Proteomic analysis reveals LRPAP1 as a key player in the micropapillary pattern metastasis of lung adenocarcinoma.

Objectives: Lung adenocarcinomas have different prognoses depending on their histological growth patterns. Micropapillary growth within lung adenocarcinoma, particularly metastasis, is related to dismal prognostic outcome. Metastasis accounts for a major factor leading to mortality among lung cancer patients. Understanding the mechanisms underlying early stage metastasis can help develop novel treatments for improving patient survival. Methods: Here, quantitative mass spectrometry was conducted for comparing protein expression profiles among various histological subtypes, including adenocarcinoma in situ, minimally invasive adenocarcinoma, and invasive adenocarcinoma (including acinar and micropapillary [MIP] types). To determine the mechanism of MIP-associated metastasis, we identified a protein that was highly expressed in MIP. The expression of the selected highly expressed MIP protein was verified via immunohistochemical (IHC) analysis and its function was validated by an in vitro migration assay. Results: Proteomic data revealed that low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) was highly expressed in MIP group, which was confirmed by IHC. The co-expressed proteins in this study, PSMD1 and HSP90AB1, have been reported to be highly expressed in different cancers and play an essential role in metastasis. We observed that LRPAP1 promoted lung cancer progression, including metastasis, invasion and proliferation in vitro and in vivo. Conclusion: LRPAP1 is necessary for MIP-associated metastasis and is the candidate novel anti-metastasis therapeutic target.

Development of a dynamic feeding strategy for continuous-flow aerobic granulation and nitrogen removal in a modified airlift loop reactor for municipal wastewater treatment.

This study investigated the aerobic sludge granulation and nitrogen removal performance in a modified airlift loop reactor treating municipal wastewater under different operation conditions. Dynamic feeding and aeration control were applied to create feast/famine conditions to facilitate microbial aggregation. Experimental results demonstrated that aerobic granular sludge could be cultivated in continuous-flow reactors fed with an optimized dynamic feeding condition. Fresh granules sizing 0.4-0.6 mm were observed in the reactors after a 61-day operation, then turned to matured granules after another 33-day operation with a compact structure, a stable size of 2-4 mm, and a low SVI of ~35 mL/g. Extracellular polymeric substances (EPS) analysis results showed that both EPS contents and the ratio of protein to polysaccharides increased with the granulation process, leading to an increase of cell hydrophobicity. Granular sludge exhibited a good nitrogen removal ability with a comparable level of specific nitrification rate and denitrification rate with those measured in state-of-the-art sequential batch reactors. Microbial population analysis showed an increase in the relative abundance of functional microbes, including Zoogloea, Nitrospira, Dechloromonas, and Thauera in the cultivated granules, suggesting a potentially crucial role of these microbes in sludge granulation and nitrogen removal. The dynamic feeding strategy and the reactor configuration are considered as critical factors for aerobic granulation under continuous-flow conditions for creating feast/famine conditions and allow sludge backflow without structure damage.

Bacterial pyridine hydroxylation is ubiquitous in environment.

Ten phenol-degrading bacterial strains were isolated from three geographically distant environments. Five of them, identified as Diaphorobacter, Acidovorax, Acinetobacter (two strains), and Corynebacterium, could additionally transform pyridine, through the transcription of phenol hydroxylase genes induced both by phenol and pyridine. HPLC-UV and LC-MS analyses indicated that one metabolite (m/e = 96.07) with the same molecular weight as monohydroxylated pyridine was produced from the five phenol-degrading strains, when pyridine was the sole carbon source. Phenol (50 mg l(-1)) could initially inhibit and later stimulate the pyridine transformation. In addition, heterologous expression of the phenol hydroxylase gene (pheKLMNOP) resulted in the detection of monohydroxylated pyridine, which confirmed the phenol hydroxylase could catalyze pyridine hydroxylation. Phylogeny of the phenol hydroxylase genes revealed that the genes from the five pyridine-hydroxylating strains form a clade with each other and with those catalyzing the hydroxylation of phenol, BTEX (acronym of benzene, toluene, ethylbenzene, and xylene), and trichloroethylene. These results suggest that pyridine transformation via hydroxylation by phenol hydroxylase may be prevalent in environments than expected.

Simultaneous degradation of phenol and n-hexadecane by Acinetobacter strains.

