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.

Quantitative Proteomics Characterization of the Effect and Mechanism of Trichostatin A on the Hippocampus of Type II Diabetic Mice.

Diabetic encephalopathy (DE) is one of the complications of diabetes mellitus with mild-to-moderate cognitive impairment. Trichostatin A (TSA) has been revealed to show protective effect on central nervous systems in Alzheimer's disease (AD) and hypoxic-ischemic brain injury. However, the effect and molecular mechanism of TSA on cognitive function of DE are unknown. Here, we demonstrated that cognitive function was damaged in diabetic mice versus normal mice and treatment with TSA improved cognitive function in diabetic mice. Proteomic analysis of the hippocampus revealed 174 differentially expressed proteins in diabetic mice compared with normal mice. TSA treatment reversed the expression levels of 111 differentially expressed proteins grouped into functional clusters, including the longevity regulating pathway, the insulin signaling pathway, peroxisomes, protein processing in the endoplasmic reticulum, and ribosomes. Furthermore, protein-protein interaction network analysis of TSA-reversed proteins revealed that UBA52, CAT, RPL29, RPL35A, CANX, RPL37, and PRKAA2 were the main hub proteins. Multiple KEGG pathway-enriched CAT and PRKAA2 levels were significantly decreased in the hippocampus of diabetic mice versus normal mice, which was reversed by TSA administration. Finally, screening for potential similar or ancillary drugs for TSA treatment indicated that HDAC inhibitors ISOX, apicidin, and panobinostat were the most promising similar drugs, and the PI3K inhibitor GSK-1059615, the Aurora kinase inhibitor alisertib, and the nucleophosmin inhibitor avrainvillamide-analog-6 were the most promising ancillary drugs. In conclusion, our study revealed that CAT and PRKAA2 were the key proteins involved in the improvement of DE after TSA treatment. ISOX, apicidin, and panobinostat were promising similar drugs and that GSK-1059615, alisertib, and avrainvillamide-analog-6 were promising ancillary drugs to TSA in the treatment of DE.

A multicenter, prospective, randomized controlled trial of intracranial hemorrhage risk of intensive statin therapy in patients with acute ischemic stroke combined with cerebral microbleeds (CHRISTMAS): Study protocol.

Background: Low serum levels of major lipid markers have been proved to be significantly associated with increased risks of hemorrhagic stroke (HS) and cerebral microbleeds (CMBs). However, there is no lipid modification guideline telling us how to maintain a balance between the prevention of ischemic stroke recurrence and the prevention of hemorrhagic events, especially in patients with acute ischemic stroke (AIS) and CMBs. Aim: The Intracranial Hemorrhage Risk of Intensive Statin Therapy in Patients with Acute Ischemic Stroke combined with Cerebral Microbleeds (CHRISTMAS) trial evaluates the risk of intracranial hemorrhage (i.e., HS and CMBs) of high-dose statin therapy in patients with AIS combined with CMBs. Methods and design: This is an investigator-initiated, multicenter, prospective, randomized controlled clinical trial design. Up to 344 eligible patients will be consecutively randomized to receive high-dose or low-dose atorvastatin in 1:1 ratio in 5 stroke centers in China. Outcomes: CHRISTMAS trial has co-primary outcomes, namely, hemorrhage risk: the incidence of HS and the changes in degree of CMBs until the end of 36-month follow-up. Discussion: The primary hypothesis of this study is that an excessive reduction in serum lipid levels by an intensive statin therapy in AIS patients with CMBs can increase the risk of intracranial hemorrhage. This study will shed light on new clinical decisions regarding the long-term serum lipid management in these patients with dilemma in clinical practice. Clinical trial registration: Clinicaltrials.gov, identifier: NCT05589454.

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.

Lycorine improves peripheral nerve function by promoting Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation in diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus and no effective therapy is approved. Here, lycorine, a natural alkaloid, was identified as a potential drug for DPN by the bioinformatics analysis of GEO datasets and Connectivity Map database. Lycorine administration improved peripheral nerve function and autophagy-associated proteins of diabetic mice. Again, in vitro high glucose-cultured rat Schwann cells (RSC96) showed enhanced autophagosome marker LC3-II with the treatment of lycorine. Additionally, beclin-1 and Atg3 were decreased in high glucose-stimulated RSC96 cells, which were reversed by lycorine treatment. Furthermore, DPN-associated differentially expressed genes (DEGs) from GEO datasets and lycorine-drug targets from PubChem and PharmMapper were visually analyzed and revealed that MMP9 was both DPN-associated DEGs and lycorine-drug target. Functional enrichment analysis of MMP9-relevant genes showed that cell energy metabolism was involved. Moreover, lycorine reduced high glucose-enhanced MMP9 expression in RSC96 cells. Overexpression of MMP9 attenuated lycorine-induced the expression of beclin-1, Atg3 and LC3-II in high glucose-cultured RSC96 cells. In addition, AMPK pathway activation was confirmed in lycorine-treated high glucose-cultured RSC96 cells. Then AMPK pathway inhibition attenuated lycorine-reduced MMP9 expression in high glucose-treated RSC96 cells. Molecular docking analysis revealed that lycorine bound the domain of AMPK containing Thr 172 site, which affected AMPK (Thr 172) phosphorylation. Finally, AMPK pathway activation and MMP9 downregulation were also revealed in the sciatic nerves of diabetic mice administrated with lycorine. Taken together, lycorine was advised to promote Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation-induced LC3-II transformation in diabetic peripheral neuropathy.

