Design and methods of the REMOVAL-HD study: a tRial Evaluating Mid cut-Off Value membrane clearance of Albumin and Light chains in HaemoDialysis patients.

BACKGROUND: Removal of uraemic toxins is inadequate using current dialysis strategies. A new class of dialysis membranes have been developed that allow clearance of larger middle molecules. The REMOVAL-HD study (a tRial Evaluating Mid cut-Off Value membrane clearance of Albumin and Light chains in HaemoDialysis patients) will address safety, efficacy and the impact on patient-centred outcomes with the use of a mid cut-off (MCO) dialyser in a chronic haemodialysis (HD) population. METHODS: REMOVAL-HD is an open label, prospective, non-randomised, single-arm, multi-centre device study in 85 chronic HD participants. All visits will be conducted during regular HD sessions and participants will undergo a 1 month wash-in period using a standardised high flux dialyser, 6 months of intervention with a MCO dialyser and 1 month of wash-out using a high flux dialyser. The primary endpoint is change in pre-dialysis concentrations of serum albumin, with secondary endpoints including the efficacy of clearance of free light chains and ß-2 microglobulin, and patient-centred outcomes including quality of life, symptom burden, functional status, nutritional status, hospitalisation and death. DISCUSSION: MCO dialysers are a novel form of HD membrane. The REMOVAL-HD study is a pivotal study designed to monitor the immediate and medium-term effects following exposure to this dialyser. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry Number (ANZCTRN) 12616000804482 . Date of registration - 21/06/2016.

Accumulation of few heavy metals in sewage sludges, soils and plants of Coimbatore, Tamil Nadu (India).

A study was carried out in Coimbatore district of Tamil Nadu (India) to assess the distribution pattern of heavy metals in the soils and plants irrigated with sewage effluent/sludge. About 69 soil samples (surface and subsurface), 65 plant samples as well as 34-sewage sludge samples were collected from various tehsils of Coimbatore. Six tehsils in Coimbatore have been identified and categorized into two groups--Class I City (densely populated tehsils) and Class II city (thinly populated tehsils). The available micronutrients like Fe, Mn, Zn and Cu; heavy metals: Cr, Cd, Ni, and Pb were within the safe limits. However, the total Cr and Cd concentrations were relatively higher in the sludge samples collected from Coimbatore and Tiruppur tehsils compared to other tehsils, while for Ni, the sequence was in the order Coimbatore > Tiruppur > Palladam > Pollachi > Avinashi > Mettupalayam and for Pb, Coimbatore > Mettupalayam > Palladam > Tiruppur > Avinashi > Pollachi. Soil analysis results indicated that heavy metal concentration recorded higher level in soils of Class I city (densely populated tehsils) compared to Class II city (thinly populated tehsils). The plant samples analyzed had also registered higher concentration of total Cd, Ni and Pb, which were classified under toxic, excessive and below excessive level, respectively. Correlation analysis revealed that iron (Fe), zinc (Zn), copper (Cu), cadmium (Cd) and lead (Pb) were significantly negatively correlated with pH of soil. EC had a significant positive correlation with available iron (Fe), zinc (Zn), cadmium (Cd) and lead (Pb). A significant positive correlation of Fe, Mn, Zn, Cu, Cd and Pb was also registered with OC. Among the plant samples collected, it was evident that heavy metal concentrations were recorded higher in grass spp followed by Amaranthus spp. It was inferred from the study that soils samples had higher levels of heavy metals even though the values recorded were below the critical value/toxic limit. However, long-term and indiscriminate application of untreated (raw) sewage sludge and/or letting of sewage effluent directly to agricultural field without prior treatment may result in accumulation of toxic metals in surface and subsurface soils and subsequent biotransfer (bioaccumlation) into the food chain, it may further lead to toxicity not only to plants and animals but also to consumers of the harvested crops.

Stochastic stability of Markovian jumping uncertain stochastic genetic regulatory networks with interval time-varying delays.

