Copper chelators promote nonamyloidogenic processing of AßPP via MT1/2 /CREB-dependent signaling pathways in AßPP/PS1 transgenic mice.

Copper is essential for the generation of reactive oxygen species (ROS), which are induced by amyloid-ß (Aß) aggregation; thus, the homeostasis of copper is believed to be a therapeutic target for Alzheimer's disease (AD). Although clinical trials of copper chelators show promise when applied in AD, the underlying mechanism is not fully understood. Here, we reported that copper chelators promoted nonamyloidogenic processing of AßPP through MT1/2 /CREB-dependent signaling pathways. First, we found that the formation of Aß plaques in the cortex was significantly reduced, and learning deficits were significantly improved in AßPP/PS1 transgenic mice by copper chelator tetrathiomolybdate (TM) administration. Second, TM and another copper chelator, bathocuproine sulfonate (BCS), promoted nonamyloidogenic processing of AßPP via inducing the expression of ADAM10 and the secretion of sAßPPα. Third, the inducible ADAM10 production caused by copper chelators can be blocked by a melatonin receptor (MT1/2 ) antagonist (luzindole) and a MT2 inhibitor (4-P-PDOT), suggesting that the expression of ADAM10 depends on the activation of MT1/2 signaling pathways. Fourth, three of the MT1/2 -downstream signaling pathways, Gq/PLC/MEK/ERK/CREB, Gs/cAMP/PKA/ERK/CREB and Gs/cAMP/PKA/CREB, were responsible for copper chelator-induced ADAM10 production. Based on these results, we conclude that copper chelators regulate the balance between amyloidogenic and nonamyloidogenic processing of AßPP via promoting ADAM10 expression through MT1/2 /CREB-dependent signaling pathways.

AßPP-tau-HAS1 axis trigger HAS1-related nuclear speckles and gene transcription in Alzheimer's disease.

As the backbone of the extracellular matrix (ECM) and the perineuronal nets (PNNs), hyaluronic acid (HA) provides binding sites for proteoglycans and other ECM components. Although the pivotal of HA has been recognized in Alzheimer's disease (AD), few studies have addressed the relationship between AD pathology and HA synthases (HASs). Here, HASs in different regions of AD brains were screened in transcriptomic database and validated in AßPP/PS1 mice. We found that HAS1 was distributed along the axon and nucleus. Its transcripts were reduced in AD patients and AßPP/PS1 mice. Phosphorylated tau (p-tau) mediates AßPP-induced cytosolic-nuclear translocation of HAS1, and negatively regulated the stability, monoubiquitination, and oligomerization of HAS1, thus reduced the synthesis and release of HA. Furthermore, non-ubiquitinated HAS1 mutant lost its enzyme activity, and translocated from the cytosol into the nucleus, forming nuclear speckles (NS). Unlike the splicing-related NS, less than 1 % of the non-ubiquitinated HAS1 co-localized with SRRM2, proving the regulatory role of HAS1 in gene transcription, indirectly. Thus, differentially expressed genes (DEGs) related to both non-ubiquitinated HAS1 mutant and AD were screened using transcriptomic datasets. Thirty-nine DEGs were identified, with 64.1 % (25/39) showing consistent results in both datasets. Together, we unearthed an important function of the AßPP-p-tau-HAS1 axis in microenvironment remodeling and gene transcription during AD progression, involving the ubiquitin-proteasome, lysosome, and NS systems.

Construction and Evaluation of a Nomogram to Predict Gallstone Disease Based on Body Composition.

