Specific Target Research Articles

Synthesis and Preclinical Evaluation of Dual-Specific Probe Targeting Glypican-3 and Prostate-Specific Membrane Antigen for Hepatocellular Carcinoma PET Imaging.

Positron emission tomography (PET) is a promising modality for early diagnosis, accurate detection, and staging of hepatocellular carcinoma (HCC). Hereby, a dual-specific probe targeting Glypican-3 (GPC3) and prostate-specific membrane antigen (PSMA) was evaluated for HCC PET imaging. The probe was prepared by conjugating TJ12P2, a GPC3-targeting peptide previously reported by our group, to a highly potent PSMA inhibitor via a polyethylene glycol linker and further tethered to the 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator. The resultant probe, NOTA-TJ12P2-PSMA, abbreviated as T2P, was labeled with gallium-68 and fluorine-18, respectively, and evaluated in murine HCC models of various levels of GPC3 and PSMA expression. Targeting specificity was confirmed by blocking studies. The synthesized [68Ga]Ga-T2P and [18F]AlF-T2P were stable in saline and fetal bovine serum for over 2 h, and bound to their respective targets with high affinity and specificity in cell assays. PET imaging at 60 min postinjection (p.i.) showed that [68Ga]Ga-T2P exhibited higher uptake (1.75 ± 0.16%ID/g) in Huh7 models with high expression of GPC3 and PSMA than gallium-68 labeled TJ12P2 (1.25 ± 0.07%ID/g, p < 0.01) or gallium-68 labeled PSMA-617 (1.07 ± 0.06%ID/g, p < 0.001). The uptake of [68Ga]Ga-T2P in Huh7 tumors was higher than that in PC-3 tumors with low expression of GPC3 or PSMA (0.55 ± 0.24%ID/g, p < 0.01). The uptake of [18F]AlF-T2P or [68Ga]Ga-T2P in the Huh7 tumor was substantially blocked by TJ12P2, TJ12P2 + PSMA, or T2P, but only partially blocked by PSMA. And the PSMA and TJ12P2 monomer blocking effect was less than that of TJ12P2 + PSMA and T2P. [18F]AlF-T2P had higher tumor-to-muscle ratios than [68Ga]Ga-T2P at 90 min postinjection (4.31 ± 0.10 vs 3.80 ± 0.17, p < 0.05) in Huh7 tumor models. To conclude, radiolabeled T2P exhibited a higher uptake and longer retention in Huh7 tumors than its monomeric counterparts. PET imaging via gallium-68 and fluorine-18 labeled T2P showed a similar imaging quality with comparable signal-to-background ratios. Our results demonstrate that T2P is a promising tool for future clinical diagnosis of HCC.

Operational field trialling of Felixer™ grooming traps for the control of feral cats in the Strzelecki Desert, Australia

Context Feral cats (Felis catus) have an impact on native wildlife populations around the world but are difficult to control because of their neophobic behaviours and preference for live prey over scavenging poisoned baits. Felixers™ address these challenges by squirting poison-gel directly onto feral cats, exploiting their fastidious oral grooming tendencies to facilitate poison ingestion. Aims This study trialled Felixers in a landscape-scale arid-ecosystem site to assess their capacity to sustainably manage cat populations in a semi-bounded in situ predator training area and to eradicate cat incursions into a feral-free safe haven. Specifically, the aims were to determine target specificity and firing rates; optimise installation sites to exploit cat behaviour and landscape features; assess the fate of individuals following Felixer interaction; and assess the overall cost, performance and efficacy of Felixers compared with conventional cat control methods. Methods Up to nine Felixers were simultaneously deployed across three trial periods, spanning 30 months (4,642 trap nights) in Sturt National Park in the Strzelecki Desert dunefields of Australia. Felixers were deployed within and outside of feral-free safe havens, at varying cat densities, with populations monitored through camera-trap activity indices and individual behaviour monitored through satellite tracking. Key results Felixers fired at 20.3–43.9% of cats that passed in front (292 of 1,144 cats), with the remainder failing to satisfy the discriminatory algorithm’s target criteria. The devices had 99.93% target specificity from 17,425 interactions with moving animals and objects. Overall cat activity was lower in a semi-bounded 10,400 ha landscape-scale area where Felixers were intensively used, relative to an adjacent unbounded area where no cat control was occurring, over a 12-month subset of the 30-month trial. Felixers also resolved one of two incursions by cats into a 2,000 ha safe haven. Optimisation of Felixer placement and upgrading of software drove improvements in technical reliability and target identification during the trial. Shortcomings included cost and high incidence of technical faults, causing significant downtime and requiring regular investment of field staff time for monitoring and resolution. Conclusions Felixers provide a safe tool for cat management and eradication at intensively managed conservation sites. Efficacy could be improved through reductions in cost and improvements in reliability. Implications Overall Felixers appear to be an effective tool for cat management, when used in an integrated approach.

