Zirui Zhu

In this work, a polydopamine-coated, camptothecin nanotube functionalized with a targeting anti-TAG-72 scFv exhibited enhanced cytotoxicity against TAG-72 positive, LS-174T colon cancer cells, compared with HT-29 cells negative for the antigen.

Background: The complementarity-determining region (CDR) of antibodies represents the most diverse region both in terms of sequence and structural characteristics, playing the most critical role in antibody recognition and binding for immune responses. Over the past decades, several numbering schemes have been introduced to define CDRs based on sequence. However, the existence of diverse numbering schemes has led to potential confusion, and a comprehensive evaluation of these schemes is lacking. Methods: We employ statistical analyses to quantify the diversity of CDRs compared to the framework regions. Results: Comparative analyses across different numbering schemes demonstrate notable variations in CDR definitions. The Kabat and AbM numbering schemes tend to incorporate more conserved residues into their CDR definitions, whereas CDRs defined by the Chothia and IMGT numbering schemes display greater diversity, sometimes missing certain loop residues. Notably, we identify a critical residue, L29, within the kappa light chain CDR1, which appears to act as a pivotal structural point within the loop. In contrast, most numbering schemes designate the topological equivalent point in the lambda light chain as L30, suggesting the need for further refinement in the current numbering schemes. Conclusions: These findings shed light on regional sequence and structural conservation within antibody sequence databases while also highlighting discrepancies stemming from different numbering schemes. These insights yield valuable guidelines for the precise delineation of antibody CDRs and the strategic design of antibody repertoires, with practical implications in developing innovative antibody-based therapeutics and diagnostics.

H3K27M diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), exhibit cellular heterogeneity comprising less-differentiated oligodendrocyte precursors (OPC)-like stem cells and more differentiated astrocyte (AC)-like cells. Here, we establish in vitro models that recapitulate DMG-OPC-like and AC-like phenotypes and perform transcriptomics, metabolomics, and bioenergetic profiling to identify metabolic programs in the different cellular states. We then define strategies to target metabolic vulnerabilities within specific tumor populations. We show that AC-like cells exhibit a mesenchymal phenotype and are sensitized to ferroptotic cell death. In contrast, OPC-like cells upregulate cholesterol biosynthesis, have diminished mitochondrial oxidative phosphorylation (OXPHOS), and are accordingly more sensitive to statins and OXPHOS inhibitors. Additionally, statins and OXPHOS inhibitors show efficacy and extend survival in preclinical orthotopic models established with stem-like H3K27M DMG cells. Together, this study demonstrates that cellular subtypes within DMGs harbor distinct metabolic vulnerabilities that can be uniquely and selectively targeted for therapeutic gain.

Lattice strain is widely investigated to improve the performance of heterogeneous catalysts, however, the effect of lattice strain is under‐explored in high‐entropy oxide based photocatalyst. In this study, noble‐metal‐free (CoCrMnFeNi)Ox with lattice strain is synthesized using a temperature‐controlled, template‐free and salt‐assisted strategy. In the presence of lattice strain, an intensive internal electric field is formed in (CoCrMnFeNi)Ox, promoting the separation of photoinduced charge carriers. The size of the (CoCrMnFeNi)Ox can be tuned by varying the calcination temperature. Specifically, (CoCrMnFeNi)Ox prepared at a higher temperature possesses a smaller grain size exposing more active sites, resulting in an enhanced CO₂ photomethanation performance. This work provides valuable insights for the rational design of the photocatalysts and highlights the promising role of high‐entropy oxides in heterogeneous photocatalysis.

