Hypoxia-responsive miR-346 promotes proliferation, migration, and invasion of renal cell carcinoma cells via targeting NDRG2.

Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. In this study, we investigated the role of miR-346 in RCC cells under hypoxia. OS-RC-2 and 786-O cells were cultured in 1% O2 or normal oxygen. Cell proliferation, migration, and invasion abilities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, transwell migration, and invasion assays, respectively. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression of miR-346 and N-myc downstream-regulated gene 2 (NDRG2). Then bioinformatics analysis, dual-luciferase reporter assay, and RNA immunoprecipitation were carried out to determine the relationship between miR-346 and NDRG2. The protein expression of NDRG2 was detected by western blot assay. Hypoxia promoted cell proliferation, migration, and invasion in OS-RC-2 and 786-O cells. Meanwhile, we found that miR-346 was upregulated in RCC cells under hypoxia as relative to normoxia. miR-346 deletion could decrease the viability, migration, and invasion abilities of RCC cells under hypoxia. Besides, our data demonstrated that NDRG2 was a target gene of miR-346. The expression of NDRG2 in OS-RC-2 and 786-O cells was lower under hypoxia than under normal oxygen conditions. Moreover, NDRG2 overexpression could inhibit cell proliferation, migration, and invasion in RCC cells under hypoxia. And NDRG2 silencing reversed the inhibitory effects of the miR-346 inhibitor on the viability, migration, and invasion abilities of RCC cells in hypoxia conditions. miR-346 promoted the viability, migration, and invasion of RCC cells under hypoxia by targeting NDRG2.

Circulating DNA for detection of gastric cancer.

OBJECTIVE: Gastric cancer (GC) is one of the most common malignant tumors worldwide, particularly, prevalent in China. Despite the decreasing incidence of GC in China, the 5-year survival rate is still not over 30% yet. Therefore, early diagnosis and therapeutic outcome evaluation of GC remains as the issue to be resolved in a clinical setting. MATERIALS AND METHODS: Recent studies have found the presence of a certain amount of circulating DNA in the peripheral blood of patients with malignant tumor and shown that these free DNA bear tumor-specific genetic information. The circulating DNA detection includes quantitative and qualitative methods and analysis. Combined monitoring of changes in circulating DNA levels and aberrant alteration of relevant tumor genes is likely to provide comprehensive real-time information to patients. RESULTS: Under normal conditions, oncogene presents in the form of proto-oncogene such as K-ras, which is in non-carcinogenic status under the influence of tumor suppressor gene. When tumor suppressor gene is damaged or mutated of oncogene itself is induced for instance P53, oncogene is then activated and induces tumorigenesis. However, compared to gene mutation detection, the detection of DNA methylation is relatively more well-developed and stable. CONCLUSIONS: This article reviews the current status of the research on circulating DNA in the diagnosis, assessment of response to therapy and prognostic evaluation in GC. In addition, the advantage, current issue and prospect of using circulating DNA as tumor marker are also analyzed.

Hip fracture after first-ever stroke: a population-based study.

OBJECTIVES: The aim of this study is to estimate the risk of hip fracture after first-ever stroke, using a nationwide population-base data set and a retrospective cohort design. MATERIALS AND METHODS: The cohort study involved 18,413 patients surviving a first-ever stroke during the 12-year period from 1997 to 2008. Another 18,413 control subjects were randomly selected with adjustment for age, gender and enrolled year. Stroke type, duration between stroke and hip fracture, six comorbidities and five categories of medication prior to hip fracture were investigated. RESULTS: This study found that 788 (4.3%) subjects in the study group suffered from hip fracture, with a 4.2 years median time frame (interquartile range = 1.8-7.1). In the control group, 492 subjects (2.7%) suffered from hip fracture during a 4.8 years median time frame (interquartile range = 2.0-7.5). The relative risk of hip fracture for stroke was increased in the first four years (1.4-2.4) and gradually declined to the level of the general population. Cox regression analysis showed osteoporosis-related factors, including ageing, female and antidepressants, significantly increased hip fracture risk (hazard ratios 1.89, 1.57, 1.92). CONCLUSIONS: These findings imply that osteoporosis may play a major role in the occurrence of hip fracture in the first four years after a first-ever stroke. Early intervention to prevent bone loss should be regarded as an important part in stroke management, especially in older females, and should be sustained for four years at least. The benefit of antidepressants in stroke patients should be weighed against the increased risk of hip fracture.

A CMOS wireless biomolecular sensing system-on-chip based on polysilicon nanowire technology.