This paper investigates the robust stability problem of stochastic genetic regulatory networks with interval time-varying delays and Markovian jumping parameters. The structure variations at discrete time instances during the process of gene regulations known as hybrid genetic regulatory networks based on Markov process is proposed. The jumping parameters considered here are generated from a continuous-time discrete-state homogeneous Markov process, which is governed by a Markov process with discrete and finite state space. The new type of Markovian jumping matrices P(i) and Q(i) are introduced in this paper. The parameter uncertainties are assumed to be norm bounded and the discrete delay is assumed to be time-varying and belong to a given interval, which means that the lower and upper bounds of interval time-varying delays are unavoidable. Based on the Lyapunov-Krasovskii functional and stochastic stability theory, delay-interval dependent stability criteria are obtained in terms of linear matrix inequalities. Some numerical examples are given to illustrate the effectiveness of our theoretical results.

Dynamics of lead (Pb) in different soil conditions.

Four soil samples were collected from various locations having wider range of pH and EC for incubation experiment. These soil samples were added with Pb (NO3)2 salts, and samples were taken on 1st, 35th, 45thand 60th day. Soils samples were fractioned by sequential extraction to estimate the concentration of lead in different fractions viz, water soluble, exchangeable +adsorbed, organic, carbonate and residual fraction. The result reveals that concentration of organic and carbonate bound-Pb was high in waterlogged condition and soil pH has been comprehensively identified as the single most important soil factor controlling the availability of lead (Pb) in soil. Low content of Pb in exchangeable + adsorbed (KNO3) and water soluble (H2O) fraction in all soils (except in S1) could signify low availability of Pb to plants. Bioavailable fractions, viz. water soluble and exchangeable + adsorbed, were low in all soils (except S1) well below critical limits, which may not pose any toxicity in the food chain.

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures.

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.

Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease.

Nucleotide excision repair plays a crucial role in removing many types of DNA adducts formed by UV light and chemical carcinogens. We have examined the interactions of Escherichia coli UvrABC nuclease proteins with three site-specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF), N:-acetyl-2-acetylaminofluorene (AAF) and 1-nitropyrene (1-NP) in a 50mer oligonucleotide. Similar to the AF and AAF adducts, the 1-NP-induced DNA adduct contains an aminopyrene (AP) moiety covalently linked to the C8 position of guanine. The dissociation constants for UvrA binding to AF-, AAF- and AP-DNA adducts, determined by gel mobility shift assay, are 33 +/- 9, 8 +/- 2 and 23 +/- 9 nM, respectively, indicating that the AAF adduct is recognized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF-DNA was cleaved approximately 2-fold more efficiently than AF- or AP-DNA (AAF > AF approximately AP), even though AP has the largest molecular size in this group. However, an opened DNA structure of six bases around the adduct increased the incision efficiency for AF-DNA (but not for AP-DNA), making it equivalent to that for AAF-DNA. These results are consistent with a model in which DNA damage recognition by the E. coli nucleotide excision repair system consists of two sequential steps. It includes recognition of helical distortion in duplex DNA followed by recognition of the type of nucleotide chemical modification in a single-stranded region. The difference in incision efficiency between AF- and AAF-DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing structural distortions in DNA. The results of this study are discussed in the light of NMR solution structures of these DNA adducts.

Mutagenicity of the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene in Escherichia coli located in a nonrepetitive CGC sequence.

1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)). Mutational spectra of randomly introduced dG(AP) in Escherichia coli included many different types of mutations. However, a prior site-specific study in a CGCG(AP)CG sequence showed only CpG deletions and +1 frame shifts. To further explore the context effects of dG(AP) in mutagenesis, in this work this adduct was incorporated into a nonrepetitive CGC sequence in single-stranded M13mp7L2 DNA. Upon replication of this construct in repair-competent E. coli, one-base deletions and base substitutions were detected. The -1 frame shifts, whose frequency increased 3-6-fold with SOS (to an average frequency of 1.5%), involved deletion of the adjacent C residues. The base substitutions ( approximately 2.2%) included targeted G-to-T and G-to-C transversions, whose frequencies did not increase with SOS. This suggests that dG(AP) mutagenesis is highly dependent on the local DNA sequence.