Enhancing non-viral DNA delivery systems: Recent advances in improving efficiency and target specificity.

DNA-based therapies are often limited by challenges such as stability, long-term integration, low transfection efficiency, and insufficient targeted DNA delivery. This review focuses on recent progress in the design of non-viral delivery systems for enhancing targeted DNA delivery and modulation of therapeutic efficiency. Cellular uptake and intracellular trafficking mechanisms play a crucial role in optimizing gene delivery efficiency. There are two main strategies employed to improve the efficiency of gene delivery vectors: (i) explore different administration routes (e.g., mucosal, intravenous, intramuscular, subcutaneous, intradermal, intratumoural, and intraocular) that best facilitates optimal uptake into the targeted cells and organs and (ii) modify the delivery vectors with cell-specific ligands (e.g., natural ligands, antibodies, peptides, carbohydrates, or aptamers) that enable targeted uptake to specific cells with higher specificity and improved biodistribution. We describe how recent progress in employing these DNA delivery strategies is advancing the field and increasing the clinical translation and ultimate clinical application of DNA therapies.

Elucidating Diurnal Patterns in Touch Desire Using Social Media Data Toward Design of Haptic Applications and Displays.

Advances in haptic technology have led researchers and engineers to seek out killer applications in which users can enjoy an experience of touch in AR/VR spaces. Such applications will respond appropriately to human desire for haptic experiences (i.e., touch desire) and thus it is essential for researchers and engineers to understand the nature of people's touch desires as they arise in the course of daily life. In this study, we employed Twitter data analysis to investigate a diurnal pattern in touch desire. Our results showed that touch desire identified in and extracted from Twitter texts did reveal a diurnal pattern. Touch desire tended to be at its lowest in the morning and increased as the day progressed. The time at which it peaked varied with the specific target of touch desire. Touch desire in relation to other people and objects reached its peak at night, but touch desire in relation to animals reached its peak at noon. These results were confirmed not only by our Twitter text analysis but also by data from other social media and an online survey. In addition, we found that the diurnal pattern of touch desire for each target shows a strong correlation with that of visual desire for the same target. This suggests that the diurnal pattern of touch desire is not limited to the sense of touch but is common to other sensory desires for each target. Our findings suggest that researchers need to take the time of day into account when investigating touch desire. Our findings also offer valuable insights for developers into the design of haptic applications and displays that takes into account the timing of daily peaks in touch desire.

Nano - Based Therapeutic Strategies in Management of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease, progressively distinctive via cartilage destruction, auto-antibody production, severe joint pain, and synovial inflammation. Nanotechnology represents as one of the utmost promising scientific technologies of the 21st century. It exhibits remarkable potential in the field of medicine, including imaging techniques and diagnostic tools, drug delivery systems and providing advances in treatment of several diseases with nanosized structures (less than 100 nm). Conventional drugs as a cornerstone of RA management including disease-modifying antirheumatic drugs (DMARDS), Glucocorticosteroids, etc are under clinical practice. Nevertheless, their low solubility profile, poor pharmacokinetics behaviour, and non-targeted distribution not only hamper their effectiveness, but also give rise to severe adverse effects which leads to the need for the emergence of nanoscale drug delivery systems. Several types of nano-diagnostic agents and nanocarriers have been identified; including polymeric nanoparticles (NPs), liposomes, nanogels, metallic NPs, nanofibres, carbon nanotubes, nano fullerene etc. Various patents and clinical trial data have been reported in relevance to RA treatment. Nanocarriers, unlike standard medications, encapsulate molecules with high drug loading efficacy and avoid drug leakage and burst release before reaching the inflamed sites. Because of its enhanced targeting specificity with the ability to solubilise hydrophobic drugs, it acts as an enhanced drug delivery system. This study explores nanoparticles potential role in RA as a carrier for site-specific delivery and its promising strategies to overcome the drawbacks. Hence, it concludes that nanomedicine is advantageous compared with conventional therapy to enhanced futuristic approach.