Three phenol- and alkanes-degrading bacterial strains were isolated from a freshwater sample. Upon the 16S rRNA gene analysis, phenotype and physiological features, the three strains were designated as Acinetobacter sp. with both phenol hydroxylase gene (phe) and alkane monooxygenase gene (alkM) detected. They could simultaneously degrade phenol and n-hexadecane for growth, but prefer phenol than n-hexadecane. Between phenol (400mgl(-1)) and n-hexadecane (400mgl(-1)), n-hexadecane enhanced phenol degradation in mineral salt medium (MSM), while phenol affects negatively the n-hexadecane degradation. However, combination of phenol (400mgl(-1)) and n-hexadecane (400mgl(-1)) in MSM led to higher growth of the strains than the phenol and n-hexadecane separately. The transcription levels of phe and alkM genes supported the physiological properties of the strains.

Efficient decentralized iterative learning tracker for unknown sampled-data interconnected large-scale state-delay system with closed-loop decoupling property.

In this paper, an efficient decentralized iterative learning tracker is proposed to improve the dynamic performance of the unknown controllable and observable sampled-data interconnected large-scale state-delay system, which consists of N multi-input multi-output (MIMO) subsystems, with the closed-loop decoupling property. The off-line observer/Kalman filter identification (OKID) method is used to obtain the decentralized linear models for subsystems in the interconnected large-scale system. In order to get over the effect of modeling error on the identified linear model of each subsystem, an improved observer with the high-gain property based on the digital redesign approach is developed to replace the observer identified by OKID. Then, the iterative learning control (ILC) scheme is integrated with the high-gain tracker design for the decentralized models. To significantly reduce the iterative learning epochs, a digital-redesign linear quadratic digital tracker with the high-gain property is proposed as the initial control input of ILC. The high-gain property controllers can suppress uncertain errors such as modeling errors, nonlinear perturbations, and external disturbances (Guo et al., 2000) [18]. Thus, the system output can quickly and accurately track the desired reference in one short time interval after all drastically-changing points of the specified reference input with the closed-loop decoupling property.

Degradation of pyridine by one Rhodococcus strain in the presence of chromium (VI) or phenol.

A Rhodococcus strain, Chr-9, which has the ability to degrade pyridine and phenol and reduce chromium (VI) (Cr (VI)) was isolated. The strain could grow with pyridine as the sole carbon and nitrogen source, and its pyridine-degradation capability was enhanced by 100 mg l(-1) phenol; however, the degradation of pyridine was inhibited when the phenol concentration was greater than 400 mg l(-1). The hydroxylation of pyridine suggested that the stimulation and inhibition of phenol to the pyridine degradation may be attributed to competition of phenol and pyridine for the hydroxylase gene. Strain Chr-9 was also able to reduce Cr (VI) when glucose and LB was used as the carbon source; however, the Cr (VI) reduction did not occur when pyridine was the sole carbon and energy source. In addition, strain Chr-9 could reduce Cr (VI) and simultaneously degrade pyridine in the presence of glucose. To the best of our knowledge, strain Chr-9 is the first Rhodococcus strain reported to degrade pyridine in the presence of Cr (VI), and the first strain with the pyridine degradation being stimulated by low concentrations of phenol.

[Discrimination of bamboo fiber and ramie fiber by near infrared spectroscopy].

The research on discrimination of natural bamboo fiber, bamboo pulp fibers and ramie fiber used in textile was demonstrated by near infrared (NIR) spectroscopy. First, the spectra of three kinds of fiber were scanned by NIR spectrometer. Then, the spectral data were pretreated by first derivatives. In the end, the databases and discriminating model of natural bamboo fiber, bamboo pulp fibers and ramie fiber were built. The kind of unknown fiber can be discriminated by the NIR model. The results indicated that the natural bamboo fiber, bamboo pulp fibers and ramie fiber can been discriminated quickly by NIR spectroscopy without destroying samples.

The nature of actinomycin D binding to d(AACCAXYG) sequence motifs.

Earlier studies by others had indicated that actinomycin D (ACTD) binds well to d(AACCATAG) and the end sequence TAG-3' is essential for its strong binding. In an effort to verify these assertions and to uncover other possible strong ACTD binding sequences as well as to elucidate the nature of their binding, systematic studies have been carried out with oligomers of d(AACCAXYG) sequence motifs, where X and Y can be any DNA base. The results indicate that in addition to TAG-3', oligomers ending with XAG-3' and XCG-3' all provide binding constants > or =1 x 10(7) M(-1) and even sequences ending with XTG-3' and XGG-3' exhibit binding affinities in the range 1-8 x 10(6) M(-1). The nature of the strong ACTD affinity of the sequences d(A1A2C3C4A5X6Y7G8) was delineated via comparative binding studies of d(AACCAAAG), d(AGCCAAAG) and their base substituted derivatives. Two binding modes are proposed to coexist, with the major component consisting of the 3'-terminus G base folding back to base pair with C4 and the ACTD inserting at A2C3C4 by looping out the C3 while both faces of the chromophore are stacked by A and G bases, respectively. The minor mode is for the G to base pair with C3 and to have the same A/chromophore/G stacking but without a looped out base. These assertions are supported by induced circular dichroic and fluorescence spectral measurements.

