Homocysteine-potentiated Kelch-like ECH-associated protein 1 promotes senescence of neuroblastoma 2a cells via inhibiting ubiquitination of ß-catenin.

Elevated serum homocysteine (Hcy) level is a risk factor for Alzheimer's disease (AD) and accelerates cell aging. However, the mechanism by which Hcy induces neuronal senescence remains largely unknown. In this study, we observed that Hcy significantly promoted senescence in neuroblastoma 2a (N2a) cells with elevated ß-catenin and Kelch-like ECH-associated protein 1 (KEAP1) levels. Intriguingly, Hcy promoted the interaction between KEAP1 and the Wilms tumor gene on the X chromosome (WTX) while hampering the ß-catenin-WTX interaction. Mechanistically, Hcy attenuated the methylation level of the KEAP1 promoter CpG island and activated KEAP1 transcription. However, a slow degradation rate rather than transcriptional activation contributed to the high level of ß-catenin. Hcy-upregulated KEAP1 competed with ß-catenin to bind to WTX. Knockdown of both ß-catenin and KEAP1 attenuated Hcy-induced senescence in N2a cells. Our data highlight a crucial role of the KEAP1-ß-catenin pathway in Hcy-induced neuronal-like senescence and uncover a promising target for AD treatment.

Acinetobacter faecalis Sp. Nov., Isolated from Elephant Faeces.

A Gram-negative coccobacillus, YIM 103518T, isolated from wild elephant feces in Xishuangbanna, Yunnan Province, West China, was characterized and identified using a polyphasic taxonomic approach. The strain was strictly aerobic, non-motile, catalase-positive and oxidase-negative, colonies were round, convex, smooth, and pale yellow. The strain growth at 4-40 ℃ (optimum, 28 ℃), pH 6.0-10.0 (optimum, pH 7.0) and 0-4% NaCl (optimum, 0%) in culture medium YIM 38. The major fatty acids of strain YIM 103518T were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:0, and C18:1 ω9c. The predominant ubiquinone was Q-9. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and phospholipids. The 16S rRNA gene sequence showed moderate level of similarity with Acinetobacter portensis AC 877T (98.7%), Acinetobacter sichuanensis CCTCC AB 2018118T (97.1%), and Acinetobacter cumulans CCTCC AB 2018119T (97.1%). The G+C content of the genomic DNA was 36.5 mol%. Strain YIM 103518T showed an average nucleotide identity value of 86.6%, 77.3% and 78.5%, a digital DNA-DNA hybridizations value of 31.2%, 21.9% and 23.0% with the type strain of A. portensis, A. sichuanensis and A. cumulans based on draft genome sequences, respectively. The results of the phenotypic, chemotaxonomic and phylogenetic analyses, showed that strain YIM 103518T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter faecalis sp. nov. is proposed. The type strain is YIM 103518T (=CCTCC AB 2019201T = NBRC 114057T).

Metabacillus kandeliae sp. nov., isolated from the rhizosphere soil of a decayed mangrove plant Kandelia candel.

In this study, we isolated a novel Gram-stain-positive, aerobic, motile, rod-shaped bacterium, named GX 13764T, from the rhizosphere soil of a decayed mangrove plant Kandelia candel collected from Beihai, Guangxi, PR China, and characterized using a polyphasic taxonomic approach. The strain exhibited yellow-orange, round, convex, shiny, smooth, opaque and 2-3 mm diameter colonies on marine agar 2216 media after 3 days of incubation at 30 °C and was capable of growth at 4-45 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, pH 7.0) and 0-4 % NaCl (w/v; optimum, 2 %). The strain was positive for catalase and negative for the oxidase. The main cellular fatty acid was anteiso-C15:0, iso-C15:0, iso-C16:0 and iso-C14:0. The cell-wall peptidoglycan comprised meso-diaminopimelic acid and the main menaquinone was MK-7. The polar lipids included one diphosphatidylglycerol, one phosphatidylglycerol, two glycolipids, two unidentified phospholipids and three unidentified lipids. Based on 16S rRNA gene analysis, GX 13764T presented the highest sequence similarity to Metabacillus mangrovi KCTC 33872T (97.04 %). The DNA G+C content of the type strain was 44.2 mol%. The average nucleotide identity values between GX 13764T and M. mangrovi KCTC 33872T, Metabacillus idriensis DSM 19097T and Metabacillus indicus LMG 22858T were 69.39, 68.87 and 68.95 %, respectively, with digital DNA-DNA hybridization values of 19.9, 19.5 and 19.5 %, respectively. Based on the polyphasic data, strain GX 13764T should be nominated as a novel species of the genus Metabacillus, for which the name Metabacillus kandeliae sp. nov. is proposed. The type strain is GX 13764T (=MCCC 1K06654T=KCTC 43366T).