Purpose: We aimed to analyze the body composition characteristics of gallstone disease (GD) patients with bioelectrical impedance analysis (BIA) and to construct a nomogram to predict GD based on body composition. Methods: Patients with or without symptomatic cholecystolithiasis or choledocholithiasis diagnosed in Inner Mongolia People's Hospital from July 2020 to December 2021 were selected as the case group, and healthy subjects during the same period were selected as the control group. The body composition of the two groups was determined by BIA. The risk predictors for GD were extracted to construct a nomogram based on regression analysis. ROC curves were used to evaluate the predictive power of the nomogram, and calibration curves were drawn to evaluate the consistency of the model. The bootstrap method was used to verify the model and evaluate the generalizability of the model. Results: A total of 1000 individuals were recruited for the study, including 500 GD cases and 500 controls, to evaluate body composition. Multivariate logistic regression analysis showed that sex (OR = 2.292, 95% CI: 1.436-3.660), BMI (OR = 1.828, 95% CI: 1.738-1.929), body fat percentage (BFP) (OR = 1.904, 95% CI: 1.811-2.205) and waist circumference (WC) (OR = 1.934, 95% CI: 1.899-1.972) were risk predictors of GD. The AUC was 0.770 (95% CI: 0.741-0.799). The calibration curve showed that the C-index was 0.767. The prediction model was validated internally with 1000 bootstrap resamples. The accurate value was 0.72, and the kappa value was 0.43. All of the indices indicated that the model was well constructed and could be used to predict the incidence of GD. Conclusion: A nomogram model of gallstone disease based on sex, BMI, BFP and WC was constructed with good discrimination, calibration and generalizability and can be used for the noninvasive and convenient prediction of gallstone disease in the general population.

Formulation, characterization and hypersensitivity evaluation of an intravenous emulsion loaded with a paclitaxel-cholesterol complex.

The objective of this paper was to develop a novel Cremophor-free, autoclave stable, intravenous emulsion for paclitaxel (PACE). A paclitaxel-cholesterol complex was used as the drug carrier to improve the solubility of paclitaxel in the oil phase of emulsions. The complex and PACE were prepared by rotary evaporation and high-pressure homogenization, respectively. Effects of oil phases, emulsifiers and pH values on the characteristics of PACE were investigated. PACE was characterized with regard to its appearance, morphology, osmolality, pH value, particle size, zeta potential, encapsulation efficiency and stability. Hypersensitivity was evaluated by guinea pig hypersensitivity reaction. The final formulation was composed of the complex, soybean oil, medium-chain triglyceridel, soybean lecithin, poloxamer 188 and glycerol. The resulting PACE had an encapsulation efficiency of 97.3% with a particle size of 135 nm and a zeta potential of -38.3 mV. Osmolality and pH of the formulation were 383 mOsmol/kg and 4.5, respectively. The formulation survived autoclaving at 115 °C for 30 min and remained stable for at least 12 months at 6 °C. PACE also exhibited a better tolerance than an equal dose of Cremophor-based paclitaxel injection in guinea pigs, as no obvious hypersensitivity reaction was observed. These results suggested that PACE has a great potential for industrial-scale production and clinical applications.

[Identification of biomarkers for early detection in gastric cancer and its clinical biological significance.].

OBJECTIVE: To systematically understand the cellular and molecular mechanism of gastric cancer (GC) development and to discover early diagnosis and predictive biomarkers, which will be used for early diagnosis and novel treatment targets. METHODS: 70 mer 22 K-oligonucleotide microarrays and bioinformatic analysis were conducted to recognize gene expression profiles in GC and normal appearing tissue (NAT). The control group was collected from non-tumor patients including 20 specimen mixture as a common reference (CR) and 5 individuals as additional control. Our results showed that 837 different expression genes (DEGs) were identified in GC while 570 DEGs were in NATs by Bayesian analysis (P<0.001, Fold change>2.0) as compared respectively with CR. An interesting finding is that we identified 67 over-expressed genes in both GC and NAT tissues, and these gene expression alterations could not be detected by comparison of GC with NATs, which were normally used in routine experiment design. Most of these genes were involved in the control of cell proliferation, metabolism and differentiation. RESULTS: These differential expressed genes were confirmed at mRNA and protein levels in primary tumors using RT-PCR and immunohistochemistry (IHC). The results showed that three genes, EGR1, CYR61 and ADAMTS1 were over expressed in both GC and NATs at mRNA level. These results were consistent with oligo microarray data. Another interesting finding is that these three genes were also over-expressed in intestinal metaplasia (IM) and dysplasia (DYS), which indicated that these three genes might be potential biomakers for early detection of GC. CONCLUSION: Through the systematic analysis of gene expression profiles in GC tissues, NAT and CR normal tissues, we identified a group of genes over-expressed both in GC and precancerous lesions, which might be potential biomarkers for early GC diagnosis.

