Molecular Dynamics and In Vitro Studies Elucidating the Tunable Features of Reconfigurable Nanodiscs for Guiding the Optimal Design of Curcumin Formulation.

In this study, we advance our exploration of Apolipoprotein A-I (apoA-I) peptide analogs (APAs) for their application in nanodisc (ND) assembly, focusing on the dynamic conformational characteristics and the potential for drug delivery. We explore APA-ND interactions with an emphasis on curcumin encapsulation, utilizing molecular dynamic simulations and in vitro assessments to evaluate the efficacy of various APA-ND formulations as drug carriers. The methodological approach involved the generation of three unique apoA-I α-11/3 helical mimics, resulting in fifteen distinct APAs. Their structural integrity was rigorously assessed using ColabFold-AF2, with particular attention to pLDDT and pTM scores. Extensive molecular dynamics simulations, covering 1.7 µs across 17 ND systems, were conducted to investigate the influence of APA sequence variations on ND stability and interactions. This study reveals that the composition of APAs, notably the presence of Proline, Serine, and Tryptophan, significantly impacts ND stability and morphology. Oligomeric APAs, in particular, demonstrated superior stability and distinct interaction patterns compared to their monomeric counterparts. Additionally, hydrodynamic diameter measurements over eight weeks indicated sequence-dependent stability, highlighting the potential of specific APA configurations for sustained colloidal stability. In vitro study successfully encapsulated curcumin in [AA]3/DMPC ND formulations, revealing concentration-dependent stability and interaction dynamics. The findings underscore the remarkable capability of APA-NDs to maintain structural integrity and efficient drug encapsulation, positioning them as a promising platform for drug delivery. The study concludes by emphasizing the tunability and versatility of APA-NDs in drug formulation, potentially revolutionizing nanomedicine by enabling customized APA sequences and ND properties for targeted drug delivery.

Melittin Tryptophan Substitution with a Fluorescent Amino Acid Reveals the Structural Basis of Selective Antitumor Effect and Subcellular Localization in Tumor Cells.

Melittin is a membrane-active peptide with strong anticancer activity against various cancers. Despite decades of research, the role of the singular Trp in the anticancer activity and selectivity of melittin remains poorly understood. Here, we propose a theranostic solution based on the substitution of Trp19 with a noncanonical fluorescent amino acid (DapAMCA). The introduction of DapAMCA residue in melittin stabilized the helical structure of the peptide, as evaluated by circular dichroism spectra and molecular dynamics simulations. In vitro hemolytic and anticancer activity assays revealed that introducing DapAMCA residue in melittin changed its mode of action with the cell membrane, resulting in reduced hemolytic toxicity and an improved the selectivity index (SI), with up to a five-fold increase compared to melittin. In vitro fluorescence imaging of DapAMCA-labeled melittin (MELFL) in cancer cells demonstrated high membrane-penetrating activity, with strong nuclear and nucleolar localization ability. These findings provide implications for novel anticancer therapies based on Trp-substituted designs and nuclear/nucleolar targeted therapy.

Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption.

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.

SNCA, a novel biomarker for Group 4 medulloblastomas, can inhibit tumor invasion and induce apoptosis.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. It contains at least four distinct molecular subgroups. The aim of this study is to explore novel diagnostic and potential therapeutic markers within each subgroup of MB, in particular within Group 4, the largest subgroup, to facilitate diagnosis together with gene therapy. One hundred and six MB samples were examined. Tumor subtype was evaluated with the NanoString assay. Several novel tumor related genes were shown to have high subgroup sensitivity and specificity, including PDGFRA, FGFR1, and ALK in the WNT group, CCND1 in the SHH group, and α-synuclein (SNCA) in Group 4. Knockdown and overexpression assays of SNCA revealed the ability of this gene to inhibit tumor invasion and induce apoptosis. Methylation-specific PCR and pyrosequencing analysis showed that epigenetic mechanisms, rather than DNA hypermethylation, might play the key role in the regulation of SNCA expression in MB tumors. In conclusion, we identify SNCA as a novel diagnostic biomarker for Group 4 MB. Some other subgroup signature genes have also been found as candidate therapeutic targets for this tumor.

Mir-449a, a potential diagnostic biomarker for WNT group of medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. The 5 year disease-free survival rate is rather low. There is a consensus that MB can be divided into at least four clinically, transcriptionally, and genetically distinct molecular variants, being designated as wingless (WNT), sonic hedgehog (SHH), Group 3 and Group 4. It poses a great challenge to the design of therapeutic strategy for MB patients. Intensive clinical intervention, including high dose radiotherapy, is commonly used in treatment of high risk MB, most of which are considered to be Group 3 patients. But such intensive therapy should be avoided to protect neurologic function of patients in the lower risk WNT group. In present study, MB subgroup assignment in formalin-fixed paraffin embedded (FFPE) specimens from 45 Chinese patients were performed by Nanostring platform using 22 well-known signature genes. Based on comparative expression profiles of miRNA real-time PCR microarray in MB cells with and without treatment of demethylation reagent, as well as MSP assay, miR-449a was demonstrated to be significantly silenced by aberrant DNA methylation in tumor cells. Real-time PCR showed that expression level of miR-449a in WNT group was significantly different from other subgroups, although it was down-regulated in most of the MB samples. In conclusion, current study demonstrates for the first time the feasibility of using the Nanostring assay for subgrouping of MBs in Chinese patients. In addition, MiR-449a, a candidate tumor suppressor regulated by hypermethylation, is a novel potential diagnostic marker for WNT group of MBs.

ATRX loss in adult supratentorial diffuse astrocytomas correlates with p53 over expression and IDH1 mutation and predicts better outcome in p53 accumulated patients.

BACKGROUND: IDH1/2 mutation, 1p/19q-codeletion and MGMT hypermethylation are well known molecular markers for gliomas. ATRX and p53 alterations are two lineage-specific genetic aberrations in diffuse astrocytic tumors. The aim of the present study is to clarify the significance of ATRX loss and its correlation with p53 overexpression, IDH1/2 mutations, 1p/19q-codeletion and MGMT hypermethylation in supertentorial astrocytoma, and to determine the prognostic value of these factors in Chinese patients. METHODS AND RESULTS: A total of 135 adult supertentorial astrocytomas were evaluated. ATRX loss was detected by immunohistochemistry (IHC) and was shown to be much less frequent in pGBs (3.5%) than in grade II, III astrocytomas and IV sGBs (31%). Direct sequencing and/or IHC analysis of the IDH1R132H gene mutation and p53 accumulation demonstrated correlation with age. Strong correlations were found between ATRX loss and IDH1R132H mutation, p53 overexpression as well as MGMT hypermethylation. 1p/19q-codeletion detected by fluorescence in situ hybridization (FISH) showed mutually exclusive with ATRX loss and p53 accumulation. In addition, patients with p53 overexpression combined with ATRX alterations demonstrated substantially longer survival than patients with wild-type ATRX. CONCLUSIONS: There may be interactions among these distinct molecules in astrocytoma development. ATRX loss may predict better clinical outcome in astrocytoma patients with p53 overexpression as compared to patients with wild-type ATRX. Tumors with astrocytoma phenotype accompanied by 1p/19q-codeletion and IDH1R132H mutation are mutually exclusive with ATRX and p53 alterations. Routine IHC can be used for evaluation of ATRX loss, p53 protein accumulation and IDH1R132H mutation, which may allow a means of classification of astrocytoma outcome.