Bone marrow transplantation increases sulfatase activity in somatic tissues in a multiple sulfatase deficiency mouse model.

BACKGROUND: Multiple Sulfatase Deficiency (MSD) is an ultra-rare autosomal recessive disorder characterized by deficient enzymatic activity of all known sulfatases. MSD patients frequently carry two loss of function mutations in the SUMF1 gene, encoding a formylglycine-generating enzyme (FGE) that activates 17 different sulfatases. MSD patients show common features of other lysosomal diseases like mucopolysaccharidosis and metachromatic leukodystrophy, including neurologic impairments, developmental delay, and visceromegaly. There are currently no approved therapies for MSD patients. Hematopoietic stem cell transplant (HSCT) has been applied with success in the treatment of certain lysosomal diseases. In HSCT, donor-derived myeloid cells are a continuous source of active sulfatase enzymes that can be taken up by sulfatase-deficient host cells. Thus, HSCT could be a potential approach for the treatment of MSD. METHODS: To test this hypothesis, we used a clinically relevant mouse model for MSD, B6-Sumf1(S153P/S153P) mice, engrafted with bone marrow cells, Sumf1+/+, from B6-PtprcK302E mice (CD45.1 immunoreactive). RESULTS: After 10 months post-transplant, flow cytometric analysis shows an average of 90% of circulating leukocytes of donor origin (Sumf1(+/+)). Enzymatic activity for ARSA, ARSB, and SGSH is significantly increased in spleen of B6-Sumf1(S153P/S153P) recipient mice. In non-lymphoid organs, only liver and heart show a significant correction of sulfatase activity and GAG accumulation. Frequency of inflammatory cells and lysosomal pathology is significantly reduced in liver and heart, while no significant improvement is detected in brain. CONCLUSIONS: Our results indicate that HSCT could be a suitable approach to treat MSD-pathology affecting peripheral organs, however that benefit to CNS pathology might be limited.

Multiple Sulfatase Deficiency (MSD) is a rare genetic disorder caused by loss-of-function variations in the SUMF1 gene. This deficiency results in the accumulation of toxic compounds, leading to developmental delays and neurological impairments. In a bone marrow transplant (BMT), donor cells are infused into the patient and secrete active proteins that can help remove those toxic compounds. We carried out BMT in a mouse model for MSD and saw beneficial effects on peripheral organs, such as the liver and heart, but less change in neurological symptoms. Our results will be useful for the design of potential cell therapy approaches that could be used clinically to treat MSD.

Hematopoietic stem cell gene therapy improves outcomes in a clinically relevant mouse model of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is a severe, lysosomal storage disorder caused by pathogenic variants in the gene SUMF1, encoding the sulfatase modifying factor formylglycine-generating enzyme. Patients with MSD exhibit functional deficiencies in all cellular sulfatases. The inability of sulfatases to break down their substrates leads to progressive and multi-systemic complications in patients, similar to those seen in single-sulfatase disorders such as metachromatic leukodystrophy and mucopolysaccharidoses IIIA. Here, we aimed to determine if hematopoietic stem cell transplantation with ex vivo SUMF1 lentiviral gene therapy could improve outcomes in a clinically relevant mouse model of MSD. We first tested our approach in MSD patient-derived cells and found that our SUMF1 lentiviral vector improved protein expression, sulfatase activities, and glycosaminoglycan accumulation. In vivo, we found that our gene therapy approach rescued biochemical deficits, including sulfatase activity and glycosaminoglycan accumulation, in affected organs of MSD mice treated post-symptom onset. In addition, treated mice demonstrated improved neuroinflammation and neurocognitive function. Together, these findings suggest that SUMF1 HSCT-GT can improve both biochemical and functional disease markers in the MSD mouse.

Cardiovascular events according to blood pressure thresholds recommended by ACC/AHA.

