The Role of Intramolecular Reactions and Chemical Degradation in the Apparent Biotransformation Pathways of a Series of SYK Inhibitors.

In vitro metabolism studies of the spleen tyrosine kinase inhibitors AZ-A and AZ-B identified four unusual metabolites. M1 (mass-to-charge ratio 411) was formed by both molecules and was common to several analogs (AZ-C to AZ-H) sharing the same core structure, appearing to derive from the complete loss of a pendent 3,4-diaminotetrahydropyran ring and pyrazole ring cleavage resulting in a nonobvious metabolite. M2-M4 were formed by AZ-A and a subset of the other compounds only and apparently resulted from a sequential loss of H2 from parent. Initial attempts to isolate M3 for identification were unsuccessful due to sample degradation, and it was subsequently found that M2 and M3 underwent sequential chemical degradation steps to M4. M4 was successfully isolated and shown by mass spectrometry and NMR spectroscopy to be a tricyclic species incorporating the pyrazole and the 3,4-diaminotetrahydropyran groups. We propose that this arises from an intramolecular reaction between the primary amine on the tetrahydropyran and a putative epoxide intermediate on the adjacent pyrazole ring, evidence for which was generated in a ß-mercaptoethanol-trapping experiment. The loss of the tetrahydropyran moiety observed in M1 was found to be enhanced in an analog that was unable to undergo the intramolecular reaction step, leading us to propose two possible reaction pathways originating from the reactive intermediate. Ultimately, we conclude that the apparently complex and unusual metabolism of this series of compounds likely resulted from a single metabolic activation step forming an epoxide intermediate, which subsequently underwent intramolecular rearrangement and/or chemical degradation to form the final observed products. SIGNIFICANCE STATEMENT: The current work provides an unusual biotransformation example showing the potential for intramolecular reactions and chemical degradation to give the appearance of complex metabolism arising from a single primary route of metabolism.

Synergistic biocementation: harnessing Comamonas and Bacillus ureolytic bacteria for enhanced sand stabilization.

Biocementation, driven by ureolytic bacteria and their biochemical activities, has evolved as a powerful technology for soil stabilization, crack repair, and bioremediation. Ureolytic bacteria play a crucial role in calcium carbonate precipitation through their enzymatic activity, hydrolyzing urea to produce carbonate ions and elevate pH, thus creating favorable conditions for the precipitation of calcium carbonate. While extensive research has explored the ability of ureolytic bacteria isolated from natural environments or culture conditions, bacterial synergy is often unexplored or under-reported. In this study, we isolated bacterial strains from the local eutrophic river canal and evaluated their suitability for precipitating calcium carbonate polymorphs. We identified two distinct bacterial isolates with superior urea degradation ability (conductivity method) using partial 16 S rRNA gene sequencing. Molecular identification revealed that they belong to the Comamonas and Bacillus genera. Urea degradation analysis was performed under diverse pH (6,7 and 8) and temperature (15 °C,20 °C,25 °C and 30 °C) ranges, indicating that their ideal pH is 7 and temperature is 30 °C since 95% of the urea was degraded within 96 h. In addition, we investigated these strains individually and in combination, assessing their microbially induced carbonate precipitation (MICP) in silicate fine sand under low (14 ± 0.6 °C) and ideal temperature 30 °C conditions, aiming to optimize bio-mediated soil enhancement. Results indicated that 30 °C was the ideal temperature, and combining bacteria resulted in significant (p ≤ 0.001) superior carbonate precipitation (14-16%) and permeability (> 10- 6 m/s) in comparison to the average range of individual strains. These findings provide valuable insights into the potential of combining ureolytic bacteria for future MICP research on field applications including soil erosion mitigation, soil stabilization, ground improvement, and heavy metal remediation.

The Disconnect in Intrinsic Clearance Determined in Human Hepatocytes and Liver Microsomes Results from Divergent Cytochrome P450 Activities.

