De novosynthesis of pH-responsive, self-assembled, and targeted polypeptide nano-micelles for enhanced delivery of doxorubicin.

Doxorubicin (DOX) is a commonly used anticancer drug, but it is inefficient as a therapeutic due to a lack of targeting. Peptide-tuned self-assembly of DOX offers a strategy to improve targeting for greater efficacy. In this work, we designed and prepared an amphiphilic tumor cell-targeting peptide, P14 (AAAAFFFHHHGRGD), able to encapsulate DOX by self-assembly to form tumor cell-targeting and pH-sensitive nano-micelles. The results showed a critical P14-micelle concentration of 1.758 mg l-1and an average particle size of micelles of 121.64 nm, with entrapment and drug-loading efficiencies of 28.02% ± 1.35% and 12.06% ± 0.59%, respectively. The prepared micelles can release 73.52 ± 1.27% DOX within 24 h in pH 4.5 medium, and the drug cumulative release profile of micelles can be described by the first-order model. Compared with free DOX, the micelles exhibited an increased ability to inhibit tumor cell growth and cause tumor apoptosisin vitro, with IC50values of DOX and P14-DOX micelles against human breast cancer cells (MCF-7) of 0.91 ± 0.07 and 0.75 ± 0.06µg ml-1, respectively, and cellular apoptotic rates of DOX and P14-DOX micelles of 70.3% and 42.4%, respectively. Cellular uptake experiments revealed high concentrations of micelles around and inside MCF-7 cells, demonstrating that micelles can target tumor cells. These results indicate the excellent potential for the application of this amphiphilic peptide as a carrier for small-molecule drugs and suggest a strategy for the design of effective anti-tumor drugs.

GanDouLing promotes proliferation and differentiation of neural stem cells in the mouse model of Wilson's disease.

Wilson's disease (WD) is an autosomal recessive disease caused by mutation of the ATPase copper transporting ß (ATP7B) gene, resulting in abnormal copper metabolism. We aimed to investigate the protective effect of GanDouLing (GDL) on neural stem cell (NSC) function in a mouse model of WD. NSCs were treated with different concentrations of GDL alone or in combination with penicillamine, following which we evaluated cellular growth, apoptosis, and differentiation. Nuclear factor E2-related factor 2 (Nrf2) pathway and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation were analyzed via Western blotting. Treatment with GDL alone or in combination with penicillamine significantly increased proliferation and inhibited apoptosis of NSCs in a dose-dependent manner. In addition, GDL treatment remarkably promoted differentiation of NSCs. Consistently, levels of class III ß-tubulin (Tuj1) and microtubule-associated protein 2 (MAP2) were significantly elevated, whereas glial fibrillary acidic protein (GFAP) levels were obviously suppressed in the presence of GDL or penicillamine. In vivo assays confirmed that GDL increased the ratio of Ki67+, Tuj1+, and MAP2+ cells and suppressed apoptosis in the hippocampal region in WD mice. Behavioral assays revealed that both GDL and penicillamine improved memory ability in WD models. Mechanistically, GDL treatment led to activation of Nrf2 signaling and suppression of the NLRP3 inflammasome in WD mice. Notably, inhibition of Nrf2 signaling reversed the protective effects of GDL on hippocampal NSCs. Collectively, these findings demonstrate that GDL exerts a protective effect on NSCs and promotes neurogenesis by targeting Nrf2 signaling and the NLRP3 inflammasome in WD.

The Effects of Sidt2 on the Inflammatory Pathway in Mouse Mesangial Cells.

In patients with chronic kidney disease, the abnormal activation of inflammatory pathways is usually an important factor leading to renal fibrosis and further deterioration of renal function. Finding effective intervention targets of the inflammatory signaling pathway is an important way to treat chronic kidney disease. As a newly discovered lysosomal membrane protein, the correlation between SID1 transmembrane family member 2 (Sidt2) and the inflammatory signaling pathway has not been reported. The aim of this study was to investigate the effect of Sidt2 on inflammation by inhibiting the expression of the Sidt2 gene in a mouse mesangial cell line mediated by a lentiviral CRISPR/Cas9 vector. Hematoxylin and eosin staining and microscopy found that the mesangial cells lost their normal morphology after inhibiting the expression of Sidt2, showing that the cell body became smaller, the edge between the cells was unclear, and part of the nucleus was pyknotic and fragmented, appearing blue-black. The expressions of IKK ß, p-IKK α/ß, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the NF-κB pathway of the Sidt2 -/- group were higher than those of the Sidt2 +/+ group. p-Jak2 and IL6 increased in the Jak/Stat pathway, and p-ERK and p-P38 increased in the MAPK pathway. The expressions of IKK ß, p-IKK α/ß, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the NF-κB pathway of the Sidt2 +/++LPS group were significantly higher than those in the Sidt2 +/+ group. The expressions of IKK ß, p-IKK α/ß, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2 -/-+LPS group were higher than those in the Sidt2 -/- group. The expressions of p-IKK α/ß, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2 -/-+LPS group were higher than those in the Sidt2 +/++LPS group. In the Jak/Stat pathway, the protein expressions of p-Jak2 and IL6 in the Sidt2 +/++LPS group were higher than those in the Sidt2 +/+ group. The expressions of p-Jak2 and IL6 in the Sidt2 -/-+LPS group were higher than those in the Sidt2 -/- group. The expressions of p-Jak2 and IL6 in the Sidt2 -/-+LPS group were higher than those in the Sidt2 +/++LPS group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the MAPK pathway in the Sidt2 +/++LPS group were higher than those in the Sidt2 +/+ group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the Sidt2 -/-+LPS group were higher than those in the Sidt2 -/- group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the Sidt2 -/-+LPS group were higher than those in the Sidt2 +/++LPS group. These data suggested that deletion of the Sidt2 gene changed the three inflammatory signal pathways, eventually leading to the damage of glomerular mesangial cells in mice.

