Sm complex assembly and 5' cap trimethylation promote selective processing of snRNAs by the 3' exonuclease TOE1.

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity toward canonical snRNAs through their Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.

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.

Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump.

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were re-synthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing, revealing a strong inhibitor of QS-regulated genes, and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump, thereby inhibiting the autogenerated positive feedback and its enhanced signal-molecule synthesis.

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.

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.

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.

Functional and in silico analysis of ATP8A2 and other P4-ATPase variants associated with human genetic diseases.

P4-ATPases flip lipids from the exoplasmic to cytoplasmic leaflet of cell membranes, a property crucial for many biological processes. Mutations in P4-ATPases are associated with severe inherited and complex human disorders. We determined the expression, localization and ATPase activity of four variants of ATP8A2, the P4-ATPase associated with the neurodevelopmental disorder known as cerebellar ataxia, impaired intellectual development and disequilibrium syndrome 4 (CAMRQ4). Two variants, G447R and A772P, harboring mutations in catalytic domains, expressed at low levels and mislocalized in cells. In contrast, the E459Q variant in a flexible loop displayed wild-type expression levels, Golgi-endosome localization and ATPase activity. The R1147W variant expressed at 50% of wild-type levels but showed normal localization and activity. These results indicate that the G447R and A772P mutations cause CAMRQ4 through protein misfolding. The E459Q mutation is unlikely to be causative, whereas the R1147W may display a milder disease phenotype. Using various programs that predict protein stability, we show that there is a good correlation between the experimental expression of the variants and in silico stability assessments, suggesting that such analysis is useful in identifying protein misfolding disease-associated variants.

Uncovering structural themes across cilia microtubule inner proteins with implications for human cilia function.

Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.

5-HT_FAsTR: a versatile, label-free, high-throughput, fluorescence-based microplate assay to quantify serotonin transport and release.

The neurotransmitter serotonin plays a pivotal role in mood and depression. It also acts as a vasoconstrictor within blood vessels and is the main neurotransmitter in the gastrointestinal system. In neurotransmission, released serotonin is taken up by serotonin transporters, which are principal targets of antidepressants and the psychostimulant, ecstasy. The investigation of serotonin transporters have relied almost exclusively on the use of radiolabeled serotonin in heterogenous end-point assays. Here we adapt the genetically encoded fluorescent biosensor, iSeroSnFR, to establish and validate the Serotonin (5-HT) Fluorescence Assay for Transport and Release (5-HT_FAsTR) for functional and pharmacological studies of serotonin transport and release. We demonstrate the applicability of the method for the study of a neuronal, high-affinity, low-capacity serotonin transporter (SERT) as well as an extraneuronal low-affinity, high-capacity organic cation transporter and mutants thereof. 5HT_FAsTR offers an accessible, versatile and reliable semi-homogenous assay format that only relies on a fluorescence plate reader for repeated, real-time measurements of serotonin influx and efflux. 5HT_FAsTR accelerates and democratizes functional characterization and pharmacological studies of serotonin transporters and genetic variants thereof in disease states such as depression, anxiety and ADHD.

Nutrition and Lifestyle-Related Factors as Predictors of Muscle Atrophy in Hematological Cancer Patients.

BACKGROUND: Cancer and side effects from cytostatic treatment commonly affect nutritional status manifested as a decrease in muscle mass. We aimed to investigate the impact of nutrition and lifestyle-related factors on muscle mass in patients with hematological cancer. METHODS: Dietary intake, food preferences, quality of life (QoL), and physical activity level (PAL) were monitored during 1-2 cytostatic treatment series. Body composition was estimated using bioelectrical impedance analysis (BIA). RESULTS: 61 patients were included. Weight loss and loss of muscle mass were detected in 64% and 59% of the patients, respectively. Muscle mass was significantly positively correlated to increasing PAL (p = 0.003), while negatively correlated to increasing age (p = 0.03), physical QoL (p = 0.007), functional QoL (p = 0.05), self-perceived health (p = 0.004), and self-perceived QoL (p = 0.007). Weight was significantly positively correlated to increased intake of soft drinks (p = 0.02) as well as the favoring of bitter grain and cereal products (p = 0.03), while negatively correlated to increasing age (p = 0.03) and increasing meat intake (p = 0.009) Conclusions: Several nutritional and lifestyle-related factors affected change in body composition. The clinical significance of these changes should be investigated in controlled, interventional studies.

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.

Na+,K+-ATPase with Disrupted Na+ Binding Sites I and III Binds Na+ with Increased Affinity at Site II and Undergoes Na+-Activated Phosphorylation with ATP.

Na+,K+-ATPase actively extrudes three cytoplasmic Na+ ions in exchange for two extracellular K+ ions for each ATP hydrolyzed. The atomic structure with bound Na+ identifies three Na+ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na+ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na+ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na+ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na+ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na+ affinity. This indicates that Na+ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E1-like configuration, allowing Na+ but not K+ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.

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.

Diet-induced ketosis in adult patients with subacute acquired brain injury: a feasibility study.

Background: Research in animal models on cerebral metabolism after brain injury highlights the potential benefits of ketosis in reducing secondary brain injury, but studies in humans are lacking. Aim: This study aimed to examine if a 6-week ketogenic diet intervention with added medium-chain triglycerides (MCT) was feasible in adult patients with acquired brain injury in the subacute phase, whether ketosis could be achieved and maintained, and to what extent serious adverse reactions, adverse reactions, serious adverse events, and adverse events occured. Methods: Patients ≥18 years of age diagnosed with subacute acquired brain injury and an expectation of hospitalisation ≥6 weeks were included in the intervention group. Patients not included in the intervention group were included in a standard care reference group. The intervention consisted of a ketogenic diet supplemented with MCT to obtain a plasma concentration of ß-hydroxybutyrate (BHB) ≥0.5 mmol/L. Patients who were enterally fed were given KetoCal® 2.5:1 LQ MCT Multi Fiber (Nutricia A/S, Allerød, Denmark), supplemented with Liquigen® (Nutricia A/S, Allerød, Denmark). Patients consuming oral nutrition were given KetoCal® 2.5:1 LQ MCT Multi Fiber supplemented with Liquigen®, in addition to ketogenic meals. Results: During a 13-week inclusion period, 12 of 13 eligible patients (92% [95% CI: 67% to 99%]) were included in the intervention group, and 17 of 18 excluded patients (94% [95% CI: 74% to 99%]) were included in the reference group. Eight patients (67%) completed the 6-week intervention. It took a median of 1 day to achieve ketosis from starting a 100% MCT ketogenic diet, and it was maintained for 97% of the intervention period after ketosis was obtained. There were no serious adverse reactions to the MCT ketogenic diet, and patients experienced adverse reactions not considered serious in 9.5% of days with the intervention. The MCT ketogenic diet was accepted by patients on all intervention days, and in the two patients transitioning from enteral feeding to oral intake, there were no complications related to transitioning. Conclusion: Intervention with MCT ketogenic diet is feasible and tolerated for 6 weeks in hospitalised adult patients with subacute acquired brain injury. Randomised controlled trials are needed to assess the benefits and harms of the MCT ketogenic diet and the effect on patients' recovery.Clinical trial registration: ClinicalTrials.gov, identifier [NCT04308577].