Moderately Heavy Atom-Substituted BODIPY Photosensitizer with Mitochondrial Targeting Ability for Imaging-Guided Photodynamic Therapy.

Advanced photodynamic therapy requires photosensitizers with targeting, diagnostic, and therapeutic properties. To fulfill this multifunctionality, we report the synthesis of two triphenylphosphonium (TPP)-functionalized boron-dipyrromethene (BODIPY) dyes, TPPB-H and TPPB-Br, which incorporate a hydrogen atom and dibrominated vinyl moiety at the 6-position of the BODIPY core, respectively. The heavy-atom effect of the moderately heavy bromine atoms allowed TPPB-Br to achieve a proper balance between the toxic singlet oxygen (1O2) production and fluorescence efficiencies. In this dye, the bromine atom-induced stimulation of the singlet-to-triplet intersystem crossing dynamics resulted in an approximately 45-fold increase in the 1O2 quantum yield with respect to that of the nonbrominated counterpart (0.0059 and 0.28 for TPPB-H and TPPB-Br, respectively). This increase was accompanied only a 2-fold reduction in the fluorescence quantum yield (0.54 and 0.22 for TPPB-H and TPPB-Br, respectively). During multicolor confocal laser scanning microscopy observations conducted using two carcinomas, MCF-7 and HeLa, both BODIPY dyes exhibited high targeting specificity toward cancer cell mitochondria owing to the TPP cation functionalization. The two dyes also showed the feasibility of fluorescence cell imaging; however, only the dibrominated BODIPY TPPB-Br manifested pronounced photocytotoxicity with half-maximal inhibitory concentrations of 0.12 and 0.77 μM obtained for MCF-7 and HeLa cells, respectively. These findings demonstrate the potential applicability of TPPB-Br as an imaging-guided photodynamic therapy agent with mitochondrial specificity.

Low-Intensity Continuous Ultrasound Enhances the Therapeutic Efficacy of Curcumin-Encapsulated Exosomes Derived from Hypoxic Liver Cancer Cells via Homotropic Drug Delivery Systems

Exosomes are extracellular nanovesicles secreted by cells that efficiently deliver therapeutic cargo for cancer treatment. However, because exosomes are present in low quantities and have limited target specificity, internal and external stress stimulation has been studied to increase exosome efficiency. Inspired by these studies, the uptake efficiency of cobalt chloride-induced hypoxic cancer cell-secreted exosomes was evaluated. Western blotting and RT-PCR data revealed increased exosome secretion and different protein compositions exhibited by hypoxic exosomes (H-Exos) compared to natural normoxic exosomes (N-Exos). Furthermore, these H-Exos were continuously stimulated using low-intensity ultrasound (LICUS) at an intensity of 360 mW/cm2 and a frequency of 3 MHz in vitro and 1 MHz in vivo. Hyperthermic and mechanical stress caused by ultrasound successfully improved exosome uptake via clathrin-mediated pathways, and confocal laser microscopy showed strong internal localization near the target cell nuclei. Finally, LICUS-equipped H-Exos were loaded with hydrophobic curcumin (H-Exo-Cur) and used to treat parent HepG2 liver cancer cells. The UV–Vis spectrophotometer displayed enhanced stability, solubility, and concentration of the encapsulated drug molecules. In MTT and FACS studies, approximately 40 times higher cell death was induced, and in animal studies, approximately 10 times higher tumor sizes were suppressed by LICUS-assisted H-Exo-Cur compared to the control. In this study, the delivery platform constructed demonstrated enormous potential for liver cancer therapy.