Unique actinomycin D binding to self-complementary d(CXYGGCCY'X'G) sequences: duplex disruption and binding to a nominally base-paired hairpin.

Actinomycin D (ACTD) has been shown to bind weakly to the sequence -GGCC-, despite the presence of a GpC site. It was subsequently found, however, that d(CATGGCCATG) binds relatively well to ACTD but exhibits unusually slow association kinetics, contrary to the strong-binding -XGCY- sites. In an effort to elucidate the nature of such binding and to delineate the origin of its interesting kinetic behavior, studies have now been extended to include oligomers with the general sequence motifs of d(CXYGGCCY'X'G)(2). It was found that analogous binding characteristics are observed for these self-duplex decamers and comparative studies with progressively base-truncated oligomers from the 5'-end led to the finding that d(GGCCY'X'G) oligomers bind ACTD considerably stronger than their parent decamers and exhibit 1:1 drug/strand binding stoichiometry. Melting profiles monitored at the drug spectral region indicated additional drug binding prior to the onset of eventual complex disruptions with near identical melting temperatures for all the oligomers studied. These results are consistent with the notion that the related oligomers share a common strong binding mode of a hairpin-type, with the 3'-terminus G folding back to base-pair with the C base of GGC. A binding scheme is proposed in which the oligomers d(CXYGGCCY'X'G) exist predominantly in the duplex form and bind ACTD initially at the central GGCC weak site but subsequently disrupt to accommodate the stronger hairpin binding and thus the slow association kinetics. Such a mechanism is supported by the observation of distinct biphasic fluorescence kinetic traces in the binding of 7-amino-ACTD to these duplexes.

Solution structure of the ActD-5'-CCGTT3GTGG-3' complex: drug interaction with tandem G.T mismatches and hairpin loop backbone.

Binding of actinomycin D (ActD) to the seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3 GTGG-3' has been studied in solution using high-resolution nuclear magnetic resonance (NMR) techniques. A strong binding constant (8 x 10(6) M(-1)) and high quality NMR spectra have allowed us to determine the initial DNA structure using distance geometry as well as the final ActD-5'-CCGTT3 GTGG-3' complex structure using constrained molecular dynamics calculations. The DNA oligomer 5'-CCGTT3GTGG-3' in the complex forms a hairpin structure with tandem G.T mismatches at the stem region next to a loop of three stacked thymine bases pointing toward the major groove. Bipartite T2O-GH1 and T2O-G2NH2 hydrogen bonds were detected for the G.T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ActD intercalates nicely between the tandem G.T mismatches in essentially one major orientation. Additional hydrophobic interactions between the ActD quinoid amino acid residues with the loop T5-T6-T7 backbone protons were also observed. The hydrophobic G-phenoxazone-G interaction in the ActD-5'-CCGTT3GTGG-3' complex is more robust than that of the classical ActD- 5'-CCGCT3GCGG-3' complex, consistent with the roughly 2-fold stronger binding of ActD to the 5'-CCGTT3GTGG-3' sequence than to its 5'-CCG CT3GCGG-3' counterpart. Stabilization by ActD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ActD or other related compounds may serve as a sequence- specific ssDNA-binding agent that inhibits human immunodeficiency virus (HIV) and other retroviruses replicating through ssDNA intermediates.

Binding of actinomycin D to single-stranded DNA of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTCT).

Our recent binding studies with oligomers derived from base replacements on d(CGTCGTCG) had led to the finding that actinomycin D (ACTD) binds strongly to d(TGTCATTG) of apparent single-stranded conformation without GpC sequence. A fold-back binding model was speculated in which the planar phenoxazone inserts at the GTC site with a loop-out T base whereas the G base at the 3'-terminus folds back to form a basepair with the internal C and stacks on the opposite face of the chromophore. To provide a more concrete support for such a model, ACTD equilibrium binding studies were carried out and the results are reported herein on oligomers of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTC). These oligomers are not expected to form dimeric duplexes and contain no canonical GpC sequences. It was found that ACTD binds strongly to d(TGTCTTTTG), d(TGTTTTGTC), and d(TGTTTTTGTC), all exhibiting 1:1 drug/strand binding stoichiometry. The fold-back binding model with displaced T base is further supported by the finding that appending TC and TCA at the 3'-terminus of d(TGTCTTTTG) results in oligomers that exhibit enhanced ACTD affinities, consequence of the added basepairing to facilitate the hairpin formation of d(TGTCTTTTGTC) and d(TGTCTTTTGTCA) in stabilizing the GTC/GTC binding site for juxtaposing the two G bases for easy stacking on both faces of the phenoxazone chromophore. Further support comes from the observation of considerable reduction in ACTD affinity when GTC is replaced by GTTC in an oligomer, in line with the reasoning that displacing two T bases to form a bulge for ACTD binding is more difficult than displacing a single base. Based on the elucidated binding principle of phenoxazone ring requiring its opposite faces to be stacked by the 3'-sides of two G bases for tight ACTD binding, several oligonucleotide sequences have been designed and found to bind well.

