Bioetics Issues of Artificial Placenta and Artificial Womb Technology.

Abstract: The worldwide infertility crisis and the increase in mortality and morbidity among infants, due to preterm births and associated complications, have stimulated research into artificial placenta (AP) and artificial womb (AW) technology as novel solutions. These technologies mimic the natural environment provided in the mother's womb, using chambers that ensure the supply of nutrients to the fetus and disposal of waste substances through an appropriate mechanism. This review aims to highlight the background of AP and AW technologies, revisit their historical development and proposed applications, and discuss challenges and bioethical and moral issues. Further research is required to investigate any negative effects of these new technologies, and ethical concerns pertaining to the structure and operation of this newly developed technology must be addressed and resolved prior to its introduction to the public sphere.

In Memory of Professor Derek Pheby.

Abstract: Professor Derek Pheby's passing in November 2022 marked a profound loss for the scientific community. Professor Derek Pheby, a stalwart figure in the fields of autoimmune diseases and bioethics, was known for his dedication to scientific research and patients' support, particularly for those affected by paraneoplastic autoimmune syndromes. Professor Pheby made significant contributions to research, especially about Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). His leadership of the ME Biobank and scientific coordination of EUROMENE demonstrated his commitment to pushing boundaries and fostering international collaborations. Professor Pheby's scientific work addressed various aspects of ME/CFS, from physician education to patient needs, the development of a post-mortem tissue bank, and effective treatments. Beyond his medical career, Professor Pheby was a crucial member of the Independent Ethics Committee of MAGI, he was a poet, humanitarian, and advocate for child protection. His generosity and boundless spirit left an enduring legacy, fostering innovative research in the pursuit of combating autoimmune diseases.

Human Cloning: Biology, Ethics, and Social Implications.

Abstract: This scholarly article delves into the multifaceted domains of human cloning, encompassing its biological underpinnings, ethical dimensions, and broader societal implications. The exposition commences with a succinct historical and contextual overview of human cloning, segueing into an in-depth exploration of its biological intri-cacies. Central to this biological scrutiny is a comprehensive analysis of somatic cell nuclear transfer (SCNT) and its assorted iterations. The accomplishments and discoveries in cloning technology, such as successful animal cloning operations and advances in the efficiency and viability of cloned embryos, are reviewed. Future improvements, such as reprogramming procedures and gene editing technology, are also discussed. The discourse extends to ethical quandaries intrinsic to human cloning, entailing an extensive contemplation of values such as human dignity, autonomy, and safety. Furthermore, the ramifications of human cloning on a societal plane are subjected to scrutiny, with a dedicated emphasis on ramifications encompassing personal identity, kinship connections, and the fundamental notion of maternity. Culminating the analysis is a reiteration of the imperative to develop and govern human cloning technology judiciously and conscientiously. Finally, it discusses several ethical and practical issues, such as safety concerns, the possibility of exploitation, and the erosion of human dignity, and emphasizes the significance of carefully considering these issues.

ß-Glucosidase from Streptomyces griseus: Nanoparticle immobilisation and application to alkyl glucoside synthesis.

A novel ß-glucosidase from Streptomyces griseus was cloned and overexpressed in E. coli. The purified ß-glucosidase (44 kDa) had a Km of 8.6 ± 0.5 mM and a Vmax of 217 ± 5.0 µmoles-1min-1mg at 37 °C, pH 7.2 with p-nitrophenyl-ß-D glucopyranoside as substrate. The enzyme was characterised in terms of pH optimum (pH 6.9), temperature optimum (69 °C) and the influence of solvents and effectors. Purified S. griseus ß-glucosidase was successfully immobilised, by simple absorption, onto zinc oxide (ZnO) nanoparticles without covalent modification. It remained tightly bound even after extensive washing and could be reused up to ten times without significant loss of activity. The immobilised enzyme had a higher optimum temperature and greater thermostability than the free enzyme. In immobilised form the enzyme readily catalysed the synthesis of alkyl glucosides.

In vitro antimicrobial activity and mechanism of action of novel carbohydrate fatty acid derivatives against Staphylococcus aureus and MRSA.

AIMS: This study investigates the antimicrobial activity and mode of action of novel carbohydrate fatty acid (CFA) derivatives against Staphylococcus aureus and methicillin-resistant Staph. aureus (MRSA). METHODS AND RESULTS: Minimum inhibitory concentrations (MICs) and the effect of CFA derivatives on lag phase were determined using a broth microdilution method. Lauric acid carbohydrate esters and corresponding ether analogues showed the greatest antimicrobial activity with MIC values between 0.04 and 0.16 mmol l(-1). Leakage studies at 260 nm following exposure to CFA derivatives at 4x MIC showed a significant increase in membrane permeability for all compounds, after c. 15 min exposure except for the lauric beta ether CFA derivative. Further assessment using both BacLight and luminescence ATP assays confirmed that an increase in membrane permeability and reduced metabolic activity was associated with CFA treatment. CONCLUSIONS: All strains were significantly inhibited by the novel compounds studied, and efficacy was related to specific structural features. Cell-membrane permeabilization was associated with CFA treatment and may account for at least a component of the mode of action of these compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the antimicrobial action of CFA compounds against a range of Staph. aureus and MRSA strains, and provides insights into their mode of action.

Aldehyde dehydrogenase activity of Drosophila melanogaster alcohol dehydrogenase: burst kinetics at high pH and aldehyde dismutase activity at physiological pH.

