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Abstract Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43 Tg -ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy.

Abstract Since the COVID-19 pandemic, the interest in RNA-lipid nanoparticle (LNP) based drug products has increased drastically. While one RNA-LNP drug product, Onpattro, was already on the market in 2018, high volume manufacturing was only initiated end of 2020 with the approval of the mRNA-LNP vaccines, Comirnaty and Spikevax. As such, deep product knowledge for RNA-LNPs is continuously increasing. In this article the effect of large-scale mixing and lab-scale shaking on mRNA-LNP drug product quality characteristics is investigated. It is shown that mixing and shaking can have a profound impact on both LNP size distribution and mRNA encapsulation, suggesting a direct correlation between both quality characteristics, and further supported by a proposed underlying mechanism. An in-depth investigation of different drug product (DP) presentations reveals a consistent effect of headspace volume and LNP content on the shaking stress sensitivity. Results reported in this study are of utter importance for both small- and large-scale manufacturers but also for care givers and patients using these products.

Abstract Background The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging. Results A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-βhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far. Conclusions In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.

Abstract Background Older patients with type 2 diabetes mellitus (T2D) have an increased risk of hypoglycaemic episodes when using sulphonylureas or insulin. In the Netherlands, guidelines exist for reducing glucose-lowering medication in older patients. However, evidence is lacking that a medication reduction in older patients can be safely pursued. Here, we will examine if promoting the deprescribing of insulin/sulphonylureas with a deprescribing programme (DPP) in general practice affects T2D-complications in older overtreated patients. Methods We will perform a 1:1 cluster randomised controlled trial in 86 general practices in the Netherlands. The DPP will consist of education sessions with general practitioners and practice nurses about reducing glucose-lowering medication in older patients (≥ 70 years). Topics of the sessions include the necessity of deprescribing, tools to initiate deprescribing and strategies to discuss deprescribing with patients (shared decision making). The DPP further includes a support programme with practice visits. The study will employ a selection tool to identify possibly overtreated older patients from the electronic medical records of the general practitioner. Eligibility for enrolment in the study will be based on HbA1c targets indicated by the Dutch guidelines, which depend on age, diabetes duration, presence of frailty, and life expectancy. The control group will provide usual care. We aim to include 406 patients. The follow-up period will be 2 years. For the primary outcome, the effect of the DPP on T2D-complications will be assessed by counting the cumulative incidence of events related to under- and overtreatment in T2D as registered in the electronic medical records. We shall perform an intention-to-treat analysis and an analysis including only patients for whom deprescribing was initiated. The implementation of the DPP in general practice will be evaluated quantitatively and qualitatively using the Extended Normalisation Process Theory (ENPT) and the Reach, Efficacy – Adoption, Implementation and Maintenance (RE-AIM) model. Other secondary outcomes include quality of life, cognitive functioning, events related to overtreatment or undertreatment, biomarkers of health, amount of blood glucose-lowering medication prescriptions, and cost-effectiveness. Discussion This study will provide insight into the safety and feasibility of a programme aimed at deprescribing sulphonylureas/insulin in older people with T2D who are treated in general practice. Trial registration ISRCTN Registry, ISRCTN50008265 , registered 09 March, 2023.

Abstract Sulfur is one of six life-essential elements, but its path from interstellar clouds to planets and their atmospheres is not well known. Astronomical observations in dense clouds have so far been able to trace only 1 percent of cosmic sulfur, in the form of gas phase molecules and volatile ices, with the missing sulfur expected to be locked in a currently unidentified form. The high sulfur abundances inferred in icy and rocky solar system bodies indicate that an efficient pathway must exist from volatile atomic sulfur in the diffuse interstellar medium to some form of refractory sulfur. One hypothesis is the formation of sulfur allotropes, particularly of the stable S8. However, experimental information about sulfur allotropes under astrochemically relevant conditions, needed to constrain their abundance, is lacking. Here, we report the laboratory far-infrared spectra of sulfur allotropes and examine their fragmentation pathways. The spectra, including that of cold, isolated S8 with three bands at 53.5, 41.3 and 21.1 µm, form a benchmark for computational modelling, which show a near-perfect match with the experiments. The experimental fragmentation pathways of sulfur allotropes, key information for astrochemical formation/destruction models, evidence a facile fragmentation of S8. These findings suggest the presence of sulfur allotropes distributions in interstellar space or in the atmosphere of planets, dependent on the environmental conditions.

