Exploring the structural landscape of DNA maintenance proteins.

Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.

Metabolic preferences of astrocytes: Functional metabolic mapping reveals butyrate outcompetes acetate.

Disruptions to the gut-brain-axis have been linked to neurodegenerative disorders. Of these disruptions, reductions in the levels of short-chain fatty acids (SCFAs), like butyrate, have been observed in mouse models of Alzheimer's disease (AD). Butyrate supplementation in mice has shown promise in reducing neuroinflammation, amyloid-ß accumulation, and enhancing memory. However, the underlying mechanisms remain unclear. To address this, we investigated the impact of butyrate on energy metabolism in mouse brain slices, primary cultures of astrocytes and neurons and in-vivo by dynamic isotope labelling with [U-13C]butyrate and [1,2-13C]acetate to map metabolism via mass spectrometry. Metabolic competition assays in cerebral cortical slices revealed no competition between butyrate and the ketone body, ß-hydroxybutyrate, but competition with acetate. Astrocytes favoured butyrate metabolism compared to neurons, suggesting that the astrocytic compartment is the primary site of butyrate metabolism. In-vivo metabolism investigated in the 5xFAD mouse, an AD pathology model, showed no difference in 13C-labelling of TCA cycle metabolites between wild-type and 5xFAD brains, but butyrate metabolism remained elevated compared to acetate in both groups, indicating sustained uptake and metabolism in 5xFAD mice. Overall, these findings highlight the role of astrocytes in butyrate metabolism and the potential use of butyrate as an alternative brain fuel source.

Funding priorities and health outcomes in Danish medical research.

External research funding is an essential component of the infrastructure of modern, academic research. Priorities in funding decisions drive what knowledge is generated, and how scientists' careers are shaped. For health research, it can ultimately have implications for health outcomes. The aim of this paper is to illustrate how funding information can be used to track priorities in health research, linking them to disease burdens and research outputs. Furthermore, funding concentrations are analysed from both researcher and disease perspectives, to estimate the influence of personal Matthew-effects on the distribution of health research funding. Denmark is used as the case, including funding information from all major public and private research foundations in the period 2004-2016. Grant information is linked to research outputs and disability-adjusted life-years (DALY rates), for 34,160 publications linked to 2630 grants, receiving DKK 4.8 billion in funding. Data show poor correlation between funding priorities, research activity and disease burdens, with several diseases receiving disproportionate amounts of funding. A research opportunity index is calculated to identify diseases with the highest potential for future investments from a burden-centred point of view. Funding is highly concentrated, both on people and on specific diseases. High funding concentrations on researchers can be a driving factor behind the observed funding-to-burden imbalances, and may risk knowledge stagnation through monopolisation of the market place of ideas. Results indicate that funders of clinical and translational research, as well as some types of biomedical research, need to supplement traditional considerations of scientific excellence with measures of societal challenges and relevance.

Is something rotten in the state of Denmark? Cross-national evidence for widespread involvement but not systematic use of questionable research practices across all fields of research.

Questionable research practices (QRP) are believed to be widespread, but empirical assessments are generally restricted to a few types of practices. Furthermore, conceptual confusion is rife with use and prevalence of QRPs often being confused as the same quantity. We present the hitherto most comprehensive study examining QRPs across scholarly fields and knowledge production modes. We survey perception, use, prevalence and predictors of QRPs among 3,402 researchers in Denmark and 1,307 in the UK, USA, Croatia and Austria. Results reveal remarkably similar response patterns among Danish and international respondents (τ = 0.85). Self-reported use indicates whether respondents have used a QRP in recent publications. 9 out of 10 respondents admitted using at least one QRP. Median use is three out of nine QRP items. Self-reported prevalence reflects the frequency of use. On average, prevalence rates were roughly three times lower compared to self-reported use. Findings indicated that the perceived social acceptability of QRPs influenced self-report patterns. Results suggest that most researchers use different types of QRPs within a restricted time period. The prevalence estimates, however, do not suggest outright systematic use of specific QRPs. Perceived pressure was the strongest systemic predictor for prevalence. Conversely, more local attention to research cultures and academic age was negatively related to prevalence. Finally, the personality traits conscientiousness and, to a lesser degree, agreeableness were also inversely associated with self-reported prevalence. Findings suggest that explanations for engagement with QRPs are not only attributable to systemic factors, as hitherto suggested, but a complicated mixture of experience, systemic and individual factors, and motivated reasoning.

