Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes

The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variations in sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content (P1-DNA, P2-DNA, P1 + P2-DNA) decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content (P1-DNA, P2-DNA, P1 + P2-DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates. During late spermatogenesis, around 85% of the histones in the sperm nucleus are replaced with protamines. This process results in sperm chromatin compaction and also transcription silencing. In the human, protamines are comprised of two types: protamine-1 (P1) and protamine-2 (P2). Variations in sperm protamine expression are associated with male infertility. Similarly, sperm DNA integrity is important for male fertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates.

The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases.

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C-1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the gamma-phosphorus of ATP.

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.

The breath test--a call for more regional use

Although the 13C-urea breath test is commonly used for detection of Helicobacter pylori infection and eradication, access to commercial testing centres for analysis may at times limit its use. We have addressed this issue by establishing a regional-based means of analysis as a Hospital-University collaboration.

A universal measure of chaotropicity and kosmotropicity

Diverse parameters, including chaotropicity, can limit the function of cellular systems and thereby determine the extent of Earth's biosphere. Whereas parameters such as temperature, hydrophobicity, pressure, pH, Hofmeister effects, and water activity can be quantified via standard scales of measurement, the chao-/kosmotropic activities of environmentally ubiquitous substances have no widely accepted, universal scale. We developed an assay to determine and quantify chao-/kosmotropicity for 97 chemically diverse substances that can be universally applied to all solutes. This scale is numerically continuous for the solutes assayed (from +361kJkg^-1mol^-1 for chaotropes to -659kJkg^-1mol^-1 for kosmotropes) but there are key points that delineate (i) chaotropic from kosmotropic substances (i.e. chaotropes =+4; kosmotropes =-4kJkg^-1mol^-1); and (ii) chaotropic solutes that are readily water-soluble (log P<1.9) from hydrophobic substances that exert their chaotropic activity, by proxy, from within the hydrophobic domains of macromolecular systems (log P>1.9). Examples of chao-/kosmotropicity values are, for chaotropes: phenol +143, CaCl₂ +92.2, MgCl₂ +54.0, butanol +37.4, guanidine hydrochloride +31.9, urea +16.6, glycerol [>6.5M] +6.34, ethanol +5.93, fructose +4.56; for kosmotropes: proline -5.76, sucrose -6.92, dimethylsulphoxide (DMSO) -9.72, mannitol -6.69, trehalose -10.6, NaCl -11.0, glycine -14.2, ammonium sulfate -66.9, polyethylene glycol- (PEG-)1000 -126; and for relatively neutral solutes: methanol, +3.12, ethylene glycol +1.66, glucose +1.19, glycerol [<5M] +1.06, maltose -1.43 (kJkg^-1mol^-1). The data obtained correlate with solute interactions with, and structure-function changes in, enzymes and membranes. We discuss the implications for diverse fields including microbial ecology, biotechnology and astrobiology.

The UK Renal Registry Advanced CKD Study: frequency of incorrect reporting of date of start of RRT

Background: A preliminary review of the UK Renal Registry (UKRR) pre-RRT study data revealed results suggesting that, for some patients, the date of start of renal replacement therapy (RRT), as reported to the UKRR, was incorrect and often significantly later than the true date of start. A more detailed study then aimed to validate a set of criteria to identify patients with an incorrect start date. Methods: Pre-RRT laboratory data were electronically extracted from 8,810 incident RRT patients from 9 UK renal centres. Any patient with a low urea (<15 mmol/L) at the start of RRT or with a substantial improvement in kidney function (either a fall in urea >10 mmol/L or rise in eGFR >2 ml/min/1.73 m) within the two months prior to RRT were considered to potentially have an incorrect date of start. In 4
selected centres, the electronic patient records of all patients flagged were reviewed to validate these criteria.
Results: Of 8,810 patients, 1,616 (18.3%) were flagged by the identification criteria as having a potentially incorrect date of start of RRT, although a single centre accounted for 41% of the total flagged cohort. Of these flagged patients, 61.7% had been assigned an incorrect date of start of haemodialysis (HD), 5.7% had evidence of acute RRT being given before the reported date of start of HD
and 9.2% had evidence of starting peritoneal dialysis exchanges prior to the reported date of start. Of
those flagged, 10.7% had a correct date of start of RRT.
Conclusions: Accurate reporting of RRT episodes is vital for the analysis of time dependent studies such as survival or time to transplantation. A proportion of patients starting RRT were assigned an incorrect start date. In order to improve the accuracy of this reporting the UK Renal Registry
must work with renal centres and clinical staff on improving data input for the start of RRT.