Abstract Parasites, such as the malaria parasite P. falciparum, are critically dependent on host nutrients. Interference with nutrient uptake can lead to parasite death and, therefore, serve as a successful treatment strategy. P. falciparum parasites cannot synthesise cholesterol, and instead source this lipid from the host. Here, we tested whether cholesterol uptake pathways could be ‘hijacked’ for optimal drug delivery to the intracellular parasite. We found that fluorescent cholesterol analogues were delivered from the extracellular environment to the intracellular parasite. We investigated the uptake and inhibitory effects of conjugate compounds, where proven antimalarial drugs (primaquine and artesunate) were attached to steroids that mimic the structure of cholesterol. These conjugated antimalarial drugs improved the inhibitory effects against multiple parasite lifecycle stages, multiple parasite species, and drug-resistant parasites, whilst also lowering the toxicity to human host cells. Steroids with introduced peroxides also displayed antimalarial activity. These results provide a proof-of-concept that cholesterol mimics can be developed as a drug delivery system against apicomplexan parasites with the potential to improve drug efficacy, increase therapeutic index, and defeat drug resistance.

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.

Abstract Mutations leading to a reduced or loss of function in genes of the leptin-melanocortin system confer a risk for monogenic forms of obesity. Yet, gain of function variants in the melanocortin-4-receptor (MC4R) gene predispose to a lower BMI. In individuals with reduced body weight, we thus expected mutations leading to an enhanced function in the respective genes, like leptin (LEP) and MC4R. Therefore, we have Sanger sequenced the coding regions of LEP and MC4R in 462 female patients with anorexia nervosa (AN), and 445 healthy-lean controls. In total, we have observed four and eight variants in LEP and MC4R, respectively. Previous studies showed different functional in vitro effects for the detected frameshift and non-synonymous variants: (1) LEP: reduced/loss of function (p.Val94Met), (2) MC4R: gain of function (p.Val103Ile, p.Ile251Leu), reduced or loss of function (p.Thr112Met, p.Ser127Leu, p.Leu211fsX) and without functional in vitro data (p.Val50Leut). In LEP, the variant p.Val94Met was detected in one patient with AN. For MC4R variants, one patient with AN carried the frameshift variant p.Leu211fsX. One patient with AN was heterozygous for two variants at the MC4R (p.Val103Ile and p.Ser127Leu). All other functionally relevant variants were detected in similar frequencies in patients with AN and lean individuals.

Abstract Attaining complete anomeric control is still one of the biggest challenges in carbohydrate chemistry. Glycosyl cations such as oxocarbenium and dioxanium ions are key intermediates of glycosylation reactions. Characterizing these highly-reactive intermediates and understanding their glycosylation mechanisms are essential to the stereoselective synthesis of complex carbohydrates. Although C-2 acyl neighbouring-group participation has been well-studied, the reactive intermediates in more remote participation remain elusive and are challenging to study. Herein, we report a workflow that is utilized to characterize rhamnosyl 1,3-bridged dioxanium ions derived from C-3 p-anisoyl esterified donors. First, we use a combination of quantum-chemical calculations and infrared ion spectroscopy to determine the structure of the cationic glycosylation intermediate in the gas-phase. In addition, we establish the structure and exchange kinetics of highly-reactive, low-abundance species in the solution-phase using chemical exchange saturation transfer, exchange spectroscopy, correlation spectroscopy, heteronuclear single-quantum correlation, and heteronuclear multiple-bond correlation nuclear magnetic resonance spectroscopy. Finally, we apply C-3 acyl neighbouring-group participation to the synthesis of complex bacterial oligosaccharides. This combined approach of finding answers to fundamental physical-chemical questions and their application in organic synthesis provides a robust basis for elucidating highly-reactive intermediates in glycosylation reactions.