AIM: The latest guidelines from ACC/AHA define hypertension at systolic blood pressure (SBP) 130-139 mmHg or diastolic blood pressure (DBP) 80-89 mmHg in contrast to guidelines from ESC/ESH defining hypertension at SBP ≥ 140 mmHg or DBP ≥ 90 mmHg. The aim was to determine whether the ACC/AHA definition of hypertension identifies persons at elevated risk for future cardiovascular outcome. METHODS: In a Danish prospective cardiovascular study, 19,721 white men and women aged 20-98 years were examined up to five occasions between 1976 and 2015. The population was followed until December 2018. The ACC/AHA definition of the BP levels were applied: Normal: SBP <120 mmHg and DBP <80 mmHg, Elevated: SBP 120-129 mmHg and DBP <80 mmHg, Stage 1: SBP 130-139 mmHg or DBP 80-89 mmHg, Stage 2: SBP ≥140 mmHg or DBP ≥90 mmHg. Absolute 10-year risk was calculated taking repeated examinations, covariates, and competing risk into account. RESULTS: For all outcomes, the 10-year risk in stage 1 hypertension did not differ significantly from risk in subjects with normal BP: The 10-year risk of cardiovascular events in stage 1 hypertension was 14.1% [95% CI 13.2;15.0] and did not differ significantly from the risk in normal BP at 12.8% [95% CI 11.1;14.5] (p = 0.19). The risk was highest in stage 2 hypertension 19.4% [95% CI 18.9;20.0] and differed significantly from normal BP, elevated BP, and stage 1 hypertension (p < 0.001). The 10-year risk of cardiovascular death was 6.6% [95% CI 5.9;7.4] in stage 1 hypertension and did not differ significantly from the risk in normal BP at 5.7% [95% CI 4.1;7.3] (p = 0.33). CONCLUSIONS: Stage 1 hypertension as defined by the ACC/AHA guidelines has the same risk for future cardiovascular events as normal BP. In contrast, the definition of hypertension as suggested by ESC/ESH identifies patients with elevated risk of cardiovascular events.

Until 2017, there was worldwide agreement on defining hypertension at systolic blood pressure (SBP) ≥ 140 mmHg or diastolic blood pressure (DBP) ≥ 90 mmHg.In 2017, the American Cardiology Societies (ACC and AHA) lowered the threshold for defining hypertension at SBP 130-139 mmHg or DBP 80-89 mmHg.Lowering the threshold might make healthy persons sick if the thresholds do not identify persons at high risk.Unnecessary medical treatment is associated with high economic cost for the health care systems.We wanted to explore whether applying the American BP definition in a Scandinavian population identified persons with elevated risk for cardiovascular disease.As part of the Copenhagen City Heart study, 19,721 men and women aged 20-98 years were followed from 1976.They went through up to five examinations between 1976 and 2018 including BP measurements.We applied the American BP thresholds and followed the persons until death or 2018.In Denmark all citizens have a unique identification number which is linked to all health care contacts and administrative registers.We used advanced statistical methods and linked the BP measurements with the data for cardiovascular disease and death date from the Danish registries for each person.The results showed that the American definition of hypertension has same risk for future cardiovascular disease as the definition of normal BP.This means that healthy persons will be diagnosed with hypertension if the US guidelines were applied in Denmark.

Beta-lactam allergy risk stratification in a maternity population in Australia: Scope for allergy de-labelling.

BACKGROUND: Unconfirmed beta-lactam allergy in pregnant people has been associated with higher morbidity, unnecessary exposure to broad-spectrum antibiotics and prolonged hospitalisation. There are no published data on beta-lactam allergies in pregnant people in Australia. AIMS: The aim was to describe patient-reported beta-lactam allergies and appropriateness for antibiotic allergy de-labelling in a maternity cohort in Australia. METHODS: Maternity patients aged ≥18 years admitted to our institution between March 2021 and June 2021 with a beta-lactam allergy documented in their electronic medical record were interviewed for details of their allergy. The documented allergies were compared to the allergy history obtained from the interview. Severity of the allergy was rated, and appropriateness for allergy de-labelling was assessed using the Victorian Therapeutics Advisory Group beta-lactam antibiotic allergy assessment tool. RESULTS: One hundred and fifty-three beta-lactam allergies (182 reactions) were reported by 145 patients. Penicillin class antibiotics were the most frequently implicated, including unspecified penicillins (95/153, 62%), amoxicillin (19/153, 13%) and amoxicillin-clavulanate (8/153, 5%). Allergy documentation required amending in 52 of 145 patients (36%); 85 of 153 (56%) of the beta-lactam allergies were considered low risk and potentially appropriate for direct oral re-challenge. CONCLUSION: Beta-lactam allergies were inaccurately documented in more than one third of the maternity patients included in our study. As such, education of maternity care providers about the importance of accurate allergy history taking remains an urgent unmet need. Furthermore, allergy assessment and de-labelling during pregnancy should be considered in maternity patients to optimise antibiotic prescribing and to improve maternal and neonatal health outcomes.