Candidate drugs may exhibit higher unbound intrinsic clearances (CLint,u) in human liver microsomes (HLMs) relative to human hepatocytes (HHs), posing a challenge as to which value is more predictive of in vivo clearance (CL). This work was aimed at better understanding the mechanism(s) underlying this 'HLM:HH disconnect' via examination of previous explanations, including passive permeability limited CL or cofactor exhaustion in hepatocytes. A series of structurally related, passively permeable (Papps > 5 × 10-6 cm/s), 5-azaquinazolines were studied in different liver fractions, and metabolic rates and routes were determined. A subset of these compounds demonstrated a significant HLM:HH (CLint,u ratio 2-26) disconnect. Compounds were metabolized via combinations of liver cytosol aldehyde oxidase (AO), microsomal cytochrome P450 (CYP) and flavin monooxygenase (FMO). For this series, the lack of concordance between CLint,u determined in HLM and HH contrasted with an excellent correlation of AO dependent CLint,u determined in human liver cytosol[Formula: see text], r2 = 0.95, P < 0.0001). The HLM:HH disconnect for both 5-azaquinazolines and midazolam was as a result of significantly higher CYP activity in HLM and lysed HH fortified with exogenous NADPH relative to intact HH. Moreover, for the 5-azaquinazolines, the maintenance of cytosolic AO and NADPH-dependent FMO activity in HH, relative to CYP, supports the conclusion that neither substrate permeability nor intracellular NADPH for hepatocytes were limiting CLint,u Further studies are required to identify the underlying cause of the lower CYP activities in HH relative to HLM and lysed hepatocytes in the presence of exogenous NADPH. SIGNIFICANCE STATEMENT: Candidate drugs may exhibit higher intrinsic clearance in human liver microsomes relative to human hepatocytes, posing a challenge as to which value is predictive of in vivo clearance. This work demonstrates that the difference in activity determined in liver fractions results from divergent cytochrome P450 but not aldehyde oxidase or flavin monooxygenase activity. This is inconsistent with explanations including substrate permeability limitations or cofactor exhaustion and should inform the focus of further studies to understand this cytochrome P450 specific disconnect phenomenon.

Before and beyond trust: reliance in medical AI.

Artificial intelligence (AI) is changing healthcare and the practice of medicine as data-driven science and machine-learning technologies, in particular, are contributing to a variety of medical and clinical tasks. Such advancements have also raised many questions, especially about public trust. As a response to these concerns there has been a concentrated effort from public bodies, policy-makers and technology companies leading the way in AI to address what is identified as a "public trust deficit". This paper argues that a focus on trust as the basis upon which a relationship between this new technology and the public is built is, at best, ineffective, at worst, inappropriate or even dangerous, as it diverts attention from what is actually needed to actively warrant trust. Instead of agonising about how to facilitate trust, a type of relationship which can leave those trusting vulnerable and exposed, we argue that efforts should be focused on the difficult and dynamic process of ensuring reliance underwritten by strong legal and regulatory frameworks. From there, trust could emerge but not merely as a means to an end. Instead, as something to work in practice towards; that is, the deserved result of an ongoing ethical relationship where there is the appropriate, enforceable and reliable regulatory infrastructure in place for problems, challenges and power asymmetries to be continuously accounted for and appropriately redressed.

Presumed Dissent? Opt-out Organ Donation and the Exclusion of Organs and Tissues.

It is often claimed that a legitimate approach to organ donation is an opt-out system, also known as 'presumed consent', 'deemed consent', or 'deemed authorisation', whereby individuals are presumed or deemed willing to donate at least some of their organs and tissues after death unless they have explicitly refused permission. While sharing a default in favour of donation, such systems differ in several key respects, such as the role and importance assigned to the family members of prospective donors and their preferences, and exclusions and safeguards which often specify the demographic groups, purposes, or organs and tissues that will remain outside the scope of the opt-out system. Using the recent shift to opt-out in England, Scotland, and Northern Ireland as case studies, and by reference to the key goals motivating this shift across the UK, this article asks whether and, if so, why, and how, opt-out systems for post-mortem organ donation should restrict the types of organs and tissues for which consent is deemed. In other words, ought opt-out systems for PMOD presume dissent regarding the donation of certain organs and tissues?