C8-guanine adduct-induced stabilization of a -1 frame shift intermediate in a nonrepetitive DNA sequence.

The mechanism of frame shift mutagenesis induced by N-(deoxyguanosin-8-yl)-1-aminopyrene, the major DNA adduct formed by the carcinogen 1-nitropyrene, was investigated by thermal melting studies of a 13-mer in which the adduct was flanked by a 5' and a 3' C. Compared to the unmodified 13-mer, the adduct destabilized the duplex by 4-5 kcal/mol, and the DeltaDeltaG value remained approximately the same regardless of which base was placed opposite the adduct. In contrast, deletion of the base opposite the adduct stabilized the duplex by nearly 4 kcal/mol. The adduct in the same sequence context was inserted into a bacteriophage M13 DNA containing the simian virus 40 origin of replication. The constructed DNA template was replicated in vitro with extracts from normal human fibroblasts. The adduct was not removed from the progeny DNA following bidirectional semiconservative replication, which suggests that it had been bypassed, rather than repaired, by the cell extract. When newly replicated bacteriophage was evaluated for mutations in the region of the modified G, most contained a G at the adduct site, indicating error-free replication. A small number of mutants ( approximately 2 x 10(-3)) were detected, all of which contained a targeted G.C base pair deletion. This suggests a relationship between the thermodynamic stability of the adduct in DNA and the errors that occurred during replicative bypass by the human DNA polymerases.

Solution structure of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dA in a DNA duplex.

Solution structural studies have been undertaken on the aminopyrene-C(8)-dG ([AP]dG) adduct in the d(C5-[AP]G6-C7). d(G16-A17-G18) sequence context in an 11-mer duplex with dA opposite [AP]dG, using proton-proton distance and intensity restraints derived from NMR data in combination with distance-restrained molecular mechanics and intensity-restrained relaxation matrix refinement calculations. The exchangeable and nonexchangeable protons of the aminopyrene and the nucleic acid were assigned following analysis of two-dimensional NMR data sets on the [AP]dG.dA 11-mer duplex in H2O and D2O solution. The broadening of several resonances within the d(G16-A17-G18) segment positioned opposite the [AP]dG6 lesion site resulted in weaker NOEs, involving these protons in the adduct duplex. Both proton and carbon NMR data are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue in the adduct duplex. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs and is in contact with dC5, dC7, dG16, dA17, and dG18 residues that form a hydrophobic pocket around it. The intercalated AP ring of [AP]dG6 stacks over the purine ring of dG16 and, to a lesser extent dG18, while the looped out deoxyguanosine ring of [AP]dG6 stacks over dC5 in the solution structure of the adduct duplex. The dA17 base opposite the adduct site is not looped out of the helix but rather participates in an in-plane platform with adjacent dG18 in some of the refined structures of the adduct duplex. The solution structures are quite different for the [AP]dG.dA 11-mer duplex containing the larger aminopyrene ring (reported in this study) relative to the previously published [AF]dG.dA 11-mer duplex containing the smaller aminofluorene ring (Norman et al., Biochemistry 28, 7462-7476, 1989) in the same sequence context. Both the modified syn guanine and the dA positioned opposite it are stacked into the helix with the aminofluorene chromophore displaced into the minor groove in the latter adduct duplex. By contrast, the aminopyrenyl ring participates in an intercalated base-displaced structure in the present study of the [AP]dG.dA 11-mer duplex and in a previously published study of the [AP]dG.dC 11-mer duplex (Mao et al., Biochemistry 35, 12659-12670, 1996). Such intercalated base-displaced structures without hydrogen bonding between the [AP]dG adduct and dC or mismatched dA residues positioned opposite it, if present at a replication fork, may cause polymerase stalling and formation of a slipped intermediate that could produce frameshift mutations, the most dominant mutagenic consequence of the [AP]dG lesion.