An enantioselective imprinted receptor for Z-glutamate exhibiting a binding induced color change

Using 1-(4-styryl)-3-(3-nitrophenyl)urea as host monomer for the imprinting of Z-(D or L)-Glu, a polymeric receptor exhibiting strong enantioselectivity and a change in color intensity upon binding of the guest was obtained.

Effects of alcohols and compatible solutes on the activity of β-galactosidase

During alcoholic fermentation, the products build up and can, ultimately, kill the organism due to their effects on the cell's macromolecular systems. The effects of alcohols on the steady-state kinetic parameters of the model enzyme ß-galactosidase were studied. At modest concentrations (0 to 2 M), there was little effect of methanol, ethanol, propanol and butanol on the kinetic constants. However, above these concentrations, each alcohol caused the maximal rate, V (max), to fall and the Michaelis constant, K (m), to rise. Except in the case of methanol, the chaotropicity of the solute, rather than its precise chemical structure, determined and can, therefore, be used to predict inhibitory activity. Compounds which act as compatible solutes (e.g. glycerol and other polyols) generally reduced enzyme activity in the absence of alcohols at the concentration tested (191 mM). In the case of the ethanol- or propanol-inhibited ß-galactosidase, the addition of compatible solutes was unable to restore the enzyme's kinetic parameters to their uninhibited levels; addition of chaotropic solutes such as urea tended to enhance the effects of these alcohols. It is possible that the compatible solutes caused excessive rigidification of the enzyme's structure, whereas the alcohols disrupt the tertiary and quaternary structure of the protein. From the point of view of protecting enzyme activity, it may be unwise to add compatible solutes in the early stages of industrial fermentations; however, there may be benefits as the alcohol concentration increases.

Heterogeneous oxidation of pyrimidine and alkyl thioethers in ionic liquids over mesoporous Ti or Ti/Ge catalysts

Heterogeneous catalytic oxidation of a series of thioethers (2-thiomethylpyrimidine, 2-thiomethyl-4,6-dimethyl-pyrimidine, 2-thiobenzylpyrimidine, 2-thiobenzyl-4,6-dimethylpyrimidine, thioanisole, and n-heptyl methyl sulfide) was performed in ionic liquids by using MCM-41 and UVM-type mesoporous catalysts containing Ti, or Ti and Ge. A range of triflate, tetrafluoroborate, trifluoroacetate, lactate and bis(trifluoromethanesulfonyl)imide-based ionic liquids were used. The oxidations were carried out by using anhydrous hydrogen peroxide or the urea-hydrogen peroxide adduct and showed that ionic liquids are very effective solvents, achieving greater reactivity and selectivity than reactions performed in dioxane. The effects of halide and acid impurities on the reactions were also investigated. Recycling experiments on catalysts were carried out in order to evaluate Ti leaching and its effect on activity and selectivity.

Mifepristone, a glucocorticoid receptor antagonist, produces clinical and metabolic benefits in patients with Cushing's syndrome

Cushing's syndrome (CS) is a disorder associated with significant morbidity and mortality due to prolonged exposure to high cortisol concentrations.

Topochemical polymerization in self-assembled rodlike micelles of bisurea bolaamphiphiles

Rod-like micelles, formed from bolaamphiphiles with oligo(ethylene oxide) hydrophilic outer segments and a hydrophobic segment with diacetylene flanked by two urea moieties, were covalently fixated by topochemical photopolymerization to high degrees of polymerization by optimizing the hydrophobic core and the hydrophilic periphery of the bolaamphiphiles. Analysis of the polymerized product with dynamic light scattering in chloroform showed degrees of polymerization of approximately 250. Cryo-TEM of bolaamphiphiles before and after UV irradiation showed that the morphology of the rods was conserved upon topochemical polymerization. © 2014 The Royal Society of Chemistry.