Ideal proportion of the population to be patch tested: How many should we be doing?

BACKGROUND: How many patients should we be patch testing? A previous study suggested that the minimum proportion of a population to be patch tested for allergic contact dermatitis was 1:700 annually. OBJECTIVES: To evaluate if the current minimum rate for patch testing has changed over the 20 years since the previous study in order to maximize the value. METHODS: In cooperation with the British Society for Cutaneous Allergy, a proforma for collation of retrospective data between January 2015 and December 2017 was sent to patch-test centers in the United Kingdom (UK) and the Republic of Ireland (ROI). The number of positive tests was analyzed against the proportion of population tested to see what proportion of the population would yield the greatest number of positive results. RESULTS: Responses from 11 centers showed that the minimum number needed to patch test had increased to 1:550 per head of population per year using the current criteria. CONCLUSIONS: In agreement with previous studies, we should be patch testing more people than we are. We could reduce the threshold for referral of patients we patch test to derive the most benefit from this investigation.

Drug metabolite identification using ultrahigh-performance liquid chromatography-ultraviolet spectroscopy and parallelized scans on a tribrid Orbitrap mass spectrometer.

RATIONALE: To capture all metabolites in metabolite identification studies, MS/MS information is required in both positive and negative ionization mode, usually involving several sample injections to gain all information about samples. A high-resolution and high mass accuracy quadrupole/linear trap/Orbitrap tribrid instrument was used to gain this information in a novel single injection 'capture-all' approach to metabolite identification. METHODS: Diclofenac, a model compound, was incubated in human and rat hepatocytes. These incubated samples were run using an ultrahigh-performance liquid chromatography/ultraviolet (UHPLC-UV) system coupled to a Thermo Fusion tribrid mass spectrometer. Five parallel scans were used: positive and negative ion full scan, data-dependent MS/MS, both high energy dissociation and collision-induced dissociation, and data-independent all ion fragmentation (AIF) spectra were collected in positive and negative ion mode. RESULTS: Nine metabolites were identified; a metabolite observed in the UV trace, but not positive ion full scan MS, was detected in the same sample injection by negative ion full scan MS. This was identified as a sulphate metabolite, and the corresponding negative ion AIF allowed for some structural elucidation. The use of a photo-diode array (PDA) detector allowed for spectral assessment in case of changes in absorbance spectra, and the subsequent semi-quantification of metabolites. CONCLUSIONS: This method provided good-quality MS/MS data across the m/z range in both positive and negative ion mode. The addition of both negative ion full scan MS and negative ion MS/MS allowed for the detection and structural elucidation of metabolites not observed in positive ion mode. The use of the PDA detector allowed for the semi-quantification of metabolites.

Improving metabolic stability and removing aldehyde oxidase liability in a 5-azaquinazoline series of IRAK4 inhibitors.

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.

Ethical and policy issues raised by uterus transplants.

INTRODUCTION: In 2014, Brännström and colleagues reported the first human live birth following uterine transplantation (UTx). Research into this treatment for absolute uterine factor infertility has since grown with clinical trials currently taking place across centers in at least thirteen countries worldwide. SOURCES OF DATA: This review summarizes and critiques the academic literature on ethical and policy issues raised by UTx. AREAS OF AGREEMENT: There is general agreement on the importance of risk reduction and, in principle, to the sharing and maintenance of patient data on an international registry. AREAS OF CONTROVERSY: There are numerous areas of controversy ranging from whether it is ethically justified to carry out uterus transplants at all (considering the associated health risks) to how deceased donor organs for transplant should be allocated. This review focuses on three key issues: the choice between deceased and living donors, ensuring valid consent to the procedure and access to treatment. GROWING POINTS: UTx is presently a novel and rare procedure but is likely to become more commonplace in the foreseeable future, given the large number of surgical teams working on it worldwide. AREAS TIMELY FOR DEVELOPING RESEARCH: Uterus transplantation requires us to re-examine fundamental questions about the ethical and social value of gestation. If eventually extended to transgender women or even to men, it may also require us to reconceptualize what it is to be a 'father' or to be a 'mother', and the definition of these terms in law.