Abstract Modern untargeted mass spectrometry (MS) analyses quickly detect and resolve thousands of molecular compounds. Although features are readily annotated with a molecular formula in high-resolution small-molecule MS applications, the large majority of them remains unidentified in terms of their full molecular structure. Collision-induced dissociation tandem mass spectrometry (CID-MS2) provides a diagnostic molecular fingerprint to resolve the molecular structure through a library search. However, for de novo identifications, one must often rely on in silico generated MS2 spectra as reference. The ability of different in silico algorithms to correctly predict MS2 spectra and thus to retrieve correct molecular structures is a topic of lively debate, for instance in the CASMI contest. Underlying the predicted MS2 spectra are the in silico generated product ion structures, which are normally not used in de novo identification, but which can serve to critically assess the fragmentation algorithms. Here we evaluate in silico generated MSn product ion structures by comparison with structures established experimentally by infrared ion spectroscopy (IRIS). For a set of three dozen product ion structures from five precursor molecules, we find that virtually all fragment ion structure annotations in three major in silico MS2 libraries (HMDB, METLIN, mzCloud) are incorrect and caution the reader against their use for structure annotation of MS/MS ions.

Abstract Background Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. Results We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. Conclusions Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.

Succinate dehydrogenase (SDH) is a protein complex that functions in the tricarboxylic acid cycle and the electron transport chain of mitochondria. In most eukaryotes, SDH is highly conserved and comprises the following four subunits: SdhA and SdhB form the catalytic core of the complex, while SdhC and SdhD anchor the complex in the membrane. Toxoplasma gondii is an apicomplexan parasite that infects one-third of humans worldwide. The genome of T. gondii encodes homologues of the catalytic subunits SdhA and SdhB, although the physiological role of the SDH complex in the parasite and the identity of the membrane-anchoring subunits are poorly understood. Here, we show that the SDH complex contributes to optimal proliferation and O2 consumption in the disease-causing tachyzoite stage of the T. gondii life cycle. We characterize a small membrane-bound subunit of the SDH complex called mitochondrial protein ookinete developmental defect (MPODD), which is conserved among myzozoans, a phylogenetic grouping that incorporates apicomplexan parasites and their closest free-living relatives. We demonstrate that TgMPODD is essential for SDH activity and plays a key role in attaching the TgSdhA and TgSdhB proteins to the membrane anchor of the complex. Our findings highlight a unique and important feature of mitochondrial energy metabolism in apicomplexan parasites and their relatives.

Widespread use of azoles in clinical and environmental settings has favored the selection of azole-resistant Aspergillus species necessitating antifungal susceptibility studies understanding their prevalence and resistance profiles in those settings. However, there is still limited information available on these strains in Africa. The objective of this study was to assess the environmental prevalence of Azole-resistance Aspergillus strains in poultry feed in Kabale, South West Uganda. The study evaluated the susceptibility profile of Aspergillus and other fungi isolates from 10 poultry feeds across 10 different commercial outlets. Isolates were first identified using rDNA 18S genomic sequencing via ITS 1 and 2 primer combination and confirmed using Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). Seventeen fungal strains were identified of which Aspergillus flavus was the most prevalent. All Aspergillus and one Penicillium isolates were further screened for azole-resistance using azole-containing agar plates (itraconazole, voriconazole, posaconazole, isavuconazole) and fluconazole antifungal susceptibility strip method at 4 mg/L, which was the first line of treatment. Aspergillus chevalieri, A. tamari, A. flavus, P. citrinum fungi isolates showed resistance to fluconazole. The EUCAST susceptibility testing method did not show any of the isolates resistant to tested azoles (itraconazole, voriconazole, posaconazole, isavoriconazole) or amphotericin B. Overall, we found resistance to fluconazole. This study is the first investigative study of Aspergillus species in poultry feed in Kabale, South West, Uganda.

