The low-molecular-weight fucosylated chondroitin sulfate (LFCS) was prepared from native fucosylated chondroitin sulfate (FCS), which was extracted and isolated from sea cucumber Cucumaria frondosa, and the anti-cancer mechanism of LFCS on mouse Lewis lung carcinoma (LLC) was investigated. The results showed that LFCS remarkably inhibited LLC growth and metastasis in a dose-dependent manner. LFCS induced cell cycle arrest by increasing p53/p21 expression and apoptosis through activation of caspase-3 activity in LLC cells. Meanwhile, LFCS suppressed the expression of vascular endothelial growth factor (VEGF), increased the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and downregulated the matrix metalloproteinases (MMPs) level. Furthermore, LFCS significantly suppressed the activation of ERK1/2/p38 MAPK/NF-κB pathway, which played a prime role in expression of MMPs. All of these data indicate LFCS may be used as anti-cancer drug candidates and deserve further study.

We aimed to investigate the influence of transient receptor potential channel 3 (TRPC3) on lipopolysaccharide-induced (LPS) preterm delivery mice.

Functional magnetic resonance imaging (fMRI) studies of motor feedback have suggested that brain activity in the ultra-low frequency band (0-0.01Hz) may be physiologically significant for various feedback conditions, i.e., real and sham feedback. However, the functional role of the ultra-low frequency band of brain activity during the feedback procedure remains unclear. Here, we carried out an fMRI study of continuous feedback (8min) of finger force and assessed two important properties of brain activity: the fluctuation amplitude and local synchronization in the ultra-low frequency band. Two intriguing results were obtained: (1) real feedback recruited a stronger fluctuation amplitude and local synchronization in the basal ganglia compared with sham feedback; however, no significant correlation was found between the two properties across subjects; and (2) the behavioral performance was significantly correlated with the fluctuation amplitude but was not correlated with local synchronization in the basal ganglia. These findings contribute to characterization of the functional role of brain activity in the ultra-low frequency band and further suggest that the fluctuation amplitude and local synchronization in the basal ganglia may contribute differently to motor feedback.

Massive parallel sequencing enables identification of numerous genetic variants in mutant organisms, but determining pathogenicity of any one mutation can be daunting. The most commonly studied preclinical model of retinitis pigmentosa called the "rodless" (rd1) mouse is homozygous for two mutations: a nonsense point mutation (Y347X) and an intronic insertion of a leukemia virus (Xmv-28). Distinguishing which mutation causes retinal degeneration is still under debate nearly a century after the discovery of this model organism. Here, we performed gene editing using the CRISPR/Cas9 system and demonstrated that the Y347X mutation is the causative variant of disease. Genome editing in the first generation produced animals that were mosaic for the corrected allele but still showed neurofunction preservation despite low repair frequencies. Furthermore, second-generation CRISPR-repaired mice showed an even more robust rescue and amelioration of the disease. This predicts excellent outcomes for gene editing in diseased human tissue, as Pde6b, the mutated gene in rd1 mice, has an orthologous intron-exon relationship comparable with the human PDE6B gene. Not only do these findings resolve the debate surrounding the source of neurodegeneration in the rd1 model, but they also provide the first example of homology-directed recombination-mediated gene correction in the visual system.

Ligand-mediated prostate cancer (PCa)-targeting gene delivery is one of the focuses of research in recent years. Our previous study reported the successful preparation of aptamer-modified nanoparticles (APT-NPs) in our laboratory and demonstrated their PCa-targeting ability in vitro. However, the mechanism underlying this PCa-targeting effect and their anticancer ability in vivo have not yet been elucidated. The objective of this study was to assess the feasibility of using APT-NPs to deliver micro RNA (miRNA) systemically to PCa cells, to testify their tumor-targeting efficiency, and to observe their biodistribution after systemic administration to a xenograft mouse model of PCa. In addition, the effect of APT depletion and endocytosis inhibitors on cellular uptake was also evaluated quantitatively in LNCaP cells to explore the internalization mechanism of APT-NPs. Finally, blood chemistry, and renal and liver function parameters were measured in the xenograft mouse model of PCa to see whether APT-NPs had any demonstrable toxicity in mice in vivo. The results showed that APT-NPs prolonged the survival duration of the PCa tumor-bearing mice as compared with the unmodified NPs. In addition, they had a potential PCa-targeting effect in vivo. In conclusion, this research provides a prototype for the safe and efficient delivery of miRNA expression vectors to PCa cells, which may prove useful for preclinical and clinical studies on the treatment of PCa.