CDK6 Immunophenotype Implicates Potential Therapeutic Application of CDK4/6 Inhibitors in Urothelial Carcinoma.

Background: Infiltrating bladder urothelial carcinoma is the most common bladder malignancy with limited therapeutic options and poor prognosis. Identifying new therapeutic targets or strategies has important clinical significance. The data from public sources indicate poor prognosis in urothelial carcinoma cases with high CDK6 mRNA levels. Furthermore, studies have shown that CDK6 expression is elevated in urothelial carcinoma tissue compared to the surrounding urothelium, thus presenting a case for performing CDK4/6 inhibitor targeted research in urothelial carcinoma. However, a phase II trial showed that CDK4/6 inhibitors are not effective for advanced urothelial carcinoma, suggesting that case screening is important for targeted therapy. Objective: Immunohistochemistry (IHC) is simple and easy to perform and can be used to screen urothelial carcinoma cases with high CDK6 expression in clinical practice. The aim of this study was to determine the CDK6 expression threshold for positive cases. Methods: We evaluated the correlation between the H-score of CDK6 protein expression and survival or CDK6 mRNA level using RNA sequencing. The effects of different CDK4/6 inhibitors were tested on bladder carcinoma cell lines with different CDK6 expression levels. Results: The H-score, which predicts poor prognosis and reflects a high CDK6 mRNA level, was determined as the selection criterion for positive cases. Furthermore, we found that urothelial carcinoma cell lines with higher CDK6 expression levels displayed greater sensitivity to CDK4/6 inhibitors than cells with lower expression levels. Conclusions: IHC staining for CDK6 protein in urothelial carcinoma is proposed as a promising screening platform for CDK4/6 inhibitor targeted therapy.

Nakamurella leprariae sp. nov., isolated from a lichen sample.

A novel actinobacterium, YIM 132084T, was isolated from Lepraria sp. lichen collected from Yunnan province, south-west PR China and identified by a polyphasic taxonomic approach. The strain was Gram-stain-positive, aerobic, catalase-positive, oxidase-negative, non-motile and coccus-shaped. Colonies were round, convex, smooth and light orange yellow in color. It grew at 10-40 °C (optimum 28 °C), at pH 6.0-11.0 (optimum pH 7.0) and in the presence of 0-4% NaCl (optimum 0%). Strain YIM 132084T comprised diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as the major polar lipids, MK-8(H4) as the predominant menaquinone, and anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0 as major fatty acids. Strain YIM 132084T had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and mannose, ribose, glucose and rhamnose as whole-cell sugars. The 16S rRNA gene sequence showed high level of similarity with Nakamurella flavida KCTC 19127T (97.7%) and Nakamurella flava CGMCC 4.7524T (97.7%). The G + C content of the genomic DNA was 72.4 mol%. Based on draft genome sequences, strain YIM 132084T showed an average nucleotide identity value of 76.1% and 74.9%, a digital DNA-DNA hybridization value of 20.9% and 20.6% with the reference strains Nakamurella flavida and Nakamurella flava, respectively. The results of the phenotypic, chemotaxonomic and phylogenetic analyses showed that strain YIM 132084T represents a novel species of the genus Nakamurella, for which the name Nakamurella leprariae sp. nov. is proposed. The type strain is YIM 132084T (= CGMCC 4.7667T = NBRC 114280T = KCTC 49367T).

Flavimobilis rhizosphaerae sp. nov., isolated from rhizosphere soil of Spartina alterniflora.