Affinity-tuned mesothelin CAR T cells demonstrate enhanced targeting specificity and reduced off-tumor toxicity.

The application of chimeric antigen receptor (CAR) T cell therapy in solid tumors is hindered by life-threatening toxicities resulting from on-target, off-tumor killing of nonmalignant cells that express low levels of the target antigen. Mesothelin (MSLN) has been identified as a target antigen for CAR T cell treatment of mesothelioma, lung, ovarian, and other cancers because of its high expression on tumor cells and limited expression on mesothelial cells. However, fatal off-tumor toxicity of high-affinity MSLN-targeting CAR T cells has been reported in multiple clinical trials. In this study, we constructed CARs using mutant variants of a single-domain nanobody that bind both human and mouse MSLN with a wide range of affinities and examined tumor responses and their toxicities from on-target, off-tumor interactions in mouse models. CAR T cells with low nanomolar affinity (equilibrium dissociation constant, KD) exhibited profound systemic expansion with no apparent infiltration into the tumor. With a gradual reduction of CAR affinity toward the micromolar KD, the expansion of CAR T cells became more restricted to tumors. Our preclinical studies demonstrated that high-affinity MSLN CARs were associated with fatal on-target, off-tumor toxicity and that affinity-tuned CARs rendered T cells more selective for MSLN-high tumors.

Fast and accurate multi-bacterial identification using cleavable and FRET-based peptide nucleic acid probes

Fast and accurate identification of pathogenic microbes in patient samples is crucial for the timely treatment of acute infectious diseases such as sepsis. The fluorescence in situ hybridization (FISH) technique allows the rapid detection and identification of microbes based on their variation in genomic sequence without time-consuming culturing or sequencing. However, the recent explosion of microbial genomic data has made it challenging to design an appropriate set of probes for microbial mixtures. We developed a novel set of peptide nucleic acid (PNA)-based FISH probes with optimal target specificity by analyzing the variations in 16S ribosomal RNA sequence across all bacterial species. Owing to their superior penetration into bacteria and higher mismatch sensitivity, the PNA probes distinguished seven bacterial species commonly observed in bacteremia with 96–99.9% accuracy using our optimized FISH procedure. Detection based on Förster resonance energy transfer (FRET) between pairs of adjacent binding PNA probes eliminated crosstalk between species. Rapid sequential species identification was implemented, using chemically cleavable fluorophores, without compromising detection accuracy. Owing to their outstanding accuracy and enhanced speed, this set of techniques shows great potential for clinical use.

A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins.

Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and - for AcrIIA5 - indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.

Porous Polymer Sorbents in Micro Solid Phase Extraction: Applications, Advantages, and Challenges.

In recent years, porous polymers have gained significant attention for their application as powerful and selective sorbents in micro solid phase extraction (µSPE). In this review we explore the preparation and utilization of various porous polymer sorbents, highlighting their impact on enhancing µSPE techniques. Molecularly imprinted polymers (MIPs), graphene oxide-modified frameworks, and zeolitic imidazole frameworks (ZIFs) are among the innovative materials discussed. These innovative materials have significantly improved µSPE methods, offering enhanced extraction efficiencies, superior selectivity, and reduced solvent consumption, all of which align with the principles of green chemistry. Key findings of this review include the demonstration that MIPs exhibit excellent target specificity, making them ideal for complex matrices, while graphene oxide frameworks and ZIFs provide increased surface area and stability for diverse analytical applications. Despite these advancements, challenges remain, particularly the high cost of certain innovative materials, limited reusability, and the absence of automation in µSPE workflows. Furthermore, controlling the precise synthesis and functionalization of these sorbents continues to be a limiting factor. To address these issues, future research should focus on developing cost-effectiveness methods, the use of biopolymer or sustainable feedstocks, and scalable synthesis methods; integrating automation into µSPE; and exploring new polymeric materials with enhanced properties. Additionally, novel hybrid materials that combine the strengths of multiple sorbents offer a promising direction for future exploration. We critically analyze the advantages and limitations of each sorbent type, providing a comprehensive overview of their applications in µSPE. This paper also examines the synthesis, characterization, and unique properties of these porous polymers, emphasizing their role in advancing analytical chemistry towards more efficient and environmentally friendly practices. The need for continued development of high-performance, low-cost, and sustainable sorbents is underscored to further enhance the effectiveness of µSPE techniques.