The complementarity determining region (CDR) of antibodies represents the most diverse region both in terms of sequence and structural characteristics, playing the most critical role in antibody recognition and binding for immune responses. Over the past decades, several numbering schemes have been introduced to define CDRs based on sequence. However, the existence of diverse numbering schemes has led to potential confusion, and a comprehensive evaluation of these schemes is lacking. We employ statistical analyses to quantify the diversity of CDRs compared to the framework regions. Comparative analyses across different numbering schemes demonstrates notable variations in CDR definitions. The Kabat and AbM numbering schemes tend to incorporate more conserved residues into their CDR definitions, whereas CDRs defined by the Chothia and IMGT numbering schemes display greater diversity, sometimes missing certain loop residues. Notably, we identify a critical residue, L29, within the kappa light chain CDR1, which appears to act as a pivotal structural point within the loop. In contrast, most numbering schemes designate the topological equivalent point in the lambda light chain as L30, suggesting the need for further refinement in the current numbering schemes. These findings shed light on regional sequence and structural conservation within antibody sequence databases while also highlighting discrepancies stemming from different numbering schemes. These insights yield valuable guidelines for the precise delineation of antibody CDRs and the strategic design of antibody repertoires, with practical implications in developing innovative antibody-based therapeutics and diagnostics. HIGHLIGHTS Amino acid distribution analysis conducted on 124,317 sequences archived in the abYsis database showed lower conservation among CDR residues, as defined by Kabat numbering, compared to the framework region. Both LCDR3 and HCDR3 exhibited particularly high levels of diversity compared to the other four CDR loops, highlighting additional functional roles of the CDR3 loops relative to the others. Two distinct residues, L29 and H30, displayed highly conserved hydrophobic side chain characteristics and played unique conformational roles within the CDR1 loop, contributing to the formation of an ‘M-shaped’ structure. This residue divides the C-terminus CDR1 loop, which was observed to be less conserved and have greater length diversity, and the N-terminus CRD1 loop. The Kabat numbering scheme appears to overestimate the LCDR2 and HCDR2 loops and underestimate HCDR1, only encompassing the C-terminal loop of the ‘M-shaped’ CDR1 loop. Structural analysis revealed that a residue within LCDR1 was numbered differently in the two subtypes (kappa and lambda) of the variable light domain. This discrepancy suggests the need for further refinement in the current major numbering schemes.

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

Nucleoli are large nuclear sub-compartments where vital cellular processes, such as rRNA production and ribosome assembly, take place. Aggressive tumor types, such as Triple Negative Breast Cancer (TNBC), require increased ribosome biogenesis to support their enhanced proliferation. Therefore, it has been suggested that the nucleolus could be a valid anti-neoplastic target. However, we still have a limited understanding of the cancer-specific functions of the nucleolar components, preventing the effective development of therapeutic agents. The characterization of nucleolar proteins is technically challenging. Several nucleolar proteins are essential, and their complete abrogation cannot be achieved through conventional RNAi or CRISPR-KO approaches. Additionally, many nucleolar proteins display peculiar biophysical properties, and their functions are connected to their abundance. Therefore, artificial overexpression systems might not fully recapitulate their endogenous biological functions. To overcome these challenges, we implemented an Auxin Inducible Degron (AID) system to acutely abrogate endogenous nucleolin, one of the most abundant nucleolar proteins, and asses its impact on the proliferation of TNBC cell lines. We used CRISPR/Cas9 editing in TNBC cell lines to modify the endogenous NCL gene, fusing it with an AID tag. Then, we stably transduced the cells with a modified Oryza Sativa TIR1 (OsTIR1-F74G) E3 ubiquitin ligase. In response to the synthetic phytohormone phenyl-auxin, OsTIR1 leads to the proteasomal degradation of AID-containing endogenous nucleolin in less than 6h, sparing any other human protein. RNA sequencing analysis showed that nucleolin degradation significantly alters the expression of genes involved in cell cycle progression. Accordingly, nucleolin degradation reduces cancer cell proliferation in vitro, associated with increased propidium iodide staining, suggestive of a G2/M cell cycle arrest. However, immunofluorescence analyses revealed that nucleolin abrogation leads to the accumulation of bi-nucleated cells with actin patches. These findings are indicative of potential cytokinesis defects due to altered spindle tension during mitosis. Notably, nucleolin abrogation increased the efficacy of chemical inhibitors of the Anaphase Promoting Complex on suppression of cell proliferation. To the best of our knowledge, this study is the first reporting the acute abrogation of an endogenous, highly abundant nucleolar protein, using the AID system. This approach allowed the characterization of nucleolin biological functions with unprecedented temporal resolution, shedding light on new biological functions in the regulation of cell division. Finally, our results suggest that nucleolin inhibition could enhance the therapeutic activities of drugs affecting cell cycle progression in TNBC. Citation Format: Nastaran Samadi Rad, Joseph Mills, Anna Tessari, Damu Sunil Kumar, Vollter Anastas, Saranya Lamba, Ashley B Reers, Ilaria Cosentini, Zirui Zhu, Thomas J Magliery, Emanuele Cocucci, Lara Rizzotto, Dario Palmieri. Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1650.