A novel Gram-stain positive, facultatively anaerobic, motile, irregularly rod-shaped bacterium, designated GY 10621T, was isolated from rhizosphere soil of Spartina alterniflora in Beihai City, Guangxi Province, PR China, and characterized using a polyphasic taxonomic approach. GY 10621T was positive for catalase and oxidase. Growth occurred at 4-42 °C (optimum 30-37 °C), at pH 5.0-9.0 (optimum pH 7.0) and in the presence of 0-5% NaCl (w/v) (optimum 1-3%). The main menaquinones were MK-9 (H4) (92.2 %) and MK-10 (7.8 %). The major cellular fatty acids were anteiso-C15 : 0 and C14 : 0. The peptidoglycan was the type A4α (l-Lys-Ser-d-Glu). The polar lipids included four phosphoglycolipids, four glycolipids, an unidentified lipid and six unidentified phospholipids. The DNA G+C content of the type strain was 71.7 mol%. On the basis of the results of 16S rRNA gene analysis, the type strain of a species with a validly published name with the highest similarity to GY 10621T was Flavimobilis soli KCTC 13155T (97.16 %), followed by Sanguibacter suarezii NBRC 16159T (96.39 %). The calculated results indicated that compared with GY 10621T, the average nucleotide identity (ANI) values of three strains closely related to GY 10621T (the two aforementioned type strains and 'S. massiliensis' Marseille-P3815) were 74.18-94.97 %, and the digital DNA-DNA hybridization (dDDH) values were 20.3-60.6 %. The results of 16S rRNA-based and genome-based phylogenetic tree analysis indicated that GY 10621T should be assigned to the genus Flavimobilis. On the basis of evidence from polyphasic studies, GY 10621T should be designated as representing a novel species of the genus Flavimobilis, for which the name Flavimobilis rhizosphaerae sp. nov. is proposed. The type strain is GY 10621T (=CGMCC 1.17411T=KCTC 49515T).

Risk factors of breast intraductal lesions in patients without pathological nipple discharge.

The majority of breast cancer arises from the ductal epithelium. It is crucial in the diagnosis and treatment of breast cancer by detecting intraductal lesions at an early stage. The typical clinical characteristic of intraductal lesions is pathological nipple discharge (PND), although many patients with intraductal lesions do not exhibit PND. It is a serious challenge for clinicians to detect patients with intraductal lesions without PND at an early stage. The aim of the present study was to investigate the risk factors associated with intraductal lesions in patients without PND. This retrospective database review, conducted between April 2016 and April 2017, included 370 lesions from 255 patients with intraductal lesions (intraductal papilloma, atypical intraductal hyperplasia, intraductal carcinoma in situ) and non-intraductal lesions (fibroadenoma, adenosis, cysts, lobular carcinoma in situ), diagnosed through surgical pathology. The patients were divided into two groups based on pathological diagnosis and clinical parameters were evaluated using univariate and multivariate analyses. Univariate analysis revealed that 9 of 14 factors were statistically significant. Five factors were identified to be associated risk factors in patients without PND through the multivariate logistic regression analysis: Age between 35 and 49 years and age ≥50 years [odds ratio (OR)=4.749, 95% confidence interval (CI)=2.371-9.513, P<0.001; OR=2.587, 95% CI=2.587-14.891, P<0.001; respectively], non-menstrual breast pain (OR=1.922, 95% CI=1.037-3.564, P=0.038), breast duct dilatation as seen using ultrasonography (OR=9.455, 95% CI=3.194-27.987, P<0.001), lesion distance from nipple ≤2 cm (OR=2.747, 95% CI=1.668-4.526, P<0.001) and lesion size ≤1 cm (OR=1.903, 95% CI=1.155-3.136, P=0.012). In conclusion, for patients without PND but with risk factors, such as the patient being >35 years, with non-menstrual breast pain, breast duct ectasia, lesion distance from nipple ≤2 cm and lesion size ≤1 cm as seen using ultrasonography, clinicians should be highly concerned about the possibility of intraductal lesions, in order to prevent misdiagnosis and reduce the misdiagnosis rate.

The modification of ferroptosis and abnormal lipometabolism through overexpression and knockdown of potential prognostic biomarker perilipin2 in gastric carcinoma.