The dynamics of biofilm development and dispersal should be taken into account when quantifying biofilm via the crystal violet microtiter plate assay.

The crystal violet microtiter plate biofilm assay is often used to compare the amount of biofilm formed by a mutant versus wild-type or a compound-treated biofilm versus the non-treatment control. In many of these studies the amount of biofilm is assessed only at one single time point. However, if the dynamics of biofilm development of the mutant (or compound-treated biofilm) is different than that of the wild-type (or non-treatment control), then biofilm quantification at a single time point may give misleading results. To overcome this shortcoming of the common biofilm quantification technique, we recommend to use a serial dilution-based crystal violet microtiter plate biofilm assay for easy assessment of the dynamics of biofilm development and dispersal. We demonstrate that the dilution-resolved crystal violet assay displays the dynamics of Pseudomonas aeruginosa biofilm development and dispersal as efficient as a time-resolved crystal violet assay. In addition, focusing on mutants of different parts of the c-di-GMP signaling system in P. aeruginosa, we provide an example illustrating the need to assess biofilm dynamics instead of quantifying biofilm biomass at a single time point.

Upscaling and Risk Evaluation of the Synthesis of the 3,5-Diamino-1H-Pyrazole, Disperazol.

This paper presents the work performed to transition a lab-scale synthesis (1 g) to a large-scale (400 g) synthesis of the 3-5-diamino-1H-Pyrazole Disperazol, a new pharmaceutical for treatment of antibiotic-resistant Pseudomonas aeruginosa biofilm infections. The potentially hazardous diazotisation step in the lab-scale synthesis was transformed to a safe and easy-to-handle flow chemistry step. Additionally, the paper presents an OSHA-recommended safety assessment of active compound E, as performed by Fauske and Associates, LLC, Burr Ridge, IL, USA.

Results from the Danish monitoring programme on pesticide residues from the period 2012-2017 - frequencies and trends in detected pesticides.

In order to assess compliance with maximum residue levels in foods and evaluate the exposure of the Danish population to pesticides, a comprehensive monitoring programme was conducted. The work from 2012-2017 involved testing pesticide residues in fruits, vegetables, cereals, animal product and processed commodities. The sampling strategy, mainly based on exposure calculations and previous findings, involved the collection of 13,492 samples primarily from fresh conventional and organic produces on the Danish market. The origin of the samples varied, with 34% being of Danish origin and 67% originating from EU and non-EU countries. The results revealed that residues in conventionally grown produce were detected in 54% of the fruit and vegetable samples, and 30% of the cereal samples. Additionally, residues above the maximum residue limits were found in 1.8% of these samples, most frequently in fruits. As previous years, more residues were found in samples of foreign origin compared to samples of Danish origin. Compared to earlier findings more than 40 pesticides were detected for the first time and including boscalid, imidacloprid, thiacloprid, etofenprox, and spinosad, all detected more than 100 times. The data shows that detection of PFAS pesticides has increased dramatically, from 24 in 2006 to 412 in 2022.

Multidimensional Outcome Parameters in a Cress Seedling-CuCl2 Crystallization Assay to Corroborate Specific Effects of Stannum metallicum 30x Compared to Lactose 30x.