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa -KO MLD mice transplanted with Arsa -KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

Chronic GLP1 therapy reduces postprandial IL6 in obese humans with prediabetes.

Single-dose glucagon-like peptide 1 (GLP1) therapy increases postprandial plasma IL6 levels in prediabetic, obese humans. GLP1-IL6 interactions underly multiple antidiabetic effects, but these may differ after acute versus chronic therapy. This study examines postprandial effects of GLP1 after chronic therapy. Seven humans (six Black) with prediabetes and obesity completed 6 weeks of exenatide extended release therapy. Then subjects returned for pre- and post-meal measurements of plasma IL6, GLP1, glucagon, and related inflammatory markers. Weight, which was measured before and after therapy, did not change. Plasma IL6 decreased from baseline to postmeal state ( = 0.016), with decreases in free fatty acids (P < 0.001) and increases in insulin (P = 0.002), glucose (P < 0.0001), triglycerides (P = 0.0178), and glucagon (P = 0.018). Baseline GLP1 levels matched 6 weeks of therapy. The fall in postprandial plasma IL6, which contrasts with the increase after acute therapy, highlights the need for more investigation regarding the mechanisms of acute versus chronic GLP1-IL6 signaling.

Biochemical signatures of disease severity in multiple sulfatase deficiency.

Sulfatases catalyze essential cellular reactions, including degradation of glycosaminoglycans (GAGs). All sulfatases are post-translationally activated by the formylglycine generating enzyme (FGE) which is deficient in multiple sulfatase deficiency (MSD), a neurodegenerative lysosomal storage disease. Historically, patients were presumed to be deficient of all sulfatase activities; however, a more nuanced relationship is emerging. Each sulfatase may differ in their degree of post-translational modification by FGE, which may influence the phenotypic spectrum of MSD. Here, we evaluate if residual sulfatase activity and accumulating GAG patterns distinguish cases from controls and stratify clinical severity groups in MSD. We quantify sulfatase activities and GAG accumulation using three complementary methods in MSD participants. Sulfatases differed greatly in their tolerance of reduction in FGE-mediated activation. Enzymes that degrade heparan sulfate (HS) demonstrated lower residual activities than those that act on other GAGs. Similarly, HS-derived urinary GAG subspecies preferentially accumulated, distinguished cases from controls, and correlated with disease severity. Accumulation patterns of specific sulfatase substrates in MSD provide fundamental insights into sulfatase regulation and will serve as much-needed biomakers for upcoming clinical trials. This work highlights that biomarker investigation of an ultra-rare disease can simultaneously inform our understanding of fundamental biology and advance clinical trial readiness efforts.

A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Inhibition of insulin-regulated aminopeptidase confers neuroprotection in a conscious model of ischemic stroke.

Stroke is a leading cause of mortality and morbidity with a paucity of effective pharmacological treatments. We have previously identified insulin-regulated aminopeptidase (IRAP) as a potential target for the development of a new class of drugs for the treatment of stroke, as global deletion of this gene in mice significantly protected against ischemic damage. In the current study, we demonstrate that small molecular weight IRAP inhibitors reduce infarct volume and improve neurological outcome in a hypertensive animal model of ischemic stroke. The effects of two structurally distinct IRAP inhibitors (HFI419 or SJM164) were investigated in a model of stroke where the middle cerebral artery was transiently occluded with endothelin-1 in the conscious spontaneously hypertensive rat. IRAP inhibitor was administered into the lateral ventricle at 2 or 6 h after stroke, with subsequent doses delivered at 24, 48 and 70 h post-stroke. Functional outcomes were assessed prior to drug treatment, and on day 1 and 3 post-stroke. Histological analyses and neuroinflammatory cytokine profiling were conducted at 72 and 24 h post-stroke respectively. IRAP inhibitor treatment following stroke significantly reduced infarct volume and improved neurological and motor deficits. These protective effects were maintained even when the therapeutic window was extended to 6 h. Examination of the cellular architecture at 72 h post-stroke demonstrated that IRAP expression was upregulated in CD11b positive cells and activated astrocytes. Furthermore, IRAP inhibitor treatment significantly increased gene expression for interleukin 6 and C-C motif chemokine ligand 2 in the ischemic core. This study provides proof-of-principle that selective inhibition of IRAP activity with two structurally distinct IRAP inhibitors reduces infarct volume and improves functional outcome even when the first dose is administered 6 h post-stroke. This is the first direct evidence that IRAP inhibitors are a class of drug with potential use in the treatment of ischemic stroke.