Exploring the correlation between Tom1L1 and the efficacy of neoadjuvant chemotherapy for locally progressive mid-low rectal cancer

ObjectiveTo investigate the specificity of Target of Myb1-Like1 (Tom1L1) expression in colorectal adenocarcinoma tissues and analyze the predictive value of Tom1L1 in the efficacy of neoadjuvant chemotherapy for patients with rectal adenocarcinoma.MethodsThe cancerous tissues and paracancerous normal tissues of 102 patients diagnosed with colorectal adenocarcinoma without treatment were selected; quantitative polymerase chain reaction (qPCR), Western blot and immunohistochemistry (IHC) were adopted to validate the expression level of Tom1L1 in the two groups. Furthermore, 34 patients with locally progressive mid-low rectal adenocarcinoma, who were treated with neoadjuvant Xelox chemotherapy prior to the operation, IHC was adopted to detect the expression of Tom1L1 protein in patients before and after neoadjuvant chemotherapy and to analyze the relationship between the expression level of Tom1L1 and the sensitivity of neoadjuvant therapy.ResultsThe results of qPCR, Western blot and IHC showed that the expression of Tom1L1 in colorectal adenocarcinoma tissues was significantly higher than that in paracancerous normal tissues, with a statistically significant difference (P < 0.01); Neoadjuvant chemotherapy was significantly more effective in patients with low expression of Tom1L1 protein than in those with high expression of Tom1L1 protein, with a statistically significant difference (P < 0.05).ConclusionsTom1L1 is highly expressed in colorectal adenocarcinoma tissues; neoadjuvant Xelox chemotherapy can have an impact on Tom1L1 expression in progressive rectal cancer; patients with locally progressive mid-low rectal adenocarcinoma who have low Tom1L1 expression are more sensitive to neoadjuvant chemotherapy.

Milk-Derived Extracellular Vesicles: A Novel Perspective on Comparative Therapeutics and Targeted Nanocarrier Application.

Milk-derived extracellular vesicles (mEVs) are emerging as promising therapeutic candidates due to their unique properties and versatile functions. These vesicles play a crucial role in immunomodulation by influencing macrophage differentiation and cytokine production, potentially aiding in the treatment of conditions such as bone loss, fibrosis, and cancer. mEVs also have the capacity to modulate gut microbiota composition, which may alleviate the symptoms of inflammatory bowel diseases and promote intestinal barrier integrity. Their potential as drug delivery vehicles is significant, enhancing the stability, solubility, and bioavailability of anticancer agents while supporting wound healing and reducing inflammation. Additionally, bovine mEVs exhibit anti-aging properties and protect skin cells from UV damage. As vaccine platforms, mEVs offer advantages including biocompatibility, antigen protection, and the ability to elicit robust immune responses through targeted delivery to specific immune cells. Despite these promising applications, challenges persist, including their complex roles in cancer, effective antigen loading, regulatory hurdles, and the need for standardized production methods. Achieving high targeting specificity and understanding the long-term effects of mEV-based therapies are essential for clinical translation. Ongoing research aims to optimize mEV production methods, enhance targeting capabilities, and conduct rigorous preclinical and clinical studies. By addressing these challenges, mEVs hold the potential to revolutionize vaccine development and targeted drug delivery, ultimately improving therapeutic outcomes across various medical fields.

Targeting T-cell costimulation to the surface of tumor cells.