Biomineralisation performance of bacteria isolated from a landfill in China.

We report an investigation of microbially induced carbonate precipitation by seven indigenous bacteria isolated from a landfill in China. Bacterial strains were cultured in a medium supplemented with 25 mmol/L calcium chloride and 333 mmol/L urea. The experiments were carried out at 30 °C for 7 days with agitation by a shaking table at 130 r/min. Scanning electron microscopic and X-ray diffraction analyses showed variations in calcium carbonate polymorphs and mineral composition induced by all bacterial strains. The amount of carbonate precipitation was quantified by titration. The amount of carbonate precipitated in the medium varied among isolates, with the lowest being Bacillus aerius rawirorabr15 (LC092833) precipitating around 1.5 times more carbonate per unit volume than the abiotic (blank) solution. Pseudomonas nitroreducens szh_asesj15 (LC090854) was found to be the most efficient, precipitating 3.2 times more carbonate than the abiotic solution. Our results indicate that bacterial carbonate precipitation occurred through ureolysis and suggest that variations in carbonate crystal polymorphs and rates of precipitation were driven by strain-specific differences in urease expression and response to the alkaline environment. These results and the method applied provide benchmarking and screening data for assessing the bioremediation potential of indigenous bacteria for containment of contaminants in landfills.

Next-generation sequencing showing potential leachate influence on bacterial communities around a landfill in China.

The impact of contaminated leachate on groundwater from landfills is well known, but the specific effects on bacterial consortia are less well-studied. Bacterial communities in a landfill and an urban site located in Suzhou, China, were studied using Illumina high-throughput sequencing. A total of 153 944 good-quality reads were produced and sequences assigned to 6388 operational taxonomic units. Bacterial consortia consisted of up to 16 phyla, including Proteobacteria (31.9%-94.9% at landfill, 25.1%-43.3% at urban sites), Actinobacteria (0%-28.7% at landfill, 9.9%-34.3% at urban sites), Bacteroidetes (1.4%-25.6% at landfill, 5.6%-7.8% at urban sites), Chloroflexi (0.4%-26.5% at urban sites only), and unclassified bacteria. Pseudomonas was the dominant (67%-93%) genus in landfill leachate. Arsenic concentrations in landfill raw leachate (RL) (1.11 × 103 µg/L) and fresh leachate (FL2) (1.78 × 103 µg/L) and mercury concentrations in RL (10.9 µg/L) and FL2 (7.37 µg/L) exceeded Chinese State Environmental Protection Administration standards for leachate in landfills. The Shannon diversity index and Chao1 richness estimate showed RL and FL2 lacked richness and diversity when compared with other samples. This is consistent with stresses imposed by elevated arsenic and mercury and has implications for ecological site remediation by bioremediation or natural attenuation.

UK trends of allergic occupational skin disease attributed to fragrances 1996-2015.

BACKGROUND: Fragrances are well known to cause allergic contact dermatitis (ACD). Occupationally related cases occur, with certain groups being at higher risk. OBJECTIVE: To investigate the incidence of occupationally related cases of cutaneous fragrance allergy and to evaluate trends. METHOD: Data on incident cases of occupational ACD caused by fragrances between 1996 and 2015 (inclusive) reported to the EPIDERM surveillance scheme were analysed. RESULTS: Of the cases reported to EPIDERM during the study period, 5.2% had ACD attributed to fragrances. The highest annual incidence rates were observed in women. Hairdressers, beauticians and people working in related occupations had a 47-fold higher incidence rate ratio than the reference category (the average of all other occupations combined). Trends analysis suggested a non-significant increase in fragrance allergy over the study period among all occupations, and beauty and food workers, and a slight decrease in healthcare workers. CONCLUSIONS: Fragrance allergy is a significant problem in an occupational setting. Although there was no significant change in the incidence of fragrance-related allergic occupational contact dermatitis (OCD) during the 20-year study period, this does not mirror the trend in OCD, which is falling. Fragrance allergy continues to be a major contributor to OCD, and contributes to a greater proportion of cases.