Cohort profile: The SmartSleep Study, Denmark, combining evidence from survey, clinical and tracking data.

PURPOSE: The SmartSleep Study is established to comprehensively assess the impact of night-time smartphone use on sleep patterns and health. An innovative combination of large-scale repeated survey information, high-resolution sensor-driven smartphone data, in-depth clinical examination and registry linkage allows for detailed investigations into multisystem physiological dysregulation and long-term health consequences associated with night-time smartphone use and sleep impairment. PARTICIPANTS: The SmartSleep Study consists of three interconnected data samples, which combined include 30 673 individuals with information on smartphone use, sleep and health. Subsamples of the study population also include high-resolution tracking data (n=5927) collected via a customised app and deep clinical phenotypical data (n=245). A total of 7208 participants are followed in nationwide health registries with full data coverage and long-term follow-up. FINDINGS TO DATE: We highlight previous findings on the relation between smartphone use and sleep in the SmartSleep Study, and we evaluate the interventional potential of the citizen science approach used in one of the data samples. We also present new results from an analysis in which we use 803 000 data points from the high-resolution tracking data to identify clusters of temporal trajectories of night-time smartphone use that characterise distinct use patterns. Based on these objective tracking data, we characterise four clusters of night-time smartphone use. FUTURE PLANS: The unprecedented size and coverage of the SmartSleep Study allow for a comprehensive documentation of smartphone activity during the entire sleep span. The study has been expanded by linkage to nationwide registers, which allow for further investigations into the long-term health and social consequences of night-time smartphone use. We also plan new rounds of data collection in the coming years.

Xanomeline displays concomitant orthosteric and allosteric binding modes at the M4 mAChR.

The M4 muscarinic acetylcholine receptor (M4 mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M4 mAChR in complex with the heterotrimeric Gi1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M4 mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M4 mAChR. These findings provide a basis for further understanding xanomeline's complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.

Role of Conserved Tyrosine Lid Residues in the Activation of the M2 Muscarinic Acetylcholine Receptor.

The development of subtype selective small molecule drugs for the muscarinic acetylcholine receptor (mAChR) family has been challenging. The design of more selective ligands can be improved by understanding the structure and function of key amino acid residues that line ligand binding sites. Here we study the role of three conserved key tyrosine residues [Y1043.33, Y4036.51, and Y4267.39 (Ballesteros and Weinstein numbers in superscript)] at the human M2 mAChR, located at the interface between the orthosteric and allosteric binding sites of the receptor. We specifically focused on the role of the three tyrosine hydroxyl groups in the transition between the inactive and active conformations of the receptor by making phenylalanine point mutants. Single-point mutation at either of the three positions was sufficient to reduce the affinity of agonists by ∼100-fold for the M2 mAChR, whereas the affinity of antagonists remained largely unaffected. In contrast, neither of the mutations affected the efficacy of orthosteric agonists. When mutations were combined into double and triple M2 mAChR mutants, the affinity of antagonists was reduced by more than 100-fold compared with the wild-type M2 receptor. In contrast, the affinity of allosteric modulators, either negative or positive, was retained at all single and multiple mutations, but the degree of allosteric effect exerted on the endogenous ligand acetylcholine was affected at all mutants containing Y4267.39F. These findings will provide insights to consider when designing future mAChR ligands. SIGNIFICANCE STATEMENT: Structural studies demonstrated that three tyrosine residues between the orthosteric and allosteric sites of the M2 muscarinic acetylcholine receptor (mAChR) had different hydrogen bonding networks in the inactive and active conformations. The role of hydroxyl groups of the tyrosine residues on orthosteric and allosteric ligand pharmacology was unknown. We found that hydroxyl groups of the tyrosine residues differentially affected the molecular pharmacology of orthosteric and allosteric ligands. These results provide insights to consider when designing future mAChR ligands.

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics.

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.