Cyr61 overexpression induced by interleukin 8 via NF-kB signaling pathway and its role in tumorigenesis of gastric carcinoma in vitro.

Cyr61 (CCN1) is a multifunctional matricellular protein in bridging inflammation and cancer, involved in many biological functions such as tumorigenesis and carcinogenesis. The role of Cyr61 in gastric cancer (GC) has not been fully understood and needs to be investigated and clarified. We examined Cyr61 expression in 6 GC cell lines and stable transfection of recombinants in to BGC823 specifically down regulated the Cyr61 mRNA and protein expression shown by the analysis with western blot, RT-PCR, western blot and immunofluorescence assay. The cells treated with siRNA shown markedly reduced activity in growth, migration and invasion compared with parental BGC823 cells as well as mock transfectants. The Cyr61 deficient cells demonstrated significantly inhibited colony formation in soft agar and reduced tumorigenicity was showed in nude mice, NF-kB pathway evidently inactivated respectively. However, under the stimulation of IL-8, the siRNA-treated cells can restore the capacity of proliferation and invasion. IL-8 can induce the high expression of Cyr61 and MMP11 through NF-kB signal pathway. Silencing of Cyr61 can inhibit or minimize the proliferation and invasiveness of gastric cancer cell. The results imply that Cyr61 enhance the proliferation and invasion of gastric cancer cells and this process is partially modulated by the IL-8 up-regulation. Cyr61 may mediate the proliferation and development of gastric carcinoma.

Expression status of ataxia-telangiectasia-mutated gene correlated with prognosis in advanced gastric cancer.

Many studies have revealed the ATM alterations involved in cancer development and progression. In order to elucidate ATM deficiency in advanced GC and its clinical significance, a total of 20 exons of ATM gene, including frequently reported variations, were screened in 40 advanced primary GC and matched normal tissues using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing analysis. Furthermore, ATM mRNA level was analyzed using Real-time RT-PCR and in situ hybridization, and protein expression and phosphorylation at Ser1981 were measured by immunohistochemical assessment in tissue microarray of GC. Five variants were identified in 6 of 40 cases (15%), but no hot spot of variation was detected. However, decreased expression and phosphorylation of ATM were consistently presented in tumors. In a cohort of 70 GC samples, low level of phosphorylated ATM was significantly correlated with poor differentiation, lymph node metastasis and poor 5-year survival (P<0.05). These results indicated that ATM phosphorylation status might be a prognostic marker for individual therapy in advanced GC patients.

[Alteration of early growth response 1 expression in gastroenterological cancers and its biological significance].

OBJECTIVE: To investigate the expression of early growth response 1 (EGR1) in gastroenterological cancers and its significance in the pathogenesis. METHODS: RT-PCR was used to determine the expression of EGR1 in normal gastric mucosa tissues from 20 non-tumor patients, gastroenterological tumor tissues and matched para-cancer tissues normal morphologically. RT-PCR and Western blotting were used to analyze the mRNA and protein expression of EGR1 in 20 cancer cell lines. Immunohistochemistry (IHC) was preformed to measure the expression level of EGR1 protein on tissue microarray including 179 tumors and 159 normal tissues. RESULTS: EGR1 was overexpressed in gastric cancer (GC) and its matched adjacent normal tissue (ANT), but not expressed or expressed at a low level in the normal gastric mucosa from the non-tumor patients, which was consistent with the GC gene expression profiling data. Overexpression of EGR1 was seen in the 20 cancer cell lines at both mRNA and protein levels. IHC showed strong positive staining of EGR1 protein in the cytoplasm of both tumor tissues and matched normal tissues and showed negative or weaker nuclear staining in the normal gastric mucosa tissues from non-tumor patients. Overexpression of EGR1 was detected in 87% (49/56) of the GC tissues and 79% (43/54) of their ANTs; 83% (43/52) of the hepatocellular carcinoma tissues and 79% (32/42) of their ANTs; 78% (41/52) of colorectal cancer tissues and 63% (22/35) of their ANTs; and 79% (15/19) of the squamous cell carcinoma tissues and 78% (14/18) of their ANTs. CONCLUSION: EGR1 may be correlated with the abnormal proliferation of cells at the early stage of malignant transformation.