BACKGROUND: Previously we developed a test system which yielded highly significant evidence for specific effects of a Stannum metallicum 30x preparation in a multi-center replication trial. This test system is based on cress seed germination in homeopathic or control samples, CuCl2 crystallization of the cress extract, and subsequent digital textural image analysis of the resulting crystallization patterns. OBJECTIVES: The current study aimed to investigate whether three novel outcome parameters could further corroborate and possibly characterize the specific effects of Stannum metallicum 30x. METHODS: To this end, (1) cress seedling length, (2) a second texture analysis parameter, entropy and (3) the local connected fractal dimension (LCFD) of crystallization patterns as a measure of complexity were considered. The stability of the experimental setup was monitored throughout the entire investigation with systematic negative control (SNC) experiments. RESULTS: Cress length and entropy revealed a time-modulated potency treatment effect, in the absence of a significant main treatment effect. This indicated that the effect of the potency treatment varied significantly across the different experimental days. LCFD yielded a highly significant potency treatment effect. In addition, a significant interaction of treatment with experimental day seems to indicate a modulation of this effect. No significant effects were observed in any of the evaluations of the SNC experiments, indicative of a stable experimental setup and a reliable and specific treatment effect. Neither significant nor strong correlations were found between the four parameters, indicating that they reflect different effects of Stannum metallicum 30x on the organism treated. CONCLUSION: This quadruple characterization of the biological effects of Stannum metallicum 30x provides an unprecedented opportunity for basic homeopathy research into, among others, the presumed specificity of homeopathic preparations.

Deficient brain GABA metabolism leads to widespread impairments of astrocyte and oligodendrocyte function.

The neurometabolic disorder succinic semialdehyde dehydrogenase (SSADH) deficiency leads to great neurochemical imbalances and severe neurological manifestations. The cause of the disease is loss of function of the enzyme SSADH, leading to impaired metabolism of the principal inhibitory neurotransmitter GABA. Despite the known identity of the enzymatic deficit, the underlying pathology of SSADH deficiency remains unclear. To uncover new mechanisms of the disease, we performed an untargeted integrative analysis of cerebral protein expression, functional metabolism, and lipid composition in a genetic mouse model of SSADH deficiency (ALDH5A1 knockout mice). Our proteomic analysis revealed a clear regional vulnerability, as protein alterations primarily manifested in the hippocampus and cerebral cortex of the ALDH5A1 knockout mice. These regions displayed aberrant expression of proteins linked to amino acid homeostasis, mitochondria, glial function, and myelination. Stable isotope tracing in acutely isolated brain slices demonstrated an overall maintained oxidative metabolism of glucose, but a selective decrease in astrocyte metabolic activity in the cerebral cortex of ALDH5A1 knockout mice. In contrast, an elevated capacity of oxidative glutamine metabolism was observed in the ALDH5A1 knockout brain, which may serve as a neuronal compensation of impaired astrocyte glutamine provision. In addition to reduced expression of critical oligodendrocyte proteins, a severe depletion of myelin-enriched sphingolipids was found in the brains of ALDH5A1 knockout mice, suggesting degeneration of myelin. Altogether, our study highlights that impaired astrocyte and oligodendrocyte function is intimately linked to SSADH deficiency pathology, suggesting that selective targeting of glial cells may hold therapeutic potential in this disease.

High efficacy treatment of murine Pseudomonas aeruginosa catheter-associated urinary tract infections using the c-di-GMP modulating anti-biofilm compound Disperazol in combination with ciprofloxacin.

Persistent urinary tract infections (UTIs) in hospitalized patients constitute an important medical problem. It is estimated that 75% of nosocomial UTIs are associated with urinary tract catheters with P. aeruginosa being a species that forms biofilms on these catheters. These infections are highly resistant to standard-of-care antibiotics, and the effects of the host immune defenses, which allows for development of persistent infections. With antibiotics losing their efficacy, new treatment options against resilient infections, such as catheter-associated urinary tract infections (CAUTIs), are critically needed. Central to our anti-biofilm approach is the manipulation of the c-di-GMP signaling pathway in P. aeruginosa to switch bacteria from the protective biofilm to the unprotected planktonic mode of life. We recently identified a compound (H6-335-P1), that stimulates the c-di-GMP degrading activity of the P. aeruginosa BifA protein which plummets the intracellular c-di-GMP content and induces dispersal of P. aeruginosa biofilm bacteria into the planktonic state. In the present study, we formulated H6-335-P1 as a hydrochloride salt (Disperazol), which is water-soluble and facilitates delivery via injection or oral administration. Disperazol can work as a monotherapy, but we observed a 100-fold improvement in efficacy when treating murine P. aeruginosa CAUTIs with a Disperazol/ciprofloxacin combination. Biologically active Disperazol reached the bladder 30 min after oral administration. Our study provides proof of concept that Disperazol can be used in combination with a relevant antibiotic for effective treatment of CAUTIs.