BACKGROUND: To investigate the biological relationship, mechanism between perilipin2 and the occurrence, advancement of gastric carcinoma, and explore the mechanism of lipid metabolism disorder leading to gastric neoplasm, and propose that perilipin2 is presumably considered as a potential molecular biomarker of gastric carcinoma. METHODS: RNA-seq was applied to analyze perilipin2 and differentially expressed genes modulated by perilipin2 in neoplastic tissues of both perilipin2 overexpression and knockdown groups in vivo. The mechanism was discovered and confirmed by Rt-qPCR, immunoblotting, immunohistochemistry, staining and microassay, respectively. Cellular function experiments were performed by flow cytometry, CCK8, clonogenic assay, etc. RESULTS: Overexpression and knockdown of perilipin2 augmented the proliferation and apoptosis of gastric carcinoma cell lines SGC7901 and MGC803, respectively. The neoplastic cells with perilipin2-overexpression obtained more conspicuously rapid growth than knockdown group in vivo, and perilipin2 affected the proliferation and apoptosis of gastric carcinoma cells by modulating the related genes:acyl-coa synthetase long-chain family member 3, arachidonate 15-lipoxygenase, microtubule associated protein 1 light chain 3 alpha, pr/set domain 11 and importin 7 that were participated in Ferroptosis pathway. Moreover, RNA-seq indicated perilipin2 was an indispensable gene and protein in the suppression of Ferroptosis caused by abnormal lipometabolism in gastric carcinoma. CONCLUSION: Our study expounded the facilitation of perilipin2 in regulating the proliferation and apoptosis of gastric carcinoma cells by modification in Ferroptosis pathway, and we interpreted that the mechanism of gastric neoplasm caused by obesity, we also discovered that pr/set domain 11 and importin 7 are novel transcription factors relevant to gastric carcinoma. Furthermore, perilipin2 probably serves not only as a diagnostic biomarker, but also a new therapeutic target.

Liraglutide Ameliorates Hyperhomocysteinemia-Induced Alzheimer-Like Pathology and Memory Deficits in Rats via Multi-molecular Targeting.

Hyperhomocysteinemia (Hhcy) is an independent risk factor for Alzheimer's disease (AD), and insulin-resistance is commonly seen in patients with Hhcy. Liraglutide (Lir), a glucagon-like peptide that increases the secretion and sensitivity of insulin, has a neurotrophic or neuroprotective effect. However, it is not known whether Lir ameliorates the AD-like pathology and memory deficit induced by Hhcy. By vena caudalis injection of homocysteine to produce the Hhcy model in rats, we found here that simultaneous administration of Lir for 2 weeks ameliorated the Hhcy-induced memory deficit, along with increased density of dendritic spines and up-regulation of synaptic proteins. Lir also attenuated the Hhcy-induced tau hyperphosphorylation and Aß overproduction, and the molecular mechanisms involved the restoration of protein phosphatase-2A activity and inhibition of ß- and γ-secretases. Phosphorylated insulin receptor substrate-1 also decreased after treatment with Lir. Our data reveal that Lir improves the Hhcy-induced AD-like spatial memory deficit and the mechanisms involve the modulation of insulin-resistance and the pathways generating abnormal tau and Aß.

Sex hormone-binding globulin levels during the first trimester may predict gestational diabetes mellitus development.

AIM: To investigate the association of sex hormone-binding globulin (SHBG) levels of early pregnancy with gestational diabetes mellitus (GDM) development. METHODS: A total of 443 pregnant women during the first trimester (<12 weeks) were enrolled. SHBG levels were measured. RESULTS: SHBG level was lower in women with GDM than in women without GDM (93.9 ± 34.4 nmol/l vs 128.1 ± 60.3 nmol/l; p = 0.001). Among the four quartiles (Q1-Q4) according to SHBG levels, GDM incidences were 17.5, 27.8, 5.1 and 2.6%, respectively. No differences were found between Q1 and Q2, and Q3 and Q4. The risk of developing GDM among women in Q1 + Q2 compared with Q3 + Q4 was 5.7. CONCLUSION: Decreased SHBG concentrations during the first trimester may predict GDM development.

The association between the CC chemokine ligand 5 -28C>G gene polymorphism and tuberculosis susceptibility.

OBJECTIVES: To assess the association between chemotactic chemokine (C-C motif) ligand 5 (CCL5) -28C>G polymorphism and tuberculosis (TB) risk.   METHODS: PubMed, Web of Science, and WanFang were searched up to April 2015 for eligible studies on CCL5 -28C>G  polymorphism. Data was extracted, and pooled odd ratios (ORs) as well as 95% confidence intervals (95% CI) were calculated.   RESULTS:  Eight case-control studies were extracted from 8 articles on the polymorphism involving 1852 TB cases and 2068 controls. The results of meta-analysis showed that significant reduced risks were found for the polymorphism with the risk of TB in Asians and Arabs as follows: OR=0.12, 95% CI=0.06-0.26, p=0.000 for mutant  homozygous (GG) versus wild-type homozygous (CC) for Asian descent, OR=0.14, 95% CI=0.07-0.28, p=0.000 for GG versus CC in the Arab descent.   CONCLUSION: Our findings demonstrated that CCL5 gene -28C>G   polymorphism might be a protective factor for the development of TB.