The Hedgehog (Hh) signaling pathway not only plays important roles in embryogenesis and adult tissue homeostasis, but also in tumorigenesis. Aberrant Hh pathway activation has been reported in a variety of malignant tumors including colon carcinoma. Here, we sought to investigate the regulation of the Hh pathway transcription factor Gli1 by arsenic trioxide and phosphoinositide 3-kinase (PI3K) inhibitor LY294002 in colon carcinoma cells. We transfected cells with siGli1 and observed a significant reduction of Gli1 expression in HCT116 and HT29 cells, which was confirmed by quantitative real-time polymerase chain reaction and Western blots. Knocking down endogenous Gli1 reduced colon carcinoma cell viability through inducing cell apoptosis. Similarly, knocking down Gli2 using short interfering RNA impaired colon carcinoma cell growth in vitro. To elucidate the regulation of Gli1 expression, we found that both Gli inhibitor arsenic trioxide and PI3K inhibitor LY294002 significantly reduced Gli1 protein expression and colon carcinoma cell proliferation. Arsenic trioxide treatment also reduced Gli1 downstream target gene expression, such as Bcl2 and CCND1. More importantly, the inhibition of Hedgehog-Gli1 by arsenic trioxide showed synergistic anticancer effect with the PI3K inhibitor LY294002 in colon carcinoma cells. Our findings suggest that the Hh pathway transcription factor Gli1 is involved in the regulation of colon carcinoma cell viability. Inhibition of Hedgehog-Gli1 expression by arsenic trioxide and PI3K inhibitor synergistically reduces colon cancer cell proliferation, indicating that they could be used as an effective anti-colon cancer combination therapy.

In our previous studies, we prepared novel oligomannuronate-chromium(III) complexes (OM2, OM4) from marine alginate, and found that these compounds sensitize insulin action better than oligomannuronate(OM), chromium, and metformin in C2C12 skeletal muscle cells. In the present study, we studied their effects on mitochondrial biogenesis, lipid metabolism, and the underlying molecular mechanisms in differentiated 3T3-L1 adipocytes.

Fabry disease (FD) is a rare X-linked α-galactosidase A (GLA) deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide (Gb3) in a variety of cell types. Here, we report a novel splicing mutation (c.801 + 1G > A) that results in alternative splicing in GLA of a FD patient with variable phenotypic presentations of renal involvement. Sequencing of the RT-PCR products from the patient's blood sample reveals a 36-nucleotide (nt) insertion exists at the junction between exons 5 and 6 of the GLA cDNA. Splicing assay indicates that the mutated minigene produces an alternatively spliced transcript which causes a frameshift resulting in an early termination of protein expression. Immunofluorescence shows puncta in cytoplasm for mutated GLA whereas uniform staining small dots evenly distributed inside cytoplasm for wild type GLA in transfected HeLa cells. The increased senescence and decreased GLA enzyme activity suggest that the abnormalities might be due to the altered localization which further might result from the lack of the C-terminal end of GLA. Our study reveals the pathogenesis of splicing mutation c.801 + 1G > A to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.