Bispecific agents targeting tumor-cell surface antigens and activating receptors on T lymphocytes are being developed for solid tumors. Effective and safe strategies depend on target specificity and at least relative tumor-tissue confinement of T-cell activation. Novel evidence suggests that constructs targeting HER2 on tumor cells with the aim of providing costimulation (signal-2) to T lymphocytes via CD137 (4-1BB) are safe and can meaningfully invigorate antitumor responses in a proportion of patients.

DDEL-02. PRECLINICAL EFFICACY OF IL13RΑ2-TARGETING POLYPEPTIDE-DRUG CONJUGATE (SELF-DEPOTTM ONCOPDC) IN GLIOBLASTOMA AND DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) are currently incurable cancers. Systemic chemotherapy shows limited efficacy because of the intact blood-brain barrier. Convection-enhanced delivery (CED) offers promise by concentrating high doses of chemotherapeutics directly within these tumors. However, drugs administered via CED often face challenges in confirming precise target delivery and are quickly cleared by increased interstitial fluid flow and drug efflux transporters. This highlights the urgent need for innovative drugs that demonstrate target specificity and prolonged retention capabilities. Intrinsically disordered polypeptides (IDPs), derived from tropoelastin, self-assemble into water-insoluble structures with extended retention properties triggered by body temperature. IL13Rα2, differentially amplified in U87MG GBM and SF8628 DIPG cells, serves as a target for therapeutic intervention. An IL13Rα2-targeting IDP (XM161) was developed by incorporating IL13Rα2 binding sequences into the IDP domains. XM161 demonstrated high selectivity in binding to IL13Rα2, with Kd values of 1,786 nM for IL13Rα1 and 12.8 nM for IL13Rα2. Conjugation of XM161 with SN38, a potent topoisomerase I inhibitor, resulted in XM161-SN38, which remained in a dissolved state up to 50°C but formed insoluble aggregates above 27°C in the presence of cerebrospinal fluid. XM161-SN38 exhibited strong cytotoxicity against U87MG and SF8628 cells, with IC50 values of 14.2 nM and 0.48 nM, respectively. Importantly, XM161-SN38 demonstrated efficacy in overcoming Temozolomide resistance in T98G GBM cells, with IC50 values of 3.35 nM compared to 10.87 nM for unconjugated SN38. In orthotopic xenograft models established in BALB/cSlc-nu/nu male mice using U87MG-Luc2 cells, three doses (3 x 0.95 μg SN38 equivalents) of XM161-SN38 delivered via CED were well tolerated and significantly extended median survival to over 65 days, providing a notable survival benefit compared to untreated controls (42 days) and Topotecan-treated group (42.5 days). These findings validate the feasibility and therapeutic potential of XM161-SN38 for GBM treatment. Ongoing studies are underway to investigate the safety and pharmacokinetics of XM161-SN38 in tumor-naive mouse brains.

The molecular basis for functional divergence of duplicated SOX factors controlling endoderm formation and left-right patterning in zebrafish.

Endoderm, one of three primary germ layers of vertebrate embryos, makes major contributions to the respiratory and gastrointestinal tracts and associated organs, including liver and pancreas. In mammals, the transcription factor SOX17 is vital for endoderm organ formation and can induce endoderm progenitor identity. Duplication of ancestral sox17 in the teleost lineage produced the paralogues sox32 and sox17 in zebrafish. Sox32 is required for specification of endoderm and progenitors of the left-right organiser (Kupffer's Vesicle, KV), with Sox17 a downstream target of Sox32 that is implicated in further KV development. Phenotypic evidence therefore suggests functional similarities between zebrafish Sox32 and Sox17 and mammalian SOX17. Here, we directly compare these orthologues and paralogues, using the early zebrafish embryo as a biological platform for functional testing. Our results indicate that, unlike Sox32, human SOX17 cannot induce endoderm specification in zebrafish. Furthermore, using hybrid protein functional analyses, we show that Sox32 specificity for the endoderm gene regulatory network is linked to evolutionary divergence in its DNA-binding HMG domain from its paralogue Sox17. Additionally, changes in the C-terminal regions of Sox32 and Sox17 underpin their differing target specificities. Finally, we establish that specific conserved peptides in the C-terminal domain are essential for the role of Sox17 in establishing correct organ asymmetry. Overall, our results illuminate the molecular basis for functional divergence of Sox32 and Sox17 in vertebrate endoderm development and left-right patterning, and the evolution of SoxF transcription factor function.

CCRG-02. MICRORNA TARGETING LOGIC IN DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMA

Abstract microRNA (miR)-124 is one of the main drivers of mitotic exit during neurogenesis, which is recapitulated in miR-9/9* and -124 (miR-9/9*-124)-mediated direct neuronal reprogramming. In the reprogramming of various cell types, miR-9/9*-124 induce non-neuronal cell fate erasure before activating the neuronal program. Regardless of the different starting transcriptome landscapes, a core gene regulatory network (GRN) is always targeted by miR-9/9*-124, with cell cycle progression as the most significant pathway. We reason that miR-9/9*-124 can induce cell fate erasure in cancerous cells by repressing similar GRNs, using glioblastoma multiforme (GBM) as a paradigm. Mechanistic insights into GBM fate erasure by miR-9/9*-124 while inducing the postmitotic state would facilitate the understanding of GRNs that govern the tumorigenic identity of GBMs and potentially offer novel therapeutic implications. Indeed, miR-9/9*-124 readily induces mitotic exit in patient-derived GBM cell lines, represses GBM cell fates, and promotes neuronal identity over time as assessed by RNA-seq. Mice receiving GBM intracerebral xenografts with miR-9/9*-124 survived significantly longer than those receiving non-specific control miRNA. Global profiling of miRNA targeting activity by AGO2-eCLIP reveals that miR-124 binds to transcripts enriched in G1/S transition and mitogen response. Conversely, miR-506—a miR-124 homolog—despite of sharing identical seed sequences, has significantly less targeting activity and fails to stop cell cycle progression. We are currently dissecting how the difference in the miRNA 3’ sequence renders contrasting biological outcomes. Preliminary data suggests that the length of miRNA alone alters targeting specificity to cell cycle transcripts. However, different permutations of miRNA 3’ sequence, though sufficient to induce mitotic exit, are not all competent to achieve neuronal reprogramming, indicating that mitotic exit and neurogenic activities of miR-124 could be decoupled. We hope that understanding the grammar logic of miRNA targeting would provide deeper insights into how miRNAs can be leveraged to manipulate GBM identity.

Chitosan-Modified Hydrogel Microsphere Encapsulating Zinc-Doped Bioactive Glasses for Spinal Cord Injury Repair by Suppressing Inflammation and Promoting Angiogenesis.

Spinal cord injury (SCI) is a common nerve injury caused by external force, resulting in sensory and motor impairments. Previous studies demonstrated that inhibiting the neuroinflammation promoted SCI repair. However, these approaches are low efficient, and lack targeting specificity, and even require repeated and high doses of systemic administration. To address such issues, in the present study, chitosan-modified hydrogel microspheres encapsulating with zinc-doped bioactive glasses (CS-MG@Zn/BGs) is constructed for targeted repair of SCI. In vitro, the CS-MG@Zn/BGs effectively inhibited the acute inflammatory response initiated by microglia and promoted angiogenic activities. In vivo, CS-MG@Zn/BGs targeted the injured site, and attenuated neuroinflammation by inhibiting microglia infiltration and modulating microglia polarization toward M2 type. Furthermore, it facilitated vascular reconstruction, neuronal differentiation, axonal regeneration and remyelination at the injured site, and thereby promoted motor function recovery of SCI mice. The in vitro and in vivo results implied that CS-MG@Zn/BGs may be a promising alternative for the rehabilitation of SCI.

C2-Linked Arabinose-Functionalized Polystyrene Microbeads Selectively Target Staphylococcus aureus.