Molecularly Imprinted and Cladded Nanoparticles Provide Better Phosphorylation Recognition.

Phosphorylation is one of the most frequently occurring post-translation modifications in mammals. Because abnormal protein phosphorylation is related to many diseases, phosphorylation analysis is essential for a sound understanding of protein phosphorylation and its relationship with diseases. Among several types of reagents for phosphorylation recognition, molecularly imprinted polymers (MIPs), as synthetic mimics of antibodies, have exhibited unique strengths that can overcome the drawbacks of biological reagents. However, the performance of current MIPs has remained unideal. Meanwhile, while the currently existing imprinting methods have permitted the production of several material formats, such as crushed particles and mesoporous nanoparticles, a general method allowing for the preparation of monodispersed molecularly imprinted nanoparticles has not been developed yet. Herein, we report a new approach called reverse microemulsion template docking surface imprinting and cladding (RMTD-SIC) for facile preparation of monodispersed imprinted nanoparticles for better phosphorylation recognition. Through rational design and controllable engineering, monodisperse imprinted and cladded nanoparticles specific to general phosphorylation and tyrosine phosphorylation were synthesized, which yield the highest imprinting factors as compared with published studies. The prepared nanomaterials exhibited excellent specificity and affinity, allowing for specific enrichment and improved mass spectrometric identification of target phosphorylated peptides from complex samples containing 100-fold more abundant interfering peptides. Therefore, the RMTD-SIC approach holds great potential for phosphorylation analysis and phosphorylation recognition-based 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.

Remediation of chromium-contaminated soil using delaminated layered double hydroxides with different divalent metals.

Soil is commonly polluted by chromium, and layered double hydroxides (LDHs) are widely used for chromium removal due to their strong adsorption capacity and the unique properties of their delaminated products. In this study, delaminated LDHs (S-Mg-LDH and S-Ca-LDH) and their original LDHs were used to remediate Cr(VI)-contaminated soil. A series of characterizations confirmed the successful synthesis of delaminated LDHs whose sheet structure was thinner with a greater surface energy than the original LDHs. The remediation results indicated that delaminated LDHs could more efficiently immobilize Cr(VI) in soil. The immobilization rate of S-Mg-LDH was 64.32%, while Mg-LDH was only 8.09%. However, at low dosages, the efficiency of S-Ca-LDH was 28.1% while Ca-LDH was 5.16%, but they had similar effects at high doses. Moreover, soil pH had little effect on their removal efficiencies. The toxicity characteristic leaching procedure (TCLP) results showed that the leaching of Cr(VI) in soil after treatment with S-Mg-LDH, S-Ca-LDH, Mg-LDH, and Ca-LDH was reduced by 75.43%, 72.43%, 86.55%, and 75.90%, respectively. The phytotoxicity tests of soil treated by S-Mg-LDH and S-Ca-LDH revealed that they effectively reduced the toxicity of chromium and lowered its bioaccumulation. Overall, this study confirms the feasibility of delaminated LDHs for Cr(VI) immobilization in soils.

[Cloning, expression and purification of novel gene Rv2742 in Mycobacterium tuberculosis H37Rv].

Rv2742 is a novel gene identified from Mycobacterium tuberculosis H37Rv by the proteogenomics strategy. The aim of this study was to establish a system of soluble expression and purification of the missing protein Rv2742 in M. tuberculosis H37Rv, to provide reference for further research on the biological function of Rv2742. The soluble protein was not successfully induced by prokaryotic expression vectors pGEX-4T-2-Rv2742, pET-32a-Rv2742, pET-28a-Rv2742 and pMAL-c2X-Rv2742. After the codon of novel gene Rv2742 was optimized according to E. coli codon usage frequency, only the recombinant strain containing plasmid pMAL-c2X-Rv2742 could produce soluble products of Rv2742 encoding gene. In addition, the expression effects of the desired fusion protein were also analyzed under different conditions including hosts, culture temperatures and IPTG concentrations. The optimum expression conditions were as follows: Rosetta (DE3) host, 16 °C culture temperature and 0.5 mmol/L IPTG. After being purified by affinity chromatography with amylose resin, the fusion protein sequence was confirmed by LC-MS/MS. These results indicated that the novel gene Rv2742 product could be successfully induced and expressed in a soluble form by the expression system pMAL-c2X with MBP tag. Our findings provide reference for studies on potential interaction and immunogenicity.

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.

Development and validation of the Chinese Version of the Adult Epilepsy Self-Management Scale (C-ESMS) in Western China.