Transforming growth factor-β1 (TGF-β1) alters the fibroblast phenotype by promoting transdifferentiation into myofibroblasts, which exhibit the ability to promote collagen synthesis and extracellular matrix (ECM) deposition, thereby playing a significant role in the pathology of silicosis. In this study, we investigated the regulatory mechanisms involved in myofibroblast transdifferentiation. Two-dimensional gel electrophoresis showed that Rho GDP-dissociation inhibitor α (RhoGDIα) was upregulated following myofibroblast transdifferentiation stimulated by TGF-β1. We hypothesised that RhoGDIα may induce myofibroblast transdifferentiation and thus result in silicosis. Accordingly, the biological significance of RhoGDIα in cell proliferation and apoptosis was investigated by deletion of RhoGDIα in MRC-5 cells. In addition, a mechanistic study showed that fasudil, an inhibitor of the RhoA/Rho kinase (ROCK) signalling pathway, reduced the levels of RhoGDIα, RhoA, and phospho-myosin phosphatase （phospho-MYPT） in MRC-5 cells and silicosis model rats. Knockdown of RhoGDIα inhibited myofibroblast transdifferentiation and collagen deposition through RhoGDIα/RhoA/ROCK signalling in silicosis model mice. Overall, downregulation of RhoGDIα may significantly promote cell apoptosis and inhibit cell growth, resulting in reversal of myofibroblast transdifferentiation by RhoA/ROCK in vitro and in vivo. These data will facilitate further exploration of the potential use of RhoGDIα as a target for silicosis therapy.

The present study aimed to investigate the expression of the forkhead box protein M1 (FOXM1) in the placenta of patients with preeclampsia, and its effect on trophoblasts. A total of 28 patients with preeclampsia and 30 patients without preeclampsia (controls) who underwent cesarean section and were admitted to the Affiliated Hospital of Qingdao University between June 2013 and September 2016 were enrolled in the present study. The expression of FOXM1 in placental tissues was examined by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. HTR8/SVneo cells were used to measure the in vitro expression of the vascular endothelial growth factor (VEGF). The results demonstrated that FOXM1 expression was downregulated in the placental tissues of patient with preeclampsia (P<0.05). Following the silencing of FOXM1 expression, the proliferation of HTR8/SVneo cells was suppressed. The results of flow cytometry demonstrated that proportion of HTR8/SVneo cells in the G0/G1 phase and the proportion of apoptotic cells increased. The expression of the apoptosis regulator BCL-2, as well as the expression of VEGF mRNA and protein expression were also downregulated following FOXM1 silencing. FOXM1 may therefore promote the development of preeclampsia via the VEGF signaling pathway.

Blood urea nitrogen (BUN) is a surrogate marker for neurohormonal activation, but the association between BUN and the post-discharge prognosis in elderly patients with acute decompensated heart failure (ADHF) is not well defined. We explored the association between BUN and post-discharge all-cause mortality in 652 elderly patients (73.9 ± 7.8 yr) with ADHF. All patients were followed for a mean duration of 32 months (12-69 months). BUN was analyzed both as a continuous variable and according to two categories: low BUN group (BUN < 15.35 mmol/L, N = 361) and high BUN group (BUN ≥ 15.35 mmol/L, N = 291). The risk of all-cause mortality increased by 1.6% per 1 mmol/L increase in BUN concentration when BUN was used as a continuous variable [hazard ratio (HR): 1.016, 95% confidence interval (CI): 1.006-1.026, p = 0.002]. BUN maintained an independent and significant positive correlation with all-cause mortality as a categorical variable (HR: 1.355, 95% CI: 1.023-1.794, p = 0.034 for the high BUN group). The BUN C-statistic for predicting all-cause mortality was 0.624 (95% CI: 0.585-0.661). The cut-off value for BUN was 15.35 mmol/L with sensitivity of 0.58 and specificity of 0.63. The prognostic performance of BUN was similar to brain natriuretic peptide (BNP) for predicting all-cause mortality (C-statistic: z = 0.044, p = 0.965). These results suggest that BUN is an independent predictor of post-discharge all-cause mortality in elderly patients with ADHF and its prognostic performance was similar to that of BNP.