Structure-activity relationship of pyrazol-4-yl-pyridine derivatives and identification of a radiofluorinated probe for imaging the muscarinic acetylcholine receptor M4.

There is an accumulating body of evidence implicating the muscarinic acetylcholine receptor 4 (M4) in schizophrenia and dementia with Lewy bodies, however, a clinically validated M4 positron emission tomography (PET) radioligand is currently lacking. As such, the aim of this study was to develop a suitable M4 PET ligand that allows the non-invasive visualization of M4 in the brain. Structure-activity relationship studies of pyrazol-4-yl-pyridine derivates led to the discovery of target compound 12 - a subtype-selective positive allosteric modulator (PAM). The radiofluorinated analogue, [18F]12, was synthesized in 28 ± 10% radiochemical yield, >37 GBq/µmol and an excellent radiochemical purity >99%. Initial in vitro autoradiograms on rodent brain sections were performed in the absence of carbachol and showed moderate specificity as well as a low selectivity of [18F]12 for the M4-rich striatum. However, in the presence of carbachol, a significant increase in tracer binding was observed in the rat striatum, which was reduced by >60% under blocking conditions, thus indicating that orthosteric ligand interaction is required for efficient binding of [18F]12 to the allosteric site. Remarkably, however, the presence of carbachol was not required for high specific binding in the non-human primate (NHP) and human striatum, and did not further improve the specificity and selectivity of [18F]12 in higher species. These results pointed towards significant species-differences and paved the way for a preliminary PET study in NHP, where peak brain uptake of [18F]12 was found in the putamen and temporal cortex. In conclusion, we report on the identification and preclinical development of the first radiofluorinated M4 PET radioligand with promising attributes. The availability of a clinically validated M4 PET radioligand harbors potential to facilitate drug development and provide a useful diagnostic tool for non-invasive imaging.

Structural basis of efficacy-driven ligand selectivity at GPCRs.

A drug's selectivity for target receptors is essential to its therapeutic utility, but achieving selectivity between similar receptors is challenging. The serendipitous discovery of ligands that stimulate target receptors more strongly than closely related receptors, despite binding with similar affinities, suggests a solution. The molecular mechanism of such 'efficacy-driven selectivity' has remained unclear, however, hindering design of such ligands. Here, using atomic-level simulations, we reveal the structural basis for the efficacy-driven selectivity of a long-studied clinical drug candidate, xanomeline, between closely related muscarinic acetylcholine receptors (mAChRs). Xanomeline's binding mode is similar across mAChRs in their inactive states but differs between mAChRs in their active states, with divergent effects on active-state stability. We validate this mechanism experimentally and use it to design ligands with altered efficacy-driven selectivity. Our results suggest strategies for the rational design of ligands that achieve efficacy-driven selectivity for many pharmaceutically important G-protein-coupled receptors.

Conversion of Phenolic Acids in Canola Fermentation: Impact on Antimicrobial Activity against Salmonella enterica and Campylobacter jejuni.

Canola meal (CM) is commonly used in poultry feeds. CM has a high protein content but also contains high levels of antimicrobial phenolic acids. Lactic acid bacteria can alter CM phenolic composition during fermentation and influence its antimicrobial activity against pathogens. Fermented CM was analyzed for phenolic composition using tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). Sinapic acid and derivatives were the major phenolic acids in CM. Growth of lactobacilli in CM was attenuated when compared to cereal substrates. Glucosides and esters of sinapic acid were extensively hydrolyzed during fermentation with Lactiplantibacillus plantarum and Furfurilactobacillus milii. Lp. plantarum transformed hydroxycinnamic acids to dihydro, 4-vinyl, and 4-ethyl derivatives, Ff. milii reduced hydroxycinnamic acids to dihydroderivatives, but Limosilactobacillus reuteri did not convert hydroxycinnamic acids. The minimum inhibitory concentration of phenolic extracts was assessed with lactobacilli, Salmonella, and Campylobacter jejuni as indicator strains. Fermentation of CM with Lp. plantarum or Ff. milii increased the antimicrobial activity of phenolic extracts against Salmonella enterica and Campylobacter jejuni. Fermentation with Lm. reuteri TMW1.656 but not fermentation with Lm. reuteri TMW1.656ΔrtcN increased the antimicrobial activity of extracts owing to the production of reutericyclin. This study demonstrates that fermentation of CM with lactobacilli converts hydroxycinammic esters and may increase the antimicrobial activity of phenolic compounds in CM against pathogens.