Looped out and perpendicular: deformation of Watson-Crick base pair associated with actinomycin D binding.

Many anticancer drugs interact directly with DNA to exert their biological functions. To date, all noncovalent, intercalating drugs interact with DNA exclusively by inserting their chromophores into base steps to form elongated and unwound duplex structures without disrupting the flanking base pairs. By using actinomycin D (ActD)-5'-GXC/CYG-5' complexes as examples, we have found a rather unusual interaction mode for the intercalated drug; the central Watson-Crick X/Y base pairs are looped out and displaced by the ActD chromophore. The looped-out bases are not disordered but interact perpendicularly with the base/chromophore and form specific H bonds with DNA. Such a complex structure provides intriguing insights into how ligand interacts with DNA and enlarges the repertoires for sequence-specific DNA recognition.

Actinomycin D binds to d(TGTCATG) with 2:1 drug to duplex stoichiometry.

Despite the apparent single-stranded conformation and the absence of a GpC site, d(TGTCATTG) has been found to bind strongly to actinomycin D (ACTD) with 1:1 drug to strand binding stoichiometry. A hairpin binding model was speculated in which the planar phenoxazone chromophore inserts at the GTC site by pushing out the T-base while the terminal G folds back to form a G small middle dotC base pair so that the 3'-sides of both G-bases stack on the opposite faces of the phenoxazone plane. However, it was also suggested that a slipped duplex binding with similar binding principle could also be operative at higher DNA concentrations. To support such a contention, ACTD binding studies were made with d(TGTCATG) and related oligomers. This heptamer differs from the parent octamer d(TGTCATTG) by a mere removal of a T-base which should result in an enhancement of dimeric duplex formation and a concomitant reduction in monomeric hairpin contribution. It was found that ACTD binds well to d(TGTCATG) with 1 drug to 1 strand (or 2 drugs to 1 duplex) binding stoichiometry. These results are consistent with a slipped duplex binding model in which a dimeric duplex is formed at the self-complementary CATG tetranucleotide sequence with extruding TGT ends. Two drug molecules are bound at both ends of the duplex by pushing out the T-bases of GTC's so that the opposite faces of each phenoxazone are stacked by the 3'-sides of the two G-bases on opposite strands. Such a model provides a ready explanation for the observed enhancement in ACTD binding to d(TGTCATGTC) and d(TGTCATGTCA), where additional base pairs at the ends will stabilize GTC/GTC binding sites, and to d(TGTCAATTG) in which two additional base pairs facilitate the slipped-duplex formation. The observed ACTD affinity reductions for oligomers containing GTTC instead of GTC are also consistent with the T-base displacement model. These findings greatly expand the repertoire of ACTD binding to DNA and may have important implications on understanding the transcription inhibitory activities of this drug.

[Prediction of eluotropic sequence of solutes in countercurrent chromatography].

According to the retention equation of countercurrent chromatography (CCC), the eluotropic sequence of solutes depends mainly on the order of their partition coefficients between the two immiscible liquid phases. The varying tendencies of partition coefficients of some compounds in aqueous or non-aqueous solvent systems by calculating the phase equilibria with universal quasichemical functional group activity coefficient (UNIFAC) (Dortmund) model are predicted. These compounds included saturated and unsaturated fatty acids ethyl esters, N-2,4-dinitrophenylamino alcohols, p-nitrophenyl glucosides and so on, with simple structures and much differences in polarity. It has been found that the predicted tendencies of partition coefficients of the analogues were consistent with the experimental ones reported in literature, and the best results were obtained for saturated fatty acid ethyl esters in hexane-acetonitrile (1:1 in volume ratio) solvent system. The establishment of a method for prediction will be helpful for the selection of non-electrolyte solvent systems in CCC.