Situational context is important: perceptual grouping modulates temporal perception.

Subjective time of an event in the sub-second range is often compressed or dilated by the situational context created by preceding and succeeding stimuli. How such context distorts psychological time is still an open question. Here, we pursued this issue by examining whether the perceptual grouping among successive visual stimuli modulates the perceived duration. Using a duration comparison task, we asked observers to judge the relative duration of a target and a comparison item, and estimated the apparent duration of the target from the corresponding psychometric function. The target was temporally flanked by a preceding item and a succeeding item. In different conditions, the target was more similar to either the preceding or the succeeding item. Results showed that perceptual grouping based on similarity modulated perceived duration. Specifically, when the target was grouped with the preceding item, its subjective duration was shorter than when it was grouped with the succeeding item. Interestingly, this pattern was observed when the preceding and target items were kept constant while the succeeding item was manipulated, suggesting that the effect depends, to some degree, on the holistic perceptual grouping rather than on fragmented processes. These results demonstrate that the situational context is an important factor in shaping temporal codes, thus bridging the seemingly independent perceptual feature processes and temporal representation.

Visual feature processing in the early visual cortex affects duration perception.

Event timing engages a distributed neural network including cortical and subcortical structures. However, it remains unclear whether the early visual cortex contributes to event timing. Here we showed that the processes of nontemporal visual features such as orientation and spatial location, which are coded by the early visual cortex, contribute to the temporal representation of a visual stimulus. Participants were presented with 2 successive Gabor patches (a prime and a target) with different orientations or spatial locations. The subjective duration of the target was significantly reduced when it was preceded by the prime compared with when presented alone. More important, this duration-compression effect varied systematically as a function of orientation similarity or spatial proximity between the prime and the target and was influenced by how the prime and the target were perceptually grouped. Our results suggest that repetition suppression of neural activity in response to orientation may contribute to the observed duration distortion and that neurons in the early visual cortex with small receptive fields and orientation selectivity may be involved in visual temporal perception. Our findings help to understand the functional role of early visual cortex in event timing in humans.

[Reversal of mdrl gene-dependent multidrug resistance in multidrug resistance human leukemia cell line K562/ADM using short hairpin RNA expression vectors].

OBJECTIVE: To explore the role of reversal multidrug resistance (MDR) using short hairpin RNA (shRNA) expression vectors in multidrug resistance human leukemia cell line K562/ADM. METHODS: The oligonucleotides with 19-mer hairpin structure were synthesized. The shRNA expression vectors were constructed and introduced into K562/ADM cells. Expression of mdr1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western blot. The apoptosis and sensitivity of the K562/ADM cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscope (LCSM). RESULTS: In positive clones of K562/ADM cells stably transfected with pSilencer 3.1-HI neo mdr1-A and mdr1-B shRNA expression vectors, RT-PCR showed that mdr1 mRNA expression was significantly reduced to 35.9% (P < 0.05), 27.5% (P < 0.01), respectively. Western blot showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 79-fold to 38-fold (P < 0.05), 30-fold (P < 0.01) respectively. Furthermore, the fluorescence intensity of K562/ADM cells was increased significantly compared with the control. shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The percent of the apoptosis cell was significantly enhanced to 18.1% (P < 0.05) , 54.4% (P < 0.01) respectively. CONCLUSIONS: shRNA expression vectors can effectively reverse MDR, and restore the sensitivity of drug-resistance K562/ADM cells to conventional chemotherapeutic agents.

Study on the bone marrow mesenchymal stem cells induced drug resistance in the U937 cells and its mechanism.