The Design, Synthesis, and Evaluation of Novel 9-Arylxanthenedione-Based Allosteric Modulators for the δ-Opioid Receptor.

Chronic pain and depression are both widely prevalent comorbid medical conditions. While efficient, µ-opioid receptor-based medications are associated with life-threatening side effects, including respiratory depression, dependence, and addiction. The δ-opioid receptor is a promising alternative biological target for chronic pain and depression due to its significantly reduced on-target side effects compared to the µ-opioid receptor. A previous study identified two δ-opioid receptor positive allosteric modulators. Herein, we report the design of five series of compounds targeting previously unexplored regions of the originally described SAR. Analogs were assessed for their ability to potentiate the agonist response of Leu-enkephalin. Of the 30 analogs, compound 6g displayed trends toward enhancing the ERK1/2 phosphorylation signaling compared to cAMP inhibition, while compound 11c exhibited a trend in shifting the signaling bias toward cAMP inhibition. Both 6g and 11c emerged as promising tool compounds toward the design of prospective therapeutics requiring specific downstream signaling attributes.

Biased Profile of Xanomeline at the Recombinant Human M4 Muscarinic Acetylcholine Receptor.

Many Food and Drug Administration (FDA)-approved drugs are structural analogues of the endogenous (natural) ligands of G protein-coupled receptors (GPCRs). However, it is becoming appreciated that chemically distinct ligands can bind to GPCRs in conformations that lead to different cellular signaling events, a phenomenon termed biased agonism. Despite this, the rigorous experimentation and analysis required to identify biased agonism are often not undertaken in most clinical candidates and go unrealized. Recently, xanomeline, a muscarinic acetylcholine receptor (mAChR) agonist, has entered phase III clinical trials for the treatment of schizophrenia. If successful, xanomeline will be the first novel FDA-approved antipsychotic drug in almost 50 years. Intriguingly, xanomeline's potential for biased agonism at the mAChRs and, in particular, the M4 mAChR, the most promising receptor target for schizophrenia, has not been assessed. Here, we quantify the biased agonism profile of xanomeline and three other mAChR agonists in Chinese hamster ovary cells recombinantly expressing the M4 mAChR. Agonist activity was examined across nine distinct signaling readouts, including the activation of five different G protein subtypes, ERK1/2 phosphorylation, ß-arrestin recruitment, calcium mobilization, and cAMP regulation. Relative to acetylcholine (ACh), xanomeline was biased away from ERK1/2 phosphorylation and calcium mobilization compared to Gαi2 protein activation. These findings likely have important implications for our understanding of the therapeutic action of xanomeline and call for further investigation into the in vivo consequences of biased agonism in drugs targeting the M4 mAChR for the treatment of schizophrenia.

Predicting stress and depressive symptoms using high-resolution smartphone data and sleep behavior in Danish adults.

STUDY OBJECTIVES: The early detection of mental disorders is crucial. Patterns of smartphone behavior have been suggested to predict mental disorders. The aim of this study was to develop and compare prediction models using a novel combination of smartphone and sleep behavior to predict early indicators of mental health problems, specifically high perceived stress and depressive symptoms. METHODS: The data material included two separate population samples nested within the SmartSleep Study. Prediction models were trained using information from 4522 Danish adults and tested in an independent test set comprising of 1885 adults. The prediction models utilized comprehensive information on subjective smartphone behavior, objective night-time smartphone behavior, and self-reported sleep behavior. Receiver operating characteristics area-under-the-curve (ROC AUC) values obtained in the test set were recorded as the performance metrics for each prediction model. RESULTS: Neither subjective nor objective smartphone behavior was found to add additional predictive information compared to basic sociodemographic factors when forecasting perceived stress or depressive symptoms. Instead, the best performance for predicting poor mental health was found in the sleep prediction model (AUC = 0.75, 95% CI: 0.72-0.78) for perceived stress and (AUC = 0.83, 95%CI: 0.80-0.85) for depressive symptoms, which included self-reported information on sleep quantity, sleep quality and the use of sleep medication. CONCLUSIONS: Sleep behavior is an important predictor when forecasting mental health symptoms and it outperforms novel approaches using objective and subjective smartphone behavior.