Chemically engineered mTOR-nanoparticle blockers enhance antitumour efficacy.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly prevalent and deadly type of cancer, and although pharmacotherapy remains the cornerstone of treatment, therapeutic outcomes are often unsatisfactory. Pharmacological inhibition of mammalian target of rapamycin (mTOR) has been closely associated with HCC regression. METHODS: Herein, we covalently conjugated AZD8055, a potent mTORC1/2 blocker, with a small panel of unsaturated fatty acids via a dynamically activating linkage to enable aqueous self-assembly of prodrug conjugates to form mTOR nanoblockers. Cell-based experiments were carried out to evaluate the effects of the nanoblocker against hepatocellular carcinoma (HCC) cells. The orthotopic and subcutaneous HCC mouse models were established to examine its antitumour activity. FINDINGS: Among several fatty acids as promoieties, linoleic acid-conjugated self-assembling nanoblocker exhibited optimal size distribution and superior physiochemical properties. Compared with free agents, PEGylated AZD8055 nanoblocker (termed AZD NB) was pharmacokinetically optimized after intravenous administration. In vivo investigations confirmed that AZD NB significantly suppressed tumour outgrowth in subcutaneous HCCLM3 xenograft, Hepatoma-22, and orthotopic Hepa1-6 liver tumour models. Strikingly, treatment with AZD NB, but not free agent, increased intratumour infiltration of IFN-γ+CD8+ T cells and CD8+ memory T cells, suggesting a potential role of the mTOR nanoblocker to remodel the tumour microenvironment. Overall, a single conjugation with fatty acid transformed a hydrophobic mTOR blocker into a systemically injectable nanomedicine, representing a facile and generalizable strategy for improving the therapeutic index of mTOR inhibition-based cancer therapy. INTERPRETATION: The mTOR inhibition by chemically engineered nanoblocker presented here had enhanced efficacy against tumours compared with the pristine drug and thus has the potential to improve the survival outcomes of patients with HCC. Additionally, this new nanosystem derived from co-assembling of small-molecule prodrug entities can serve as a delivery platform for the synergistic co-administration of distinct pharmaceutical agents. FUNDING: This work was supported by the National Natural Science Foundation of China (32171368,81721091), the Zhejiang Provincial Natural Science Foundation of China (LZ21H180001), the Jinan Provincial Laboratory Research Project of Microecological Biomedicine (JNL-2022039c and JNL-2022010B), State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (zz202310), and Natural Science Foundation of Shandong Province (ZR2023ZD59).

Association between plasma CTRPs with cognitive impairment and neurodegeneration of Alzheimer's disease.

AIMS: Recent evidence indicated the biological basis of complement 1q (C1q)/tumor necrosis factor (TNF)-related protein (CTRP) 3, 4, and 14 for affecting brain structure and cognitive function. Thus, we aimed to investigate the association between plasma CTRPs with Alzheimer's disease (AD). METHODS: A multicenter, cross-sectional study recruited patients with AD (n = 137) and cognitively normal (CN) controls (n = 140). After the data collection of demographic characteristics, lifestyle risk factors, and medical history, plasma levels of tau phosphorylated at threonine 217 (pT217), pT181, neurofilament light (NfL), CTRP3, 4, and 14 were examined and compared. Multivariate logistic regression analysis was applied to determine associations of plasma CTPRs with the presence of AD. The correlation analysis was used to explore correlations between plasma CTPRs with scores of Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), Activities of Daily Living (ADL) scale, and Clinical Dementia Rating Sum of Boxes (CDR-SB), and levels of plasma pT217, pT181, and NfL. Receiver-operating characteristic (ROC) analysis and Delong's test were used to determine the diagnostic power of plasma CTPRs. RESULTS: Plasma levels of CTRP3, 4, and 14 were higher in AD group than those in CN group. After adjusting for conventional risk factors, CTRP3, CTRP4, and CTRP14 were associated with the presence of AD. In AD patients, CTRP3 was negatively correlated with scores of MMSE and MoCA, while positively correlated with ADL score, CDR-SB score, pT217, and pT181; CTRP4 was positively correlated with CDR-SB score, pT181, and NfL; CTRP14 was negatively correlated with MMSE score, while positively correlated with CDR-SB score, pT217, and NfL. An independent addition of CTRP3 and 4 to the basic model combining age, sex, years of education, APOE4 status, BMI, TG, and HDL-C led to a significant improvement in diagnostic power for AD, respectively. CONCLUSIONS: All the findings preliminarily uncovered associations between plasma CTRPs and AD and suggested the potential of CTRPs as a blood-derived biomarker for AD.

Double-edged effect of frequent freeze-thaw on the stability of zero-valent iron after heavy metal remediation.