Tetrathiomolybdate Treatment Leads to the Suppression of Inflammatory Responses through the TRAF6/NFκB Pathway in LPS-Stimulated BV-2 Microglia.

Although the positive relationship between copper and Alzheimer's disease (AD) was reported by a lot of epidemiological data, the mechanism is not completely known. Copper is a redox metal and serves as a mediator of inflammation. Because the homeostasis of copper is altered in Aß precursor protein (APP) and presenilin 1 (PS1) transgenic (Tg) mice, the using of copper chelators is a potential therapeutic strategy for AD. Here we report that a copper chelator, tetrathiomolybdate (TM), is a potential therapeutic drug of AD. We investigated whether TM treatment led to a decrease of pro-inflammatory cytokines in vivo and in vitro, and found that TM treatment reduced the expression of iNOS and TNF-α in APP/PS1 Tg mice through up-regulating superoxide dismutase 1 (SOD1) activity. In vitro, once stimulated, microglia secretes a variety of proinflammatory cytokines, so we utilized LPS-stimulated BV-2 cells as the inflammatory cell model to detect the anti-inflammatory effects of TM. Our results indicated that TM-pretreatment suppressed the ubiquitination of TRAF6 and the activation of NFκB without affecting the expression of TLR4 and Myd88 in vitro. By detecting the activity of SOD1 and the production of reactive oxygen species (ROS), we found that the anti-inflammatory effects of TM could be attributed to its ability to reduce the amount of intracellular bioavailable copper, and the production of ROS which is an activator of the TRAF6 auto-ubiquitination. Hence, our results revealed that TM-treatment could reduce the production of inflammatory cytokines by the suppression of ROS/TRAF6/AKT/NFκB signaling pathway.

[Relations of transforming growth factor-beta1 expression to differentiation and prognosis of advanced gastric cancer].

OBJECTIVE: To clarify the correlation of transforming growth factor-beta1 (TGF-beta1) expression with the differentiation and prognosis of advanced gastric cancer (GC). METHODS: Whole genome expression chip hybridization, was used to detect the expression of TGF-beta1 and TGF-betaR1 in 20 specimens of intestinal-type GC and para-cancer tissues. RT-PCR and immunohistochemistry (IHC) analysis were used to detect the mRNA and protein expression of TGF-beta1 and TGF-betaR1 in 30 specimens of intestinal-type GC tissue and para-cancer tissues. The mixture of gastric mucosa tissues from 20 non-tumor patients was used as common reference. RESULTS: The expression level of TGF-beta1 and TGF-betaR-1 genes was higher in the GC tissues than in the para-cancer tissues. However, the expression of Smad gene family was not significantly different between the GC tissues and para-tumor normal tissues. TGF-beta1 gene expression and TGF-betaR1 gene expression were higher in the GC tissues. RT-PCR showed that both TGF-beta1 and TGF-betaR-1 genes were highly expressed in the mRNA level in 21 of the 30 CC patients IHC showed that TGF-beta1 protein was expressed mainly in the cytoplasm. 32 of the 90 specimens of GC tissue were highly positive in TGF-beta1 protein (64%), in comparison with the positive rate of 5% (1/20) in the para-cancer normal tissues. The TGF-beta1 protein expression rate of the highly and moderately differentiated GC tissues was 59% (59%, 23/39), significantly higher than that of the lowly differentiated GC tissues (18%, 9/51, P < 0.01). IHC showed that the TGF-beta R-I rate was 57% (42/74) in the well differentiated specimens, particularly 68% (26/38) in the highly differentiated specimens, and was 44% in the poorly differentiated GC (6/20, P < 0.05). Log rank test showed that the prognosis of the patients positive in TGF-beta1 was significantly better than those negative in TGF-beta1 (P = 0.0058). However, the survival rate did not differ significantly according to TGF-beta R-I expression (P = 0.8453). CONCLUSION: TGF-beta1 expression is significantly correlated with the differentiation degree of GC. Moreover, positive expression of TGF-beta1 is a favorable prognostic factor in advanced GC. Expression of TGF-beta1 may be an important preoperative prognostic variable for advanced GC.