As developments of modern societies, an on-field and personalized diagnosis has become important for disease prevention and proper treatment. To address this need, in this work, a polysilicon nanowire (poly-Si NW) based biosensor system-on-chip (bio-SSoC) is designed and fabricated by a 0.35 µm 2-Poly-4-Metal (2P4M) complementary metal-oxide-semiconductor (CMOS) process provided by a commercialized semiconductor foundry. Because of the advantages of CMOS system-on-chip (SoC) technologies, the poly-Si NW biosensor is integrated with a chopper differential-difference amplifier (DDA) based analog-front-end (AFE), a successive approximation analog-to-digital converter (SAR ADC), and a microcontroller to have better sensing capabilities than a traditional Si NW discrete measuring system. In addition, an on-off key (OOK) wireless transceiver is also integrated to form a wireless bio-SSoC technology. This is pioneering work to harness the momentum of CMOS integrated technology into emerging bio-diagnosis technologies. This integrated technology is experimentally examined to have a label-free and low-concentration biomolecular detection for both Hepatitis B Virus DNA (10 fM) and cardiac troponin I protein (3.2 pM). Based on this work, the implemented wireless bio-SSoC has demonstrated a good biomolecular sensing characteristic and a potential for low-cost and mobile applications. As a consequence, this developed technology can be a promising candidate for on-field and personalized applications in biomedical diagnosis.

Analysis of the regulatory region of the lysC gene of Escherichia coli.

A lysC-lac'Z fusion plasmid was constructed to study the regulatory region of the lysC gene. Analysis by deletion mutations confirmed the existence of an alternative promoter, P2, located upstream of the previously identified promoter, P1. The transcription start site of promoter P2 was located 85 base pairs upstream the transcription start site of promoter P1. Both promoters are regulated by lysine.

Molecular structure of kanamycin nucleotidyltransferase determined to 3.0-A resolution.

Kanamycin nucleotidyltransferase, as originally isolated from Staphylococcus aureus, inactivates the antibiotic kanamycin by catalyzing the transfer of a nucleotidyl group from nucleoside triphosphates such as ATP to the 4'-hydroxyl group of the aminoglycoside. The molecular structure of the enzyme described here was determined by X-ray crystallographic analysis to a resolution of 3.0 A. Crystals employed in the investigation belonged to the space group P4(3)2(1)2 with unit cell dimensions of a = b = 78.9 A and c = 219.2 A. An electron density map phased with seven heavy-atom derivatives revealed that the molecules packed in the crystalline lattice as dimers exhibiting local 2-fold rotation axes. Subsequent symmetry averaging and solvent flattening improved the quality of the electron density such that it was possible to completely trace the 253 amino acid polypeptide chain. Each monomer is divided into two distinct structural domains: the N-terminal motif composed of residues Met 1-Glu 127 and the C-terminal half delineated by residues Ala 128-Phe 253. The N-terminal region is characterized by a five-stranded mixed beta-pleated sheet whereas the C-terminal domain contains five alpha-helices, four of which form an up-and-down alpha-helical bundle very similar to that observed in cytochrome c'. The two subunits wrap about one another to form an ellipsoid with a pronounced cleft that could easily accommodate the various aminoglycosides known to bind to the enzyme.

Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents.

A series of variants of kanamycin nucleotidyltransferase (KNTase), isolated previously on the basis of enhanced thermostability by cloning and selection for enzymatic activity in the thermophile Bacillus stearothermophilus, was used to systematically test the hypothesis that thermostable enzymes would also be more resistant to other forms of protein denaturation. The purified KNTases were treated with proteinase K or assayed at 37 degrees C in the presence of urea, N-lauroylsarcosine, Triton X-100, tetrahydrofuran, ethanol, or dimethylformamide. With all these agents, the KNTases displayed increasing resistance to denaturation in the order: wild type, mutant TK9 (with a Thr130-->Lys substitution), TK1 (Asp80-->Tyr), and TK101 (both substitutions). This is the same order in which their thermostability increases, indicating that the structural mechanism(s) whereby the mutations yield enhanced resistance to heat denaturation also yield stabilization towards chemical forms of enzymatic inactivation. These results suggest that selection in thermophiles is a useful method to obtain enzyme variants with increased overall stability, even at nonthermophilic temperatures.

Crystallization and preliminary crystallographic analysis of a thermostable mutant of kanamycin nucleotidyltransferase.

A thermostable mutant of kanamycin nucleotidyltransferase isolated by cloning and selection for kanamycin resistance in Bacillus stearothermophilus at 70 degrees C has been crystallized in a form suitable for high-resolution diffraction analysis. This enzyme catalyzes nucleotidyl group transfer from nucleoside triphosphates such as ATP to hydroxyl groups of various aminoglycosides, thus inactivating the antibiotic. The kanamycin nucleotidyltransferase gene, originally encoded on plasmid pUB110 from the mesophile Staphylococcus aureus, was transferred to the thermophile B. stearothermophilus via shuttle plasmids and the mutant carrying the substitutions D80Y and T130K was isolated from kanamycin-resistant colonies grown at 70 degrees C. The thermostable enzyme was crystallized in two forms from solutions of polyethylene glycol 8000 (PEG8000) using batch and vapor diffusion methods. Type I crystals grown from 19% (w/v) PEG8000 and 200 mM NaCl belong to the orthorhombic space group C222(1), have unit cell dimensions of a = 128.4, b = 156.8, c = 155.8 A, and diffract to at least 2.4-A resolution. The type II form of the crystals were grown from 10% PEG8000, 200 mM KCl, and 3 mM CoCl2, and belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 78.9, and c = 220.4 A; these crystals diffract to at least 2.5-A resolution.