Freeze-thaw cycles (FTCs) cause dynamic microscale changes in ions and solvents. During freezing, heavy metals adsorbed on zero-valent iron (M-ZVI) and protons are excluded by ice crystals and concentrated in the liquid-like grain boundary region. The high proton concentration in this region leads to the dissolution of the passivation layer of ZVI. To assess the environmental risks of M-ZVI during FTCs, this study evaluated the stability of M-ZVI in this scenario from both microscale and macroscale perspectives. The results showed that the dissolution of the passivation layer had a dual effect on the stability of M-ZVI, which depends on the by-products of M-ZVI. The dissolution of the passivation layer was accompanied by the leaching of heavy metals, such as Ni-ZVI, but it also enhanced the reactivity of ZVI, causing it to re-react with desorbed heavy metals. The stability of Cr-ZVI and Cd-ZVI was improved due to frequent FTCs. Furthermore, changes in the surrounding environment (water dipole moment, ion concentration, etc.) of ZVI affected the crystallization of Fe oxides, increasing the content of amorphous Fe oxide. As low-crystallinity Fe oxides could facilitate ion doping, Ni2+ was doped into Fe3O4 lattice during FTCs, which reduced the mobility of heavy metals. Contrary to traditional views that freezing temperatures slow chemical reactions, this study provides new insights into the application of iron-based materials in cold environments.

Molecularly Imprinted and Cladded Nanotags Enable Specific SERS Bioimaging of Tyrosine Phosphorylation.

Tyrosine phosphorylation is an important post-translational modification of proteins, and its accurate analysis is of vital importance. However, due to limited abundance of tyrosine phosphorylation as well as severe interference of serine/threonine phosphorylation and other phosphate-containing species, approaches that can directly analyse tyrosine phosphorylation on the cell membrane still remain limited. Herein, we report the rational development of molecularly imprinted and cladded Raman nanotags and their successful application in surface enhanced Raman spectroscopy (SERS) imaging of tyrosine phosphorylation on cancerous cells and tissues. The prepared molecularly imprinted and cladded SERS nanotags could specifically recognize phosphotyrosine and thereby allowed for distinguishing phosphotyrosine from other phosphate-containing species on cancerous cells and tissues by SERS imaging. Therefore, the molecularly imprinted and cladded nanotags-based SERS imaging can be a promising tool for tyrosine phosphorylation analysis and tyrosine phosphorylation-related studies, showing great potential for biomedical applications.

Multiplexed Single-Cell Plasmonic Immunoassay of Intracellular Signaling Proteins Enables Non-Destructive Monitoring of Cell Fate.

It is of significant importance in cancer biology to identify signaling pathways that play key roles in cell fate determination. Dissecting cellular signaling pathways requires the measurement of a large number of signaling proteins. However, tools for simultaneously monitoring multiple signaling pathway components in single living cells remain limited at present. Herein, we describe an approach, termed multiplexed single-cell plasmonic immunosandwich assay (mxscPISA), for simultaneous detection of multiple signaling proteins in individual living cells. This approach enabled simultaneous non-destructive monitoring of multiple (up to five, currently the highest multiplexing capacity in living cells) cytoplasmic and nucleus signaling proteins in individual cells with ultrahigh detection sensitivity. As a proof of principle, the epidermal growth factor receptor (EGFR) pathway, which plays a central role in cell fate determination, was investigated using this approach in this study. We found that there were differential attenuation rate of pro-survival and accumulation rate of pro-death signaling protein of the EGFR pathway in response to EGFR inactivation. These findings implicate that, after EGFR inactivation, a transient imbalance between survival and apoptotic signaling outputs contributed to the final cell fate of death. The mxscPISA approach can be a promising tool to reveal a signaling dynamic pattern at the single-cell level and to identify key components of signaling pathways that contribute to the final cell fate using only a limited number of cells.

Digging for Missing Proteins Using Low-Molecular-Weight Protein Enrichment and a "Mirror Protease" Strategy.

In 2012, the Chromosome-centric Human Proteome Project (C-HPP) launched an investigation for missing proteins (MPs) to complete the Human Proteome Project (HPP). The majority of the MPs were distributed in low-molecular-weight (LMW) ranges, especially from 0 to 40 kDa. LMW protein identification is challenging, owing to their short length, low abundance, and hydrophobicity. Furthermore, many sequences from trypsin digestion are unlikely to yield detectable peptides or a reasonable quality of MS2 spectrum. Therefore, we focused on small MPs by combining LMW protein enrichment and a pair of complementary proteases strategy with trypsin and LysargiNase for human testis samples. In-depth testis LMW protein profiling resulted in the identification of 4063 proteins, of which 2565 were LMW proteins and 1130 had pairs of peptides generated from both trypsin and LysargiNase. This provided additional mass spectral evidence of further verification of small MPs. Finally, two MPs were verified from the seven MP candidates. One of them, Q8N688 , was verified with two series of continuous and complementary b/y-product ions from the pairs of spectra for tryptic and LysargiNase digested peptides after the "mirror spectrum" matching. This make the confident identification of the representative peptides for the target MPs. On the contrary, the two verified peptides for Q86WR6 were identified with the same strategy from the gel-separation and gel-elution samples, respectively. Although the other five MP candidates showed high-quality spectra, they could not be sufficiently distinguished as PE1s and require further verification. All MS data sets have been deposited in the ProteomeXchange with identifier PXD010093.