The ability of Drosophila alcohol dehydrogenase (D-ADH) to catalyze the oxidation of aldehydes to carboxylic acids has been re-examined. Prior studies are shown to have been compromised by a nonenzymic reaction between the aldehydic substrates and amine-containing buffers, e.g., glycine or Tris, and an amine-catalyzed addition of aldehyde to NAD+. These reactions interfere with spectrophotometric assays for monitoring aldehyde oxidation and obscure the nature and scope of D-ADH-catalyzed aldehyde oxidation, particularly at physiological pH. Use of nonreactive buffers, such as pyrophosphate or phosphate, and 1H NMR spectroscopy to monitor all the components of the reaction mixture reveals the facile dismutation of aldehydes into equimolar quantities of the corresponding acids and alcohols at both neutral and high pH. At high pH, dismutation is accompanied by a small burst of NADH production to a steady-state concentration ( < 10 microM) that represents a partitioning between NADH dissociation and aldehyde reduction. The increase in A340 is therefore not a direct measure of the aldehyde oxidation reaction, and the resulting kinetic values cannot be compared to those for alcohol dehydrogenation. The present results for D-ADH, combined with data from the literature, establish that aldehyde oxidation, manifest as dismutation, is a widespread property of alcohol dehydrogenases with potential physiological importance in alcohol metabolism and aldehyde detoxification.

Purification and crystallization of benzoylformate decarboxylase.

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.

The oxidation of aldehydes by horse liver alcohol dehydrogenase.

A lag phase in the spectrophotometric assay progress curve of aldehyde oxidation by HL-ADH was observed and characterised. The aldehyde oxidation and aldehyde dismutation reactions were shown to be related, and a mechanism to explain net aldehyde oxidation was proposed. The spectrophotometric assay was shown to be unsuitable for measurement of kinetic parameters for aldehyde oxidation by HL-ADH, and kinetic constants previously determined were shown to be in error. Existing data on the aldehyde dismutation reaction are insufficient to discount a role for HL-ADH in aldehyde transformation in vivo.

Horse liver alcohol dehydrogenase-catalyzed oxidation of aldehydes: dismutation precedes net production of reduced nicotinamide adenine dinucleotide.

The oxidation of aldehydes by horse liver alcohol dehydrogenase (HL-ADH) is more complex than previously recognized. At low enzyme concentrations and/or high aldehyde concentrations, a pronounced lag in the assay progress curve is observed when the reaction is monitored for NADH production at 340 nm. When the progress of the reaction is followed by 1H NMR spectroscopy, rapid dismutation of the aldehyde substrate into the corresponding acid and alcohol is observed during the lag phase. Steady-state production of NADH commences only after aldehyde concentrations drop below 5% of their initial value; thereafter, NADH production occurs with continuous adjustment of the equilibrium between aldehyde, alcohol, NADH, and NAD+. The steady-state NADH production exhibits normal Michaelis-Menten kinetics and is in accord with earlier studies using much higher enzyme concentrations where no lag phase was reported. These results establish that the ability of HL-ADH to oxidize aldehydes is much greater than previously thought. The relationship between aldehyde dismutase and aldehyde dehydrogenase activities of HL-ADH is discussed.

Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes.

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear 'mixed inhibition' pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.

The effects of assay temperature on the complex kinetics of acetaldehyde oxidation by aldehyde dehydrogenase from human erythrocytes.

Several studies have shown preparations of the cytosolic aldehyde dehydrogenase (EC 1.2.1.3) from sheep and human liver and from human erythrocytes to exhibit complex kinetic behaviour in which the dependence of the initial velocity on the concentration of acetaldehyde gives rise to downwardly curving double-reciprocal plots. This behaviour has often been analysed in terms of a sharp discontinuity in the double-reciprocal plots and its possible implications for the oxidation of acetaldehyde and other pharmacologically important aldehydes has been a subject of speculation. In the present work, it is shown that the purified, apparently homogeneous, enzyme from human erythrocytes exhibits such complex kinetic behaviour when initial rates are determined at 25 degrees, although the double-reciprocal plots describe a smooth curve with no sharp discontinuity. However, when the assays were performed at 37 degrees there was no significant deviation from Michaelis-Menten kinetics over a wide range of acetaldehyde concentrations (0.2-30 mM). At higher concentrations of acetaldehyde inhibition occurred which was competitive with respect to NAD+. These results, which indicate that the complex kinetic behaviour of aldehyde dehydrogenase is not important at physiological temperature, are interpreted in terms of the mechanisms that have been advanced to explain the phenomena.

Subcellular distribution of aldehyde dehydrogenase activities in human liver.

The subcellular distributions of aldehyde dehydrogenase activities towards acetaldehyde have been determined in wedge-biopsy samples of human liver. A form with Km values of less than 1 microM and 285 microM towards acetaldehyde and NAD+ respectively was present in the mitochondrial fraction. This enzyme had no detectable activity towards N-tele-methylimidazole acetaldehyde, the aldehyde derived from the oxidation of N-tele-methylhistamine. The activity in the cytosol was more sensitive to inhibition by disulfiram and had Km values of 270 microM and 25 microM for acetaldehyde and NAD+, respectively. It was active towards N-tele-methylimidazole acetaldehyde with a Km value of 2.5 microM and a maximum velocity that was 40% of that determined with acetaldehyde. Both these cytosolic activities had alkaline pH optima.