The cotton-melon aphid, Aphis gossypii Glover, is a worldwide-spreading species, and pesticide-resistant populations are increasing rapidly. In this study, investigations were performed based on Illumina HiSeq sequencing of the 16S rDNA V4 region for the bacterial communities embodied as intracellular symbionts under natural and in pesticide-treated populations of A. gossypii. The results revealed that more than 82% of bacterial communities belonged to the phylum Proteobacteria in which the maximum proportion (53.24%) was of the genus Arsenophonus; Hamiltonella composed 22.31; and 1.37% was of the genus Acinetobacter. The relative abundance of Hamiltonella was obvious, vertically transmitted, divided into two groups, and its infection influenced the bacterial communities in A. gossypii. Symbiont density and composition were changed in samples tested on different days. Azadirachtin and phoxim influenced on the composition of bacterial communities. Different biomarkers were used for pesticide-treated samples with LEfSe results. These findings will increase awareness regarding bacterial communities in naturally occurring populations of A. gossypii and pave the way to study the relationship between symbionts and pesticide resistance.

The present study aimed to investigate the role of leptin in the growth of breast cancer. In the present study, expression of leptin in tumor tissues and adjacent tissues was detected by reverse transcription-quantitative polymerase chain reaction. Serum leptin in patients with breast cancer and normal healthy controls was detected by ELISA. Different concentrations of leptin were used to treat breast cancer cells, and cell proliferation was detected by cell proliferation assay. Interactions between leptin and Wnt/β-catenin were evaluated by western blotting. It was demonstrated that expression of leptin mRNA was significantly higher in tumor tissues than in adjacent tissues in 43 out of 48 patients. Serum level of leptin was significantly higher in patients with breast cancer than in normal controls. Leptin promoted the proliferation of breast cancer cells in a dose-dependent manner. Leptin significantly increased the expression level of β-catenin in breast cancer cells. Treatment with Wnt inhibitor reduced the enhancing effects of leptin on proliferation of breast cancer cells. The results of the present study therefore indicate that leptin may promote the growth of breast cancer by upregulating the Wnt/β-catenin pathway.

Molecular characterization of the MSP2/P44 protein of Anaplasma phagocytophilum may determine not only if the bacterium is capable of invading hosts but also whether it generates antigenic variation for the purpose of escaping the host immune response, resulting in various pathologic injuries and serious clinical outcomes. Chinese anaplasmosis patients usually present with serious manifestations, and the fatality rate is as high as 26.5%. In this study, we amplified, cloned and sequenced the msp2/p44 genes of three Chinese A. phagocytophilum isolates from Laizhou Bay, Shandong Province, where human granulocytic anaplasmosis (HGA) patients present severe clinical manifestations, and analyzed their genetic characterization and structural features. We also compared them with the HZ and Webster A. phagocytophilum strains. The sequences for both strains are available in GenBank. Analyses indicated that Chinese A. phagocytophilum isolates were significantly different from the HZ and Webster strains in terms of nucleotide sequences, amino acid sequences and protein secondary and tertiary structures. Moreover, the number of immunologic B-cell epitopes (19) of the MSP2 protein of the Chinese isolates was higher than that of the A. phagocytophilum strains HZ (16) and Webster (9). This genetic diversity of the MSP2/P44 protein of Chinese A. phagocytophilum isolates might be relevant and might have serious clinical outcomes. This observation could provide a clue to further understand the pathogenesis of Chinese A. phagocytophilum.

Comparative proteomics of the multiple strains within the same species can reveal the genetic variation and relationships among strains without the need to assess the genomic data. Similar to comparative genomics, core proteome and pan proteome can also be obtained within multiple strains under the same culture conditions. In this study we present the core proteome and pan proteome of four epidemic Salmonella Paratyphi A strains cultured under laboratory culture conditions. The proteomic information was obtained using a Two-dimensional gel electrophoresis (2-DE) technique. The expression profiles of these strains were conservative, similar to the monomorphic genome of S. Paratyphi A. Few strain-specific proteins were found in these strains. Interestingly, non-core proteins were found in similar categories as core proteins. However, significant fluctuations in the abundance of some core proteins were also observed, suggesting that there is elaborate regulation of core proteins in the different strains even when they are cultured in the same environment. Therefore, core proteome and pan proteome analysis of the multiple strains can demonstrate the core pathways of metabolism of the species under specific culture conditions, and further the specific responses and adaptations of the strains to the growth environment.