BackgroundNegative body image and adverse body self-evaluation represent key psychological constructs within the realm of weight bias (WB), potentially intertwined with the negative self-evaluation characteristic of depressive symptomatology. Although WB encapsulates an implicit form of self-critical assessment, its exploration among people with mood disorders (MD) has been under-investigated. Our primary goal is to comprehensively assess both explicit and implicit WB, seeking to reveal specific dimensions that could interconnect with the symptoms of MDs.MethodsA cohort comprising 25 MD patients and 35 demographically matched healthy peers (with 83% female representation) participated in a series of tasks designed to evaluate the congruence between various computer-generated body representations and a spectrum of descriptive adjectives. Our analysis delved into multiple facets of body image evaluation, scrutinizing the associations between different body sizes and emotionally charged adjectives (e.g., active, apple-shaped, attractive).ResultsNo discernible differences emerged concerning body dissatisfaction or the correspondence of different body sizes with varying adjectives. Interestingly, MD patients exhibited a markedly higher tendency to overestimate their body weight (p = 0.011). Explicit WB did not show significant variance between the two groups, but MD participants demonstrated a notable implicit WB within a specific weight rating task for BMI between 18.5 and 25 kg/m2 (p = 0.012).ConclusionsDespite the striking similarities in the assessment of participants’ body weight, our investigation revealed an implicit WB among individuals grappling with MD. This bias potentially assumes a role in fostering self-directed negative evaluations, shedding light on a previously unexplored facet of the interplay between WB and mood disorders.

To reduce the number of sedentary people, an improved understanding of effects of exercise in this specific group is needed. The present project investigates the impact of regular aerobic exercise uptake on body image, and how this effect is associated with differences in interoceptive abilities and affective response to exercise. Participants were 29 sedentary adults who underwent a 12-week aerobic physical activity intervention comprised of 30-36 sessions. Body image was improved with large effect sizes. Correlations were observed between affective response to physical activity and body image improvement, but not with interoceptive abilities. Explorative mediation models suggest a neglectable role of a priori interoceptive abilities. Instead, body image improvement was achieved when positive valence was assigned to interoceptive cues experienced during exercise., Author(s): Duangkamol Srismith [sup.1] [sup.2] [sup.3] , Katja Dierkes [sup.4] , Stephan Zipfel [sup.1] , Ansgar Thiel [sup.4] , Gorden Sudeck [sup.4] , Katrin E. Giel [sup.1] , Simone C. [...]

Abstract Background Patients with eating disorders (ED) typically report delays between the onset of symptoms and engagement with treatment services. Personal barriers including stigma, shame, and guilt, as well as the availability of social support may influence patients’ decisions to engage with treatment services. Patient narratives are personalized stories discussing the illness and recovery of previously affected persons. Such narratives can reduce self-stigma and provide current patients with hope for their own recovery. Method This pilot study will examine the effects of patient narrative videos on the treatment motivation and uptake of treatment services for patients with ED. Three narrative videos were developed from the perspectives of (a) a former patient with an ED, (b) an ED specialist, and (c) the same former patient discussing a somatic condition unrelated to ED. Patients will be randomized into three video viewing and one treatment-as-usual group. Effects on treatment motivation will be assessed using the University of Rhode Island Change Assessment Scale (URICA-S) immediately after viewing the videos, as well as one-week and three-month follow-ups. Treatment uptake will be assessed during follow-up using a questionnaire listing possible treatment interactions. A post-intervention questionnaire and semi-structured interviews will be used to assess the feasibility and acceptability of patient narrative videos for this population. Discussion There is an urgent need to encourage patients with ED to engage with specialized treatments as soon as possible. Patient narratives may be a pivotal approach to implementing cost effective and easy to disseminate early intervention programs to future patients with ED.