Assessing winter oilseed rape freeze injury based on Chinese HJ remote sensing data.

The winter oilseed rape (Brassica napus L.) accounts for about 90% of the total acreage of oilseed rape in China. However, it suffers the risk of freeze injury during the winter. In this study, we used Chinese HJ-1A/1B CCD sensors, which have a revisit frequency of 2 d as well as 30 m spatial resolution, to monitor the freeze injury of oilseed rape. Mahalanobis distance-derived growing regions in a normal year were taken as the benchmark, and a mask method was applied to obtain the growing regions in the 2010-2011 growing season. The normalized difference vegetation index (NDVI) was chosen as the indicator of the degree of damage. The amount of crop damage was determined from the difference in the NDVI before and after the freeze. There was spatial variability in the amount of crop damage, so we examined three factors that may affect the degree of freeze injury: terrain, soil moisture, and crop growth before the freeze. The results showed that all these factors were significantly correlated with freeze injury degree (P<0.01, two-tailed). The damage was generally more serious in low-lying and drought-prone areas; in addition, oilseed rape planted on south- and west-oriented facing slopes and those with luxuriant growth status tended to be more susceptible to freeze injury. Furthermore, land surface temperature (LST) of the coldest day, soil moisture, pre-freeze growth and altitude were in descending order of importance in determining the degree of damage. The findings proposed in this paper would be helpful in understanding the occurrence and severity distribution of oilseed rape freeze injury under certain natural or vegetation conditions, and thus help in mitigation of this kind of meteorological disaster in southern China.

Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells.

AIM: To elucidate molecular mechanism of chemopreventive efficacies of garlic against human gastric cancer (HGC). METHODS: HGC cell line BGC823 was treated with Allitridi (a kind of garlic extract) and Allitridi-treated and parental BGC823 cDNA libraries were constructed respectively by using lambdaZAP II vector. cDNA Representational Difference Analysis (cDNA RDA) was performed using Bam H I cutting-site and abundant cDNA messages provided by the libraries. Northern blot analysis was applied to identify the obtained difference products. RESULTS: Two specific cDNA fragments were obtained and characterized to be derived from homo sapiens folate receptor alpha (FRalpha) gene and calcyclin gene respectively. Northern blot results showed a 4-fold increase in FRalpha gene expression level and 9-fold increase in calcyclin mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: The method of cDNA RDA based on cDNA libraries combines the high specificity of cDNA RDA with abundant cDNA messages in cDNA library; this expands the application of cDNA library and increases the specificity of cDNA RDA. Up-regulation of FRalpha gene and calcyclin gene expressions induced by Allitridi provide valuable molecular evidence for the efficacy garlic in treating HGC as well as other diseases.

Alteration of the ATM gene occurs in gastric cancer cell lines and primary tumors associated with cellular response to DNA damage.

Ataxia telangiectasia mutated (ATM) is the gene mutated in the genetic disorder ataxia telangiectasia (AT), the symptoms of which include sensitivity to radiation and an increased risk of cancer. ATM is a kinase involved in activating the appropriate damage-response pathway, leading to either cell-cycle arrest or apoptosis, and is therefore a key checkpoint molecule in regulating cell-cycle response to DNA damage and responsible for maintenance of genome integrity. However, little is known about the association of ATM mutations with human gastric cancer (HGC). In order to determine the mutation and mRNA expression changes of the ATM gene in HGC, we performed analyses by denaturing high-performance liquid chromatography (DHPLC), DNA sequencing and RT-PCR technique on 13 human gastric tumor cell lines and 30 cases of fresh tumor specimens matched normal tissue. We compared the potential effect of the ATM gene mutation and cell behavior including cell-cycle arrest and induction of apoptosis in the tumor cell lines MGC803 and BGC823 with and without ionizing radiation (IR) exposure. Our data show that frequent variations were observed at 10 exons and 2 cDNA fragments which covered 8 other exons of the ATM gene as 5 out of 13 on the cell lines (38.5%) and 2 out of 30 cases in the tissue specimens (6.7%). All point mutations were confirmed as base substitutions (5982T-C; 6620A-G; 8684G-G/A; 9389C-G) and deletions (1079delC) by use of DNA sequencing. Among the mutations, one was reported previously in breast cancer, the other five have not yet been reported. The expression of ATM was significantly lower in five cell lines (MGC803; MKN45; SGC7901; GES and SUN-1) than in two others (BGC823 and RF48). G2/M cell-cycle arrest and apoptosis were observed in ATM-deficient MGC803 cells challenged with IR. A transient up-regulation of p53 occurred 1h post-IR in BGC823 cells but not in MGC803 cells. Our findings suggest that ATM mutations might be a pathogenic factor for an increased risk of gastric cancer, and the dysfunction of ATM may lead to a hypersensitivity to ionizing radiation in gastric cancer cells, possibly by a p53-dependent pathway.

Spatio-temporal reconstruction of air temperature maps and their application to estimate rice growing season heat accumulation using multi-temporal MODIS data.

The accumulation of thermal time usually represents the local heat resources to drive crop growth. Maps of temperature-based agro-meteorological indices are commonly generated by the spatial interpolation of data collected from meteorological stations with coarse geographic continuity. To solve the critical problems of estimating air temperature (T(a)) and filling in missing pixels due to cloudy and low-quality images in growing degree days (GDDs) calculation from remotely sensed data, a novel spatio-temporal algorithm for T(a) estimation from Terra and Aqua moderate resolution imaging spectroradiometer (MODIS) data was proposed. This is a preliminary study to calculate heat accumulation, expressed in accumulative growing degree days (AGDDs) above 10 °C, from reconstructed T(a) based on MODIS land surface temperature (LST) data. The verification results of maximum T(a), minimum T(a), GDD, and AGDD from MODIS-derived data to meteorological calculation were all satisfied with high correlations over 0.01 significant levels. Overall, MODIS-derived AGDD was slightly underestimated with almost 10% relative error. However, the feasibility of employing AGDD anomaly maps to characterize the 2001-2010 spatio-temporal variability of heat accumulation and estimating the 2011 heat accumulation distribution using only MODIS data was finally demonstrated in the current paper. Our study may supply a novel way to calculate AGDD in heat-related study concerning crop growth monitoring, agricultural climatic regionalization, and agro-meteorological disaster detection at the regional scale.

Matrix metalloproteinase 11 depletion inhibits cell proliferation in gastric cancer cells.

Our previous study has shown that matrix metalloproteinase 11 (MMP11) is highly expressed in tumor cell lines and primary tumor of gastric cancer (GC). In order to reveal the correlation between expression of MMP11 and biological features of GC cell, we have constructed the recombinant plasmids producing hairpin small interfering RNA (siRNA) to target MMP11 mRNA using a vector-based RNA interference technology. Stable transfection of recombinants into GC cell line BGC823 specifically depleted the mRNA and protein of MMP11 as demonstrated by RT-PCR and Western blotting analysis. The siRNA-treated cells exhibited significantly decreased growth ability compared with mock transfectants and parental BGC823 cells. Furthermore, colony formation of MMP11 deficient cells was dramatically inhibited in soft agar and tumorigenicity was reduced in nude mice, respectively. These results provide new insights into the function of MMP11 and suggest that MMP11 may play an important role in the control of cell proliferation and tumor development in GC.