What are the support experiences and needs of patients who have received bariatric surgery?

OBJECTIVE: To explore the support needs and experiences of patients who had received publicly or privately funded bariatric surgery and the importance of this support in mediating outcomes of surgery. METHODS: Seven semi-structured focus groups were conducted. A broad interview schedule guided the discussions which were audio-recorded and transcribed verbatim. Data were analysed thematically. RESULTS: Twenty-six women and 15 men with a mean age of 54 years (range 24-72) participated in the study. Participants described support needs from health professionals, significant others (family and friends), peers (bariatric surgery recipients) and the general community. Peer, dietetic and psychological support were identified as important factors influencing the outcomes (e.g. weight reduction or health improvement) or experience of bariatric surgery but were identified as infrequently received or inadequately provided. Psychological support was proposed as one of the most significant but commonly overlooked components of care. Support needs appeared higher in the first year post-surgery, when subsequent related or unrelated surgeries were required and following significant life change such as worsening health. For some participants, deficits in support appeared to negatively influence the experience or outcomes of surgery. CONCLUSION: Providers of bariatric surgery should discuss support needs and accessibility regularly with patients especially in the first year post-surgery and following significant change in a patient's life (e.g. declined health or childbirth). Nutrition, psychological and peer support (e.g. through support groups) may be especially important for some patients.

Germline Genetic Modification and Identity: the Mitochondrial and Nuclear Genomes.

In a legal 'first', the UK removed a prohibition against modifying embryos in human reproduction, to enable mitochondrial replacement techniques (MRTs), a move the Government distanced from 'germline genetic modification', which it aligned with modifying the nuclear genome. This paper (1) analyzes the uses and meanings of this term in UK/US legal and policy debates; and (2) evaluates related ethical concerns about identity. It shows that, with respect to identity, MRTs and nuclear genome editing techniques such as CRISPR/Cas-9 (now a policy topic), are not as different as has been supposed. While it does not follow that the two should be treated exactly alike, one of the central reasons offered for treating MRTs more permissively than nuclear genetic modification, and for not regarding MRTs as 'germline genetic modification', is thereby in doubt. Identity cannot, by itself, do the work thus far assigned to it, explicitly or otherwise, in law and policy.

Should uterus transplants be publicly funded?

Since 2000, 11 human uterine transplantation procedures (UTx) have been performed across Europe and Asia. Five of these have, to date, resulted in pregnancy and four live births have now been recorded. The most significant obstacles to the availability of UTx are presently scientific and technical, relating to the safety and efficacy of the procedure itself. However, if and when such obstacles are overcome, the most likely barriers to its availability will be social and financial in nature, relating in particular to the ability and willingness of patients, insurers or the state to pay. Thus, publicly funded healthcare systems such as the UK's National Health Service (NHS) will eventually have to decide whether UTx should be funded. With this in mind, we seek to provide an answer to the question of whether there exist any compelling reasons for the state not to fund UTx. The paper proceeds as follows. It assumes, at least for the sake of argument, that UTx will become sufficiently safe and cost-effective to be a candidate for funding and then asks, given that, what objections to funding there might be. Three main arguments are considered and ultimately rejected as providing insufficient reason to withhold funding for UTx. The first two are broad in their scope and offer an opportunity to reflect on wider issues about funding for infertility treatment in general. The third is narrower in scope and could, in certain forms, apply to UTx but not other assisted reproductive technologies (ARTs). The first argument suggests that UTx should not be publicly funded because doing so would be inconsistent with governments' obligations to prevent climate change and environmental pollution. The second claims that UTx does not treat a disorder and is not medically necessary. Finally, the third asserts that funding for UTx should be denied because of the availability of alternatives such as adoption and surrogacy.