BACKGROUND: The hematopoietic microenvironment (HM) plays a critical role in malignant cell growth, patient survival, and response to chemotherapy in hematologic malignancies. However, mechanisms associated with this environmental influence remain unclear. In this study, we investigated the role of bone marrow derived mesenchymal stem cells (MSCs) in U937 cell line, to find out the relations between leukemia drug resistance and the MSCs. METHODS: U937 cells were cultured in suspension or grew adherently with MSCs. The cell growth curve was drawn and the cell cycle was measured by flow cytometry. Apoptosis and sensitivity of U937 to daunoblastina (DNR) were quantified by DNA ladder detection and trypan blue exclusion assays, respectively. The gene expression profile chip technology was used to determine and analyze the changes in apoptosis-related gene expression after adherent culture and the expression of MDR1 mRNA was assessed by reverse transcriptional polymerase chain reaction (RT-PCR) at the same time. RESULTS: In the adherent culture, the proliferation of the U937 cells was inhibited, the G0/G1 phase cells increased (F = 64.9726, P < 0.0001), G2/M phase cells were decreased (F = 98.1361, P < 0.0001) and the natural apoptosis rate was decreased (F = 24.0866, P < 0.0001) compared with those in the suspended culture. U937 cell viability was enhanced and cell apoptosis was blocked during DNR treatment in adherent culture with MSCs. Thirty-nine differently expressed genes were screened from the 487 apoptosis related genes in the adherent culture U937 cells. Among the 37 upregulated genes, Bcl-XL was upregulated most significantly. Two genes were downregulated. Adherent culture did not induce MDR1 mRNA expression in U937 cells. CONCLUSIONS: MSCs play a role in modulating the proliferation of U937 cells and response of U937 cells to DNR, and Bcl-XL apoptosis-inhibiting gene may be most important in determining the sensitivity of leukemic cells to treatment, which is not related to MDR1.

[Influence of human mesenchymal stem cells on cell proliferation and chemo-sensitivity of K562 cells].

This study was aimed to compare K562 cell proliferation, chemo-sensitivity and alteration of MDR1 before and after adhesive culture with MSC, so as to evaluate the relationship between chemodrug-resistance of leukemia cells and hemopoietic microenvironment. K562 cell cultivated in suspension and adhesively cultivated with MSC were collected respectively and cell proliferation curves were drawn; the cell cycle was determined by flow cytometry; the effect of chemotherapy on cellular viability and apoptosis of K562 cell was investigated, the MDR1 gene expression was determined by RT-PCR. The results showed that K562 cells adhesively cultivated with MSC were inhibited and cells in G0/G1 increased (P < 0.05), cells in S phase decreased (P < 0.05) and those in G0/G1 increased (P < 0.01), compared with that cultivated in suspension. In process of daunomycin-inducing apoptosis, K562 cell apoptosis in the adhesive culture with MSC was inhibited (P < 0.05). MDR1 gene expression in K562 cells was not induced or altered by adhesive co-cultivation. It is concluded that by co-culture of cell-cell contact with MSC, growth suppression and induction of chemo-resistance of K562 cells take place. The mechanism, however, seems not relevant with MDR1.

Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors.

BACKGROUND: RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). METHODS: The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P < 0.05 was considered statistically significant. RESULTS: In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P < 0.05), 30.1% (P < 0.01) (transient transfection) and 37.6% (P < 0.05), 28.0% (P < 0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P < 0.05), 54-fold (P < 0.01) (transient transfection) and to 108-fold (P < 0.05), 50-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. CONCLUSIONS: shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.

[Effects of donor-specific bone marrow cell infusion on chimerism and acute rejection in kidney transplantation].

OBJECTIVE: To evaluate the effect of donor-specific bone marrow cell infusion on the production of chimerism and acute rejection in kidney transplantation. METHODS: Sixty-one patients, 48 males and 13 females, aged 38.4 (23 - 45), underwent transplantation of cadaveric kidneys, 24 of which underwent kidney transplantation combined with donor-specific bone marrow cell infusion and 37 of which underwent pure transplantation of kidney. During the kidney transplantation combined with donor-specific bone marrow cell infusion the donor bone marrow cells (DBMC) were isolated from the thoracic vertebrae and iliac bones of the donors-cadavers and infused into the recipients of the kidneys of the same donors. After operation the peripheral blood was collected from the 61 recipients every 2 - 3 months for at least 1 year to undergo PCR to detect the presence of chimerism, and the rates of chimerism and acute rejection were compared between these 2 groups. RESULTS: During the follow-up the presence rate of chimerism was 87.5% (21/24) in the marrow recipients, significantly higher than that in the control group (40.5%, 15/37, P < 0.001). The prevalence of acute rejection in the chimerism positive patients was 19.4% (7/16), significantly lower than that in the chimerism negative patients (44%, 11/25, P < 0.05). CONCLUSION: Donor-specific bone marrow cell infusion in cadaver kidney transplantation induces the production of chimerism, and increases the immune tolerance to the donor organs, thus finally decreasing the incidence of acute rejection. Chimerism is correlated with immune tolerance.