Polymerisable squaramide receptors for anion binding and sensing

A novel series of polymerisable squaramides has been synthesised in high yields using simple chemical reactions, and evaluated in the binding of anionic species. These vinyl monomers can be used as functional building blocks in co-polymerisations with a plethora of co-monomers or cross-linkers, grace to their compatibility with free-radical polymerisation reactions. Aromatic substituted squaramides were found to be the strongest receptors, while binding of certain anions was accompanied by a strong colour change, attributed to the de-protonation of the squaramide. The best performing squaramide monomers were incorporated in molecularly imprinted polymers (MIPs) targeting a model anion and their capacities and selectivity were evaluated by rebinding experiments. Polymers prepared using the new squaramide monomers were compared to urea based co-polymers, and were found to contain up to 80% of the theoretical maximum number of binding sites, an exceptionally high value compared to previous reports. Strong polymer colour changes were observed upon rebinding of certain anions, equivalent to those witnessed in solution, paving the way for application of such materials in anion sensing devices.



Graphical abstract: Polymerisable squaramide receptors for anion binding and sensing

Exploring molecular recognition pathways within a family of gelators with different hydrogen bonding motifs

We report the synthesis of a family of gelators in which alkyl chains are connected to the amino groups of L-lysine methyl ester using a range of different hydrogen bonding linking groups (carbamate, amide, urea, thiourea and diacylhydrazine) using simple synthetic methodology based on isocyanate or acid chloride chemistry. The ability of these compounds to gelate organic solvents such as toluene or cyclohexane can be directly related to the ability of the linking group to form intermolecular hydrogen bonds. In general terms, the ability to structure solvents can be considered as: thiourea <carbamate <amide <urea similar to diacylhydrazine. This process has been confirmed by thermal measurements, scanning electron microscopy (SEM) and infrared and circular dichroism spectroscopies. By deprotecting the methyl ester group, we have demonstrated that a balance between hydrophobic and hydrophilic groups is essential-if the system has too much hydrophilicity (e. g., diacylhydrazine, urea) it will not form gels due to low solubility in the organic media. However, the less effective gelators based on amide and carbamate linkages are enhanced by converting the methyl ester to a carboxylic acid. Furthermore, subsequent mixing of the acid with a second component (diaminododecane) further enhances the ability to form networks, and, in the case of the amide, generates a two-component gel, which can immobilise a wide range of solvents of industrial interest including petrol and diesel (fuel oils), olive oil and sunflower oil (renewable food oils) and ethyl laurate, isopropyl myristate and isopropyl palmitate (oils used in pharmaceutical formulation). The gels are all thermoreversible, and may therefore be useful in controlled release/formulation applications.

Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228–259, 2015a, doi:10.​1016/​j.​copbio.​2015.​02.​010; Ball and Hallsworth in Phys Chem Chem Phys 17:8297–8305, 2015, doi:10.​1039/​C4CP04564E; Cray et al. in Environ Microbiol 15:287–296, 2013a, doi:10.​1111/​1462-2920.​12018). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight−1. Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0–85.7 kJ kg−1). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as ‘halophile’ and ‘polyextremophile’; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.

Friedel–Crafts alkylation catalysed by GaCl3-based liquid coordination complexes

Friedel–Crafts alkylation of benzene with 1-decene was catalysed by a new family of liquid Lewis acids: liquid coordination complexes (LCCs). LCCs are prepared by mixing a metal halide (e.g. GaCl3) and a donor molecule (e.g. N,N-dimethylacetamide, urea, or trioctylphosphine oxide), with the metal halide typically used in excess. This leads to the formation of a eutectic mixture comprised of charged and neutral species in a dynamic equilibrium. GaCl3-based LCCs were used in catalytic amounts, giving high reaction rates under ambient conditions, with selectivities to 2-phenyldecane superior to those previously reported in the literature. The influence of reaction conditions and catalyst composition on the reaction rate and selectivity was investigated. Optimised reaction conditions were suggested. This exploratory study offers promise with regard to the development of safer, LCC-based alternatives to HF in industrial alkylations.