Carbohydrates play pivotal roles in the first stages of microbial infections and can be exploited as decoys to hijack the interactions between bacteria and the host cell. Multivalent glycan probes mimicking the natural presentation of glycans in living cells have been successfully employed to study fundamental carbohydrate/protein interactions in microbial systems; however, most pathogenic glycan receptors exhibit a shared specificity for commonly found sugars present in both healthy and pathogenic cells, posing a challenge for target selectivity. In this study, we report the synthesis of a small library of d-arabinose multivalent probes, a sugar absent in human physiology, and their evaluation in a bacteria agglutination assay using cluster analysis. Our findings reveal preferential binding to Staphylococcus aureus of C2-linked arabinose moieties over C1- or C5-linked probes, underscoring the importance of glycan presentation in targeting specificity. Furthermore, we demonstrate the selectivity of the C2-linked probe toward S. aureus across a panel of common bacterial pathogens. Additionally, these probes are able to disrupt biofilm formation in S. aureus SH1000, thereby further proving the cell surface interactions with S. aureus.

Novel Peptide-Based 68Ga-Labeled Radiotracer for Preclinical Studies of TIM3 Expression.

T-cell immunoglobulin and mucin domain-3 (TIM3) is an immune checkpoint that plays a negative regulatory role in the immune response. TIM3-targeted drugs inhibit this negative regulation, thereby modulating the level of immune response activation. In the previous investigation, several peptides targeting TIM3 were identified through screening from a phage peptide library. In this research, three peptides were selected to construct the radioactive molecular probes according to the characteristic that targeting TIM3 drugs would lead to the increase of interferon-γ (IFN-γ) secretion. Molecular docking was performed to assess the binding properties of the selected peptides with the TIM3 protein. To further enhance the targeting properties, one of the peptides with a higher-affinity peptide was structurally modified. Then, 68Ga was used to construct the peptide probe 68Ga-DOTA-peptide by labeling the six peptides with 68Ga riboprobes, and the binding affinity and specificity were assessed using TIM3 overexpressing cell line A549TIM3 and the parental A549 cells. In addition, in Micro-PET/CT imaging, transfected model mice were dynamically imaged for 30 min after injection of 3.7-7.4 MBq 68Ga-DOTA-peptides via the tail vein. Meanwhile, the same dose of molecular probes was injected in the MC38 model (colorectal cancer in mice) and the CCRCC (clear cell renal cell carcinoma) xenografted model, followed by static scans at 15, 30, and 60 min postinjection. Finally, immunohistochemical (IHC) staining was performed to assess TIM3 expression in the dissected tumor tissues. The molecular docking results showed that the binding energy of P26 to TIM3 protein was -6.5 kcal/mol, which was lower than that of P24 to TIM3 protein, -3.6 kcal/mol, indicating that the affinity of P26 peptide to TIM3 protein was higher than that of P24 and P20 peptide. After structural modification of the P26 peptide, P26NH2, r-NH2, and P26X2 were obtained, and the above peptides were successfully constructed into six targeting TIM3 peptide probes by 68Ga labeling. Cellular uptake experiments demonstrated that 68Ga-DOTA-P26, 68Ga-DOTA-P26NH2, and 68Ga-DOTA-r-NH2 showed significantly higher uptake in A549TIM3 cells than in A549 cells and could be blocked by the unlabeled peptide. Micro-PET imaging experiments showed that the uptake of each probe in the A549TIM3 model tumor tissue was significantly higher than that in the A549 model tumor tissue, and a comparison of the tumor-to-cardiac uptake ratios of each group showed that the 68Ga-DOTA-P26 had a better tumor-to-cardiac uptake ratio in the A549TIM3 model than several other molecular probes, and in the MC38 model, similar results were obtained, with the difference that the 68Ga-DOTA-P26NH2 had the highest tumor-to-cardiac uptake ratio in the CCRCC model. Finally, validation by IHC showed that A549TIM3, MC38, and CCRCC tumor tissues had varying degrees of TIM3 expression. Upon comparison of ex vivo and in vivo studies, one of them, the 68Ga-DOTA-P26 probe, demonstrated significant target specificity for TIM3. These results suggest that studying peptide probes targeting TIM3 will promote the process of TIM3-targeted drug research and is expected to guide the application of TIM3 immune checkpoint drugs in immunotherapy.