PURPOSE: To develop and validate the Epilepsy Self-Management Scale (ESMS) for Chinese persons with epilepsy (PWE). METHODS: On the basis of ESMS, the standard translation procedure was used to set up the Chinese version of the ESMS (C-ESMS). A consecutive cohort of PWE admitted in Sichuan Provincial People's Hospital were recruited randomly from May 2017 to December 2017 and required to complete the C-ESMS. Project analysis was employed to test the homogeneity of each dimension. Content validity was evaluated by experts. Exploratory factor analysis and confirmatory factor analysis (CFA) were applied to assess the validity. Cronbach's alpha was used to evaluate the reliability. RESULTS: Of the 400 completed C-ESMS forms, only 394 (98.5%) were suitable for analysis. The C-ESMS included 34 items and five dimensions, after removing four and modifying three items. The correlation coefficient of all 34 items was greater than 0.4. Each item level (I-CVI) and scale level CVI (S-CVI) was equal to 1. Five factors were extracted and together they explained 51.24% of the data's variance. The factor load of each item was 0.446-0.843. The CFA showed that CMIN/DF was 1.325, goodness of fit was 0.835, comparative fit index was 0.921, and root mean square error of approximation was 0.041. The total Cronbach's alpha of the scale was 0.848, and Cronbach's alpha in each dimension was 0.784-0.845. CONCLUSION: The C-ESMS exhibited good reliability and validity for adult PWE in western China.

Protein Sequence Comparison and DNA-binding Protein Identification with Generalized PseAAC and Graphical Representation.

AIM AND OBJECTIVE: The rapid increase in the amount of protein sequence data available leads to an urgent need for novel computational algorithms to analyze and compare these sequences. This study is undertaken to develop an efficient computational approach for timely encoding protein sequences and extracting the hidden information. METHODS: Based on two physicochemical properties of amino acids, a protein primary sequence was converted into a three-letter sequence, and then a graph without loops and multiple edges and its geometric line adjacency matrix were obtained. A generalized PseAAC (pseudo amino acid composition) model was thus constructed to characterize a protein sequence numerically. RESULTS: By using the proposed mathematical descriptor of a protein sequence, similarity comparisons among ß-globin proteins of 17 species and 72 spike proteins of coronaviruses were made, respectively. The resulting clusters agreed well with the established taxonomic groups. In addition, a generalized PseAAC based SVM (support vector machine) model was developed to identify DNA-binding proteins. Experiment results showed that our method performed better than DNAbinder, DNA-Prot, iDNA-Prot and enDNA-Prot by 3.29-10.44% in terms of ACC, 0.056-0.206 in terms of MCC, and 1.45-15.76% in terms of F1M. When the benchmark dataset was expanded with negative samples, the presented approach outperformed the four previous methods with improvement in the range of 2.49-19.12% in terms of ACC, 0.05-0.32 in terms of MCC, and 3.82- 33.85% in terms of F1M. CONCLUSION: These results suggested that the generalized PseAAC model was very efficient for comparison and analysis of protein sequences, and very competitive in identifying DNA-binding proteins.

Quantitative assessment and correlation analysis of cerebral microbleed distribution and leukoaraiosis in stroke outpatients.

OBJECTIVE: Cerebral microbleeds (CMBs) are bleeding events associated with cerebral small vessel disease (SVD). Strictly lobar CMBs and strictly deep CMBs are likely caused by cerebral amyloid angiopathy (CAA) and hypertensive arteriopathy, respectively. Leukoaraiosis (LA) reflects an ischaemic change in SVD, and LA severity has been correlated with CMBs. However, whether different locations (aetiologies) of CMBs correlate with LA is unknown. METHODS: Patients receiving brain MRI and tbl2*-weighted gradient-recalled echo scans in a stroke outpatient department were screened for CMBs. The MRI results of the patients with CMBs were sent to investigators for further review and were evaluated using the Microbleed Anatomical Rating Scale. Cerebral microbleed severity was graded using a numerical scale. Leukoaraiosis severity was assessed using the Fazekas scale. RESULTS: Cerebral microbleeds were observed in 14.6% of the 1289 screened patients. The CMB incidence increased with age (in years, < 50: 1.3%; 50-59: 10.7%; 60-69: 17.6% and ≥ 70: 23.6%; P = 0.000). The CMB locations were distributed as follows: 23.4% strictly lobar, 12.2% strictly deep, 6.4% strictly infratentorial and 58.0% mixed. Cerebral microbleed severity correlated with the total Fazekas scale score. The numbers of lobar, deep and infratentorial CMBs correlated with the total Fazekas scale score. The mixed CMB group displayed a significantly higher Fazekas scale score than the strictly lobar CMB group (P = 0.000). DISCUSSION: Cerebral microbleed incidence increased with age. Mixed CMB type displayed the highest incidence. The severity and number of CMBs at any location correlated with LA severity. These results suggested interactions between hypertension and CAA during LA progression.