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.

Effect of temperature on the expression of wild-type and thermostable mutants of kanamycin nucleotidyltransferase in Escherichia coli.

The expression of kanamycin nucleotidyltransferase (KNTase) in Escherichia coli results in different forms of the protein, depending on the temperature; soluble active enzyme is synthesized at 23 degrees C but the protein is mostly aggregated and inactive in inclusion bodies when made at 37 degrees C. However, active enzyme can be recovered by solubilization of the inclusion bodies with 8 M urea followed by dilution of the denaturant, indicating that the polypeptide is not damaged covalently but is present in a misfolded state. The availability of thermostable mutants of KNTase allows a test of the hypothesis that formation of inclusion bodies when proteins are highly expressed in E. coli is due to the accumulation of a folding intermediate that is prone to temperature-dependent aggregation. Because these mutants were isolated by cloning the KNTase gene into the thermophile Bacillus stearothermophilus and selecting for kanamycin resistance at high growth temperatures, they must be thermostable for both synthesis and activity and must have folding intermediates that are less susceptible to the formation of aggregates. Indeed, whereas decreasing the temperature from 37 to 23 degrees C increased the KNTase specific activity 10-fold in cells expressing the wild-type enzyme, this change resulted in only a 2.1-fold increase for the TK1 (Asp80----Tyr) mutant and a 1.7-fold increase for the TK101 (Asp80----Tyr and Thr130----Lys) double mutant. The strategy of cloning in thermophiles and selecting or screening for mutants that fold correctly to yield biological activity at high growth temperatures may be useful in overcoming the problem of the insolubility of some proteins when expressed in heterologous hosts.

Quasi-static electric-field-enhanced diffraction effects in a nematic liquid-crystal film.

A quasi-static electric field can enhance laser-induced diffraction rings from a nematic liquid-crystal film. This phenomenon is shown to be the combined result of the critical behavior of the sample at the Freedericksz transition and the nonlinear coupling of the optical and quasi-static electric fields.

Adenosine 3',5'-phosphate phosphodiesterase and pheromone response in the yeast Saccharomyces cerevisiae.

Theophylline, aminophylline, and isobutylmethylxanthine, compounds reported to be inhibitors of adenosine 3',5'-phosphate (cAMP) phosphodiesterase, prevented the alpha-factor-induced cell cycle arrest of Saccharomyces cerevisiae a cells. To determine whether the in vivo effect of these methylxanthines on yeast pheromone response was related to their known biochemical mode of action, two assays for cAMP phosphodiesterase based on affinity of the product of the reaction (5'-AMP) for boronate groups were developed and were used to monitor the activity of the low Km cAMP phosphodiesterase present in yeast extracts. It was found that the relative efficacy of the methylxanthines as inhibitors of this enzyme in vitro was correlated with the degree to which they antagonized alpha-factor action in vivo. These results were consistent with our previous proposal that pheromone action involves a lowering of cAMP level in the target cell.

Clostridial apoferredoxin messenger ribonucleic acid. Assay and partial purification.

An assay for Clostridium pasteurianum apoferredoxin messenger ribonucleic acid (mRNA) was developed, based on the synthesis of the protein in vitro. Quantitation of apoferredoxin synthesis was accomplished by trypsinization of the cell-free incubation labeled with 3H- or 14C-labeled amino acids, separation of the products by SDS-urea polyacrylamide gel electrophoresis, and excising and counting the NCS-solubilized gel band corresponding to the unique 52-amino acid tryptic peptide derived from apoferredoxin. Its synthesis was shown to be RNA dependent, and was optimized with respect to several parameters of the in vitro protein-synthesizing system. The specificity of the assay was examined with RNA from Clostridium acidi urici, a related species the ferredoxin of which does not yield the 52-amino acid tryptic peptide, and by the use of [3H]leucine, which is not present in C. pasteurianum apoferredoxin. By these methods, the overestimation of apoferredoxin synthesis due to the comigration of fragments from other in vitro products with the legitimate apoferredoxin-derived peptide could be accounted for. The apoferredoxin mRNA was partially purified by the sequential zonal sucrose gradient centrifugation of total RNA followed by Sephadex G-200 chromatography of the enriched RNA, after which a fraction was obtained in which apoferredoxin mRNA was 20-fold enriched. The enriched RNA fraction can now be used for further purification of the apoferredoxin-coding sequences by cloning procedures.