BACKGROUND This study was proposed to compare the efficacy and safety of GTM-1, Rapamycin (Rap), and Carbamazepine (CBZ) in managing Alzheimer disease (AD). The impact of the above mentioned therapeutic drugs on autophagy was also investigated in our study. MATERIAL AND METHODS Firstly, 3×Tg AD mice were randomly allocated into 4 groups (each group with 10 mice), in which AD mice were separately treated with dimethylsulfoxide (DMSO, vehicle group), GTM-1 (6 mg/kg), Rap (1 mg/kg), and CBZ (100 mg/kg). Then spatial memory and learning ability of mice was tested using the Morris water maze. Routine blood tests were performed to evaluate the toxicity of these drugs. Amyloid-β42 (Aβ42) concentration was detected by ELISA and immunohistochemistry. Proteins related to autophagy were detected by Western blot. RESULTS GTM-1, Rap, and CBZ significantly improved the spatial memory of 3×Tg AD mice compared to that in the vehicle group (all P<0.05). Moreover, this study revealed that CBZ dosage was related to toxicity in mice. All of the above drugs significantly increased the expression of LC3-II and reduced Aβ42 levels in hippocampi of 3×Tg AD mice (all P<0.05). On the other hand, neither GTM-1 nor CBZ had significant influence on the expression of proteins on the mTOR pathway. CONCLUSIONS GTM-1 can alleviate the AD syndrome by activating autophagy in a manner that is dependent on the mTOR pathway and it therefore can be considered as an alternative to Rap.

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG-PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.

Biologically inert gases play important roles in the biological functionality of proteins. However, researchers lack a full understanding of the effects of these gases since they are very chemically stable only weakly absorbed by biological tissues. By combining X-ray fluorescence, particle sizing and molecular dynamics (MD) simulations, this work shows that the aggregation of these inert gases near the hydrophobic active cavity of pepsin should lead to protein deactivation. Micro X-ray fluorescence spectra show that a pepsin solution can contain a high concentration of Xe or Kr after gassing, and that the gas concentrations decrease quickly with degassing time. Biological activity experiments indicate a reversible deactivation of the protein during this gassing and degassing. Meanwhile, the nanoparticle size measurements reveal a higher number of "nanoparticles" in gas-containing pepsin solution, also supporting the possible interaction between inert gases and the protein. Further, MD simulations indicate that gas molecules can aggregate into a tiny bubble shape near the hydrophobic active cavity of pepsin, suggesting a mechanism for reducing their biological function.

Q fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP) assay to identify C. burnetii rapidly and sensitively.

Inflammasome activation plays key roles in host defense, but also contributes to the pathogenesis of auto-inflammatory, and neurodegenerative diseases. As autophagy is connected with both the innate and adaptive immune systems, autophagic dysfunction is also closely related to inflammation, infection, and neurodegeneration. Here we identify that lincRNA-Cox2, previously known as a mediator of both the activation and repression of immune genes expression in innate immune cells, could bind NF-κB p65 and promote its nuclear translocation and transcription, modulating the expression of inflammasome sensor NLRP3 and adaptor ASC. Knockdown of lincRNA-Cox2 inhibited the inflammasome activation and prevented the lincRNA-Cox2-triggered caspase-1 activation, leading to decreased IL-1β secretion and weakened TIR-domain-containing adapter-inducing interferon-β (TRIF) cleavage, thereby enhancing TRIF-mediated autophagy. Elucidation of the link between lincRNA-Cox2 and the inflammasome-autophagy crosstalk in macrophage and microglia reveals a role for lncRNAs in activation of NLRP3 inflammasome and autophagy, and provides new opportunities for therapeutic intervention in neuroinflammation-dependent diseases.