clinical formulation (Taxol) in an orthotopic TNBC model. These results provide new insights into approaches for precise drug delivery of TNBC.

A maytansin derivative, DM1, is a promising therapeutic compound for treating tumors, but is also a highly poisonous substance with various side effects. For clinical expansion, we tried to develop novel peptide-drug conjugates (PDCs) with DM1. In the study, a one-bead one-compound (OBOC) platform was used to screen and identify a novel, highly stable, non-natural amino acid peptide targeting the tyrosine receptor FGFR2. Then, the identified peptide, named LLC2B, was conjugated with the cytotoxin DM1. Our results show that LLC2B has high affinity for the FGFR2 protein according to an isothermal titration calorimetry (ITC) test. LLC2B-Cy5.5 binding to FGFR2-positive cancer cells was confirmed by fluorescent microscopic imaging and flow cytometry in vitro. Using xenografted nude mouse models established with breast cancer MCF-7 cells and esophageal squamous cell carcinoma KYSE180 cells, respectively, LLC2B-Cy5.5 was observed to specifically target tumor tissues 24 h after tail vein injection. Incubation assays, both in aqueous solution at room temperature and in human plasma at 37 °C, suggested that LLC2B has high stability and strong anti-proteolytic ability. Then, we used two different linkers, one of molecular disulfide bonds and another of a maleimide group, to couple LLC2B to the toxin DM1. The novel peptide-drug conjugates (PDCs) inhibited tumor growth and significantly increased the maximum tolerated dose of DM1 in xenografted mice. In brief, our results suggest that LLC2B-DM1 can be developed into a potential PDC for tumor treatment in the future.

Abstract Since mid‐1990s, concerns have increased about a human‐induced “pollination crisis.” Threats have been identified to animals that act as plant pollinators, plants pollinated by these animals, and consequently human well‐being. Threatening processes include loss of natural habitat, climate change, pesticide use, pathogen spread, and introduced species. However, concern has mostly been during last 10–15 years and from Europe and North America, with Australasia, known as Down‐Under, receiving little attention. So perhaps Australasia has “dodged the bullet”? We systematically reviewed the published literature relating to the “pollination crisis” via Web of Science, focusing on issues amenable to this approach. Across these issues, we found a steep increase in publications over the last few decades and a major geographic bias towards Europe and North America, with relatively little attention in Australasia. While publications from Australasia are underrepresented, factors responsible elsewhere for causing the “pollination crisis” commonly occur in Australasia, so this lack of coverage probably reflects a lack of awareness rather than the absence of a problem. In other words, Australasia has not “dodged the bullet” and should take immediate action to address and mitigate its own “pollination crisis.” Sensible steps would include increased taxonomic work on suspected plant pollinators, protection for pollinator populations threatened with extinction, establishing long‐term monitoring of plant–pollinator relationships, incorporating pollination into sustainable agriculture, restricting the use of various pesticides, adopting an Integrated Pest and Pollinator Management approach, and developing partnerships with First Nations peoples for research, conservation and management of plants and their pollinators. Appropriate Government policy, funding and regulation could help.

BackgroundCurrently, there are no effective medications to reverse periodontal disease (PD)-induced bone loss. The objective of this study was to test a new anabolic compound, LLP2A-Ale, or with the combination treatment of mesenchymal stromal cell (MSC), in the treatment of bone loss secondary to PD.MethodsPD was induced in mice by placing a ligature around the second right molar. At one week after disease induction, the mice were treated with placebo, LLP2A-Ale, MSCs, or combination of LLP2A-Ale + MSCs, and euthanized at week 4.ResultsWe found that PD induced alveolar bone loss that was associated with reduced bone formation. LLP2A-Ale alone or in combination with MSCs sustained alveolar bone formation and reversed alveolar bone loss. Additionally, PD alone caused systemic inflammation and increased the circulating levels of G-CSF, IP-10, MIP-1a, and MIP2, which were suppressed by LLP2A-Ale +/- MSCs. LLP2A-Ale +/- MSCs increased bone formation at the peripheral skeletal site (distal femur), which was otherwise suppressed by PD.ConclusionOur findings indicated that LLP2A-Ale treatment rescued alveolar bone loss caused by PD, primarily by increasing bone formation. LLP2A-Ale also attenuated the circulating levels of a series of inflammatory cytokines and reversed the PD-induced suppression of systemic bone formation.

AimsOne of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardio-protective effects of DS-IkL after acute myocardial infarction.Methods and resultsIn vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 minutes of left anterior descending coronary artery ligation induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischemia/reperfusion at the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischemia/reperfusion and 24 hours later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls.ConclusionsOur findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.Translational perspectiveCardiovascular disease remains the leading cause of mortality worldwide. Acute inflammation from myocardial infarction (MI) results in damaged endothelial cells, leading to the loss of glycocalyx. Here, we designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. DS-IkL limits neutrophil binding and platelet activation on inflamed endothelial cells. Treatment with DS-IkL in a preclinical model of MI reduces infarct size by preventing neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis. Our findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

Membrane active peptides (MAPs) have gained wide interest due to their far reaching applications in drug discovery and drug delivery. The search for new MAPs, however, has been largely skewed with bias selecting for physicochemical parameters believed to be important for membrane activity, such as alpha helicity, cationicity and hydrophobicity. Here we carry out a search-and-find strategy to screen a 100,000-membered one-bead-one-compound (OBOC) combinatorial peptide library for lead compounds, agnostic of those physicochemical constraints. Such a synthetic strategy also permits expansion of our peptide repertoire to include unnatural amino acids. Using this approach, we discovered a structurally unique lead peptide LBF14, a linear 14-mer peptide, that induces gross morphological disruption of membranes, irrespective of membrane composition. Further, we demonstrate that the unique insertion mechanism of the peptide, visualized by spinning disc confocal microscopy and further analyzed by electron paramagnetic resonance measurements, may be the cause of this large scale membrane deformation. We also demonstrate the robustness, reproducibility, and potential application of this technique to discover and characterize new membrane active peptides that display activity by local insertion and subsequent allosteric effects leading to global membrane disruption.

Although the efficacy of cancer radiotherapy (RT) can be enhanced by targeted immunotherapy, the immunosuppressive factors induced by radiation on tumor cells remain to be identified. Here, we report that CD47-mediated anti-phagocytosis is concurrently upregulated with HER2 in radioresistant breast cancer (BC) cells and RT-treated mouse syngeneic BC. Co-expression of both receptors is more frequently detected in recurrent BC patients with poor prognosis. CD47 is upregulated preferentially in HER2-expressing cells, and blocking CD47 or HER2 reduces both receptors with diminished clonogenicity and augmented phagocytosis. CRISPR-mediated CD47 and HER2 dual knockouts not only inhibit clonogenicity but also enhance macrophage-mediated attack. Dual antibody of both receptors synergizes with RT in control of syngeneic mouse breast tumor. These results provide the evidence that aggressive behavior of radioresistant BC is caused by CD47-mediated anti-phagocytosis conjugated with HER2-prompted proliferation. Dual blockade of CD47 and HER2 is suggested to eliminate resistant cancer cells in BC radiotherapy.

Activation and migration of endogenous mesenchymal stromal cells (MSCs) are critical for bone regeneration. Here, we report a combinational peptide screening strategy for rapid discovery of ligands that not only bind strongly to osteogenic progenitor cells (OPCs) but also stimulate osteogenic cell Akt signaling in those OPCs. Two lead compounds are discovered, YLL3 and YLL8, both of which increase osteoprogenitor osteogenic differentiation in vitro. When given to normal or osteopenic mice, the compounds increase mineral apposition rate, bone formation, bone mass, and bone strength, as well as expedite fracture repair through stimulated endogenous osteogenesis. When covalently conjugated to alendronate, YLLs acquire an additional function resulting in a "tri-functional" compound that: (i) binds to OPCs, (ii) targets bone, and (iii) induces "pro-survival" signal. These bone-targeted, osteogenic peptides are well suited for current tissue-specific therapeutic paradigms to augment the endogenous osteogenic cells for bone regeneration and the treatment of bone loss.

Distance-dependent magnetic resonance tuning (MRET) technology enables the sensing and quantitative imaging of biological targets in vivo, with the advantage of deep tissue penetration and fewer interactions with the surroundings as compared with those of fluorescence-based Förster resonance energy transfer. However, applications of MRET technology in vivo are currently limited by the moderate contrast enhancement and stability of T1-based MRET probes. Here we report a new two-way magnetic resonance tuning (TMRET) nanoprobe with dually activatable T1 and T2 magnetic resonance signals that is coupled with dual-contrast enhanced subtraction imaging. This integrated platform achieves a substantially improved contrast enhancement with minimal background signal and can be used to quantitatively image molecular targets in tumours and to sensitively detect very small intracranial brain tumours in patient-derived xenograft models. The high tumour-to-normal tissue ratio offered by TMRET in combination with dual-contrast enhanced subtraction imaging provides new opportunities for molecular diagnostics and image-guided biomedical applications.

Establishing and maintaining a healthy endothelium on vascular and intravascular devices is crucial for the prevention of thrombosis and stenosis. Generating a biofunctional surface on vascular devices to recruit endothelial progenitor cells (EPCs) and endothelial cells (ECs) has proven efficient in promoting in situ endothelialization. However, molecules conventionally used for EPC/EC capturing generally lack structural stability, capturing specificity, and biological functionalities, which have limited their applications. Discovery of effective, specific, and structurally stable EPC/EC capturing ligands is desperately needed. Using the high-throughput One-Bead One-Compound combinatorial library screening technology, we recently identified a disulfide cyclic octa-peptide LXW7 (cGRGDdvc), which possesses strong binding affinity and functionality to EPCs/ECs, weak binding to platelets, and no binding to inflammatory cells. Because LXW7 is cyclic and 4 out of the 8 amino acids are unnatural D-amino acids, LXW7 is highly proteolytically stable. In this study, we applied LXW7 to modify small diameter vascular grafts using a Click chemistry approach. In vitro studies demonstrated that LXW7-modified grafts significantly improved EPC attachment, proliferation and endothelial differentiation and suppressed platelet attachment. In a rat carotid artery bypass model, LXW7 modification of the small diameter vascular grafts significantly promoted EPC/EC recruitment and rapidly achieved endothelialization. At 6 weeks after implantation, LXW7-modified grafts retained a high patency of 83%, while the untreated grafts had a low patency of 17%. Our results demonstrate that LXW7 is a potent EPC/EC capturing and platelet suppressing ligand and LXW7-modified vascular grafts rapidly generate a healthy and stable endothelial interface between the graft surface and the circulation to reduce thrombosis and improve patency. STATEMENT OF SIGNIFICANCE: In this study, One-Bead One-Compound (OBOC) technology has been applied for the first time in discovering bioactive ligands for tissue regeneration applications. Current molecules used to modify artificial vascular grafts generally lack EPC/EC capturing specificity, biological functionalities and structural stability. Using OBOC technology, we identified LXW7, a constitutionally stable disulfide cyclic octa-peptide with strong binding affinity and biological functionality to EPCs/ECs, very weak binding to platelets and no binding to inflammatory cells. These characteristics are crucial for promoting rapid endothelialization to prevent thrombosis and improve patency of vascular grafts. LXW7 coating technology could be applied to a wide range of vascular and intravascular devices, including grafts, stents, cardiac valves, and catheters, where a "living" endothelium and healthy blood interface are needed.

The αvβ3 integrin, a receptor for many extracellular matrix proteins with RGD-sequence motif, is involved in multiple physiological processes and highly expressed in tumor cells, therefore making it a target for cancer therapy and tumor imaging. Several RGD-containing cyclic octapeptide (named LXW analogs) were screened as αvβ3 antagonists with dramatically different binding affinity, and their structure-activity relationship (SAR) remains elusive. We performed systematic SAR studies and optimized LXW analogs to improve antagonistic potency. The NMR structure of LXW64 was determined and docked to the integrin. Structural comparison and docking studies suggested that the hydrophobicity and aromaticity of the X7 amino acid are highly important for LXW analogs binding to the integrin, a potential hydrophobic pocket on the integrin surface was proposed to play a role in stabilizing the peptide binding. To develop a cost-efficient and fast screening method, computational docking was performed on LXW analogs and compared with in vitro screening. A consistency within the results of both methods was found, leading to the continuous optimization and testing of LXW mutants via in silico screening. Several new LXW analogs were predicted as the integrin antagonists, one of which-LXZ2-was validated by in vitro examination. Our study provides new insight into the RGD recognition specificity and valuable clues for rational design of novel αvβ3 antagonists.