[Methylation of CHD5 Gene Promoter Regulates p19Arf/p53/p21Cip1 Pathway to Facilitate Pathogenesis of Acute Myeloid Leukemia].

OBJECTIVE: To investigate the methylation status of CHD5 gene promoter in bone marrow from acute myeloid leukemia (AML) patients, and the underlying mechanism for initiating the pathogenesis of AML via p19Arf/p53/p21Cip1 pathway. METHODS: Methylation status of the CHD5 gene promoter was detected by using methylation-specific polymerase chain reaction (MSPCR) in bone marrow from AML patients, and the iron-deficiency anemia (IDA) samples were served as control. The expression of CHD5, p19Arf, p53 and p21Cip1 was determined by real-time quantitative reverse transcriptase PCR and Western blot. RESULTS: The methylation of CHD5 gene in bone marrow from AML patients increased significantly (39.06%) as compared with control group (6.67%). The methylation of CHD5 gene significantly correlated with chromosome karyotype differentiation (P＜0.01), but did not correlate with the patient's sex, age and clinical classification (P＞0.05). The mRNA expression of CHD5 gene in AML decreased, compared with control group, the mRNA and protein expression of p19Arf, p53 and p21Cip1 in AML with CHD5 methylation promoter decreased. CONCLUSION: The hypermeltylation of CHD5 gene promoter in AML patients can lead to decrease of CHD5, p19Arf, p53 and p21Cip1 expression levels which may reduce the inhibitory effect on proliferation of leukemia cells through the regulation of p19Arf, p53 and p21Cip1 pathway, thus promotes the occurence of AML.

Microstructure changes in whiter matter relate to cognitive impairment in Wilson's disease.

Purpose: Wilson's disease (WD) is a genetic disorder of copper metabolism with pathological copper accumulation in the brain. The purpose of the present study was to evaluate the relationship between the damaged white matter and the impaired cognitive function in WD patients. Materials and methods: Thirty WD adolescents and thirty age- and sex-matched healthy controls (HC) were enrolled. All subjects had received brain MRI, including conventional and diffusion-tensor imaging (DTI) scans. The DTI parameter of fractional anisotropy (FA) was calculated by diffusion kurtosis estimator software. The t test was used to compare the differences between two groups. The correlation between cognitive function and whiter matter disorders were analyzed by linear regression. The results of FA parameter and MD parameter intergroup analysis were both corrected with False Discovery Rate (FDR) simulations by SPSS. Results: WD adolescents showed significantly lower scores of time-based prospective memory (TBPM) and verbal fluency test (VFT) compared with HC. We found significantly higher FA in the right thalamus, right lentiform nucleus, left thalamus, left lentiform nucleus, and brain stem in WD adolescents. Besides, WD adolescents exhibited significantly lower FA in right cerebellum and cingulum and left middle frontal lobe compared with controls (P<0.05). There were significantly negative correlations between FA in bilateral lentiform and thalamus and cognitive impairment in WD adolescents (P<0.05). Conclusion: The whiter matter of WD adolescents was impaired and mainly distributed in subcortical brain regions. The impaired cognitive function was affected by the damaged whiter matter. The present study may be helpful for recognition and understanding of WD.

A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71.

In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD+-specific (NAD-IDH) or NADP+-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD+-specific and showed no detectable activity with NADP+. The Km values of the enzyme for NAD+ were 262.6±7.4 µM or 309.1±11.2 µM with Mg2+ or Mn2+ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP+ was approximately 24-fold higher than that for NAD+, suggesting that ClIDH is an NAD+-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn2+. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD+-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs.