Bacteroidota inhibit microglia clearance of amyloid-beta and promote plaque deposition in Alzheimer's disease mouse models.

The gut microbiota and microglia play critical roles in Alzheimer's disease (AD), and elevated Bacteroides is correlated with cerebrospinal fluid amyloid-ß (Aß) and tau levels in AD. We hypothesize that Bacteroides contributes to AD by modulating microglia. Here we show that administering Bacteroides fragilis to APP/PS1-21 mice increases Aß plaques in females, modulates cortical amyloid processing gene expression, and down regulates phagocytosis and protein degradation microglial gene expression. We further show that administering Bacteroides fragilis to aged wild-type male and female mice suppresses microglial uptake of Aß1-42 injected into the hippocampus. Depleting murine Bacteroidota with metronidazole decreases amyloid load in aged 5xFAD mice, and activates microglial pathways related to phagocytosis, cytokine signaling, and lysosomal degradation. Taken together, our study demonstrates that members of the Bacteroidota phylum contribute to AD pathogenesis by suppressing microglia phagocytic function, which leads to impaired Aß clearance and accumulation of amyloid plaques.

Preclinical development of EXT608, an investigational parathyroid hormone derivative with extended half-life for the treatment of hypoparathyroidism.

Hypoparathyroidism, a deficiency of parathyroid hormone (PTH), results in hypocalcemia, hyperphosphatemia, and hypercalciuria. The disease is poorly controlled by calcium and vitamin D supplements or native PTH(1-84) replacement therapy. A version of PTH is being developed using D-VITylation technology, whereby vitamin D is conjugated to a therapeutic peptide, which confers a long plasma half-life by virtue of binding to the abundant vitamin D binding protein (DBP). D-VITylation of PTH caused no reduction in activity at the PTHR1 receptor, and resulted in a plasma elimination half-life of 7-15 h in rats and 24-32 h in cynomolgus monkeys. Analysis of steady-state pharmacokinetics as a function of dose showed flat profiles with smaller peak:trough ratios at low doses, indicative of slower subcutaneous absorption. In thyroparathyroidectomized (TPTx) rats, PTH(1-34)-vitamin D conjugates restored serum calcium and phosphate levels into the normal range over the 24 h dosing period, and increased bone turnover markers and reduced bone mineral density. Urinary calcium was initially elevated, but normalized by the end of treatment on day 27. In healthy monkeys, a single dose of PTH(1-34)-vitamin D conjugates elevated serum calcium levels above the normal range for a period of 24-48 h while simultaneously reducing urinary calcium. Therefore, the lead compound, EXT608, is a promising candidate as a therapeutic that can truly mimic the endogenous activity of PTH and warrants further study in patients with hypoparathyroidism.

Bortezomib promotes the TRAIL-mediated killing of resistant rhabdomyosarcoma by ErbB2/Her2-targeted CAR-NK-92 cells via DR5 upregulation.

Treatment resistance and immune escape are hallmarks of metastatic rhabdomyosarcoma (RMS), underscoring the urgent medical need for therapeutic agents against this disease entity as a key challenge in pediatric oncology. Chimeric antigen receptor (CAR)-based immunotherapies, such as the ErbB2 (Her2)-CAR-engineered natural killer (NK) cell line NK-92/5.28.z, provide antitumor cytotoxicity primarily through CAR-mediated cytotoxic granule release and thereafter-even in cases with low surface antigen expression or tumor escape-by triggering intrinsic NK cell-mediated apoptosis induction via additional ligand/receptors. In this study, we showed that bortezomib increased susceptibility toward apoptosis in clinically relevant RMS cell lines RH30 and RH41, and patient-derived RMS tumor organoid RMS335, by upregulation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor DR5 in these metastatic, relapsed/refractory (r/r) RMS tumors. Subsequent administration of NK-92/5.28.z cells significantly enhanced antitumor activity in vitro. Applying recombinant TRAIL instead of NK-92/5.28.z cells confirmed that the synergistic antitumor effects of the combination treatment were mediated via TRAIL. Western blot analyses indicated that the combination treatment with bortezomib and NK-92/5.28.z cells increased apoptosis by interacting with the nuclear factor κB, JNK, and caspase pathways. Overall, bortezomib pretreatment can sensitize r/r RMS tumors to CAR- and, by upregulating DR5, TRAIL-mediated cytotoxicity of NK-92/5.28.z cells.

Determinants of mosaic chromosomal alteration fitness.

Clonal hematopoiesis (CH) is characterized by the acquisition of a somatic mutation in a hematopoietic stem cell that results in a clonal expansion. These driver mutations can be single nucleotide variants in cancer driver genes or larger structural rearrangements called mosaic chromosomal alterations (mCAs). The factors that influence the variations in mCA fitness and ultimately result in different clonal expansion rates are not well understood. We used the Passenger-Approximated Clonal Expansion Rate (PACER) method to estimate clonal expansion rate as PACER scores for 6,381 individuals in the NHLBI TOPMed cohort with gain, loss, and copy-neutral loss of heterozygosity mCAs. Our mCA fitness estimates, derived by aggregating per-individual PACER scores, were correlated (R2 = 0.49) with an alternative approach that estimated fitness of mCAs in the UK Biobank using population-level distributions of clonal fraction. Among individuals with JAK2 V617F clonal hematopoiesis of indeterminate potential or mCAs affecting the JAK2 gene on chromosome 9, PACER score was strongly correlated with erythrocyte count. In a cross-sectional analysis, genome-wide association study of estimates of mCA expansion rate identified a TCL1A locus variant associated with mCA clonal expansion rate, with suggestive variants in NRIP1 and TERT.

Dual-step pharmacological intervention for traumatic-like memories: implications from D-cycloserine and cannabidiol or clonidine in male and female rats.

RATIONALE: Therapeutic approaches to mitigating traumatic memories have often faced resistance. Exploring safe reconsolidation blockers, drugs capable of reducing the emotional valence of the memory upon brief retrieval and reactivation, emerges as a promising pharmacological strategy. Towards this objective, preclinical investigations should focus on aversive memories resulting in maladaptive outcomes and consider sex-related differences to enhance their translatability. OBJECTIVES: After selecting a relatively high training magnitude leading to the formation of a more intense and generalized fear memory in adult female and male rats, we investigated whether two clinically approved drugs disrupting its reconsolidation remain effective. RESULTS: We found resistant reconsolidation impairment by the α2-adrenergic receptor agonist clonidine or cannabidiol, a major non-psychotomimetic Cannabis sativa component. However, pre-retrieval administration of D-cycloserine, a partial agonist at the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor complex, facilitated their impairing effects on reconsolidation. A similar reconsolidation blockade by clonidine or cannabidiol was achieved following exposure to a non-conditioned but generalized context after D-cycloserine administration. This suggests that sufficient memory destabilization can accompany generalized fear expression. Combining clonidine with cannabidiol without potentiating memory destabilization by D-cycloserine was ineffective. CONCLUSIONS: These findings highlight the importance of NMDA receptor signaling in memory destabilization and underscore the efficacy of a dual-step pharmacological intervention in attenuating traumatic-like memories, even in a context different from the original learning environment.

Whole genome sequencing based analysis of inflammation biomarkers in the Trans-Omics for Precision Medicine (TOPMed) consortium.

Inflammation biomarkers can provide valuable insight into the role of inflammatory processes in many diseases and conditions. Sequencing based analyses of such biomarkers can also serve as an exemplar of the genetic architecture of quantitative traits. To evaluate the biological insight, which can be provided by a multi-ancestry, whole-genome based association study, we performed a comprehensive analysis of 21 inflammation biomarkers from up to 38 465 individuals with whole-genome sequencing from the Trans-Omics for Precision Medicine (TOPMed) program (with varying sample size by trait, where the minimum sample size was n = 737 for MMP-1). We identified 22 distinct single-variant associations across 6 traits-E-selectin, intercellular adhesion molecule 1, interleukin-6, lipoprotein-associated phospholipase A2 activity and mass, and P-selectin-that remained significant after conditioning on previously identified associations for these inflammatory biomarkers. We further expanded upon known biomarker associations by pairing the single-variant analysis with a rare variant set-based analysis that further identified 19 significant rare variant set-based associations with 5 traits. These signals were distinct from both significant single variant association signals within TOPMed and genetic signals observed in prior studies, demonstrating the complementary value of performing both single and rare variant analyses when analyzing quantitative traits. We also confirm several previously reported signals from semi-quantitative proteomics platforms. Many of these signals demonstrate the extensive allelic heterogeneity and ancestry-differentiated variant-trait associations common for inflammation biomarkers, a characteristic we hypothesize will be increasingly observed with well-powered, large-scale analyses of complex traits.

Heart rate variability and vasovagal tone index in brachycephalic dogs.

Brachycephalic breeds of dogs, most of which show signs of the brachycephalic syndrome may have greater parasympathetic stimulation than other breeds, leading to higher values of heart rate variability and vagal tone index. The aim of this study was to establish a computerized electrocardiographic study and an assessment of the vagus sympathetic balance through heart rate variability and vagal tone index of five brachycephalic breeds compared to mesocephalic dogs. Sixty dogs were used, divided into groups made up of Boxers, English Bulldogs, French Bulldogs, Pugs, Shih-Tzu and no defined breed mesocephalic dogs. Statistical analysis was carried out using the Shapiro-Wilk test, Kruskal-Wallis and Dunn's test or ANOVA and Bonferroni (p<0.05). In the evaluation of vagal sympathetic balance among all the dogs, there was a negative correlation between heart rate and HRV 10RR (r = - 0.7678; p < 0.0001), HRV 20RR (r = - 0.8548, p < 0.0001) and VVTI (r = - 0.2770; p = 0.0321). It can therefore be concluded that the dog's breed and morphology did not alter its electrocardiographic parameters or heart rate variability. The vagal tone index, which in other studies differed in brachycephalic dogs, showed no difference when compared separately in brachycephalic breeds.

Locally administered nanosuspension increases delivery of estradiol for the treatment of vaginal atrophy in mice.

Vaginal atrophy affects up to 57% of post-menopausal women, with symptoms ranging from vaginal burning to dysuria. Estradiol hormone replacement therapy may be prescribed to alleviate these symptoms, though many vaginal products have drawbacks including increased discharge and local tissue toxicity due to their hypertonic nature. Here, we describe the development and characterization of a Pluronic F127-coated estradiol nanosuspension (NS) formulation for improved vaginal estradiol delivery. We compare the pharmacokinetics to the clinical comparator vaginal cream (Estrace) and demonstrate increased delivery of estradiol to the vaginal tissue. We utilized ovariectomized (OVX) mice as a murine model of post-menopausal vaginal atrophy and demonstrated equivalent efficacy in vaginal re-epithelialization when dosed with either the estradiol NS or Estrace cream. Further, we demonstrate compatibility of the estradiol NS with vaginal bacteria in vitro. We demonstrate that a Pluronic F127-coated estradiol NS may be a viable option for the treatment of post-menopausal vaginal atrophy.

Evaluation of the Safer Use of Antipsychotics in Youth Study on Population Level Antipsychotic Initiation: An Interrupted Time Series Analysis.

Background: Antipsychotics carry a higher-risk profile than other psychotropic medications and may be prescribed for youth with conditions in which other first-line treatments are more appropriate. This study aimed to evaluate the population-level effect of the Safer Use of Antipsychotics in Youth (SUAY) trial, which aimed to reduce person-days of antipsychotic use among participants. Methods: We conducted an interrupted time series analysis using segmented regression to measure changes in prescribing trends of antipsychotic initiation rates pre-SUAY and post-SUAY trial at four U.S. health systems between 2013 and 2020. Results: In our overall model, adjusted for age and insurance type, antipsychotic initiation rates decreased by 0.73 (95% confidence interval [CI]: 0.30, 1.16, p = 0.002) prescriptions per 10,000 person-months before the SUAY trial. In the first quarter following the start of the trial, there was an immediate decrease in the rate of antipsychotic initiations of 6.57 (95% CI: 0.99, 12.15) prescriptions per 10,000 person-months. When comparing the posttrial period to the pretrial period, there was an increase of 1.09 (95% CI: 0.32, 1.85) prescriptions per 10,000 person-months, but the increasing rate in the posttrial period alone was not statistically significant (0.36 prescriptions per 10,000 person-months, 95% CI: -0.27, 0.99). Conclusion: The declining trend of antipsychotic initiation seen between 2013 and 2018 (pre-SUAY trial) may have naturally reached a level at which prescribing was clinically warranted and appropriate, resulting in a floor effect. The COVID-19 pandemic, which began in the final three quarters of the posttrial period, may also be related to increased antipsychotic medication initiation.

Antibodies expand the scope of angiotensin receptor pharmacology.

G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.

Cardiac function in a large animal model of myocardial infarction at 7 T: deep learning based automatic segmentation increases reproducibility.

Cardiac magnetic resonance (CMR) imaging allows precise non-invasive quantification of cardiac function. It requires reliable image segmentation for myocardial tissue. Clinically used software usually offers automatic approaches for this step. These are, however, designed for segmentation of human images obtained at clinical field strengths. They reach their limits when applied to preclinical data and ultrahigh field strength (such as CMR of pigs at 7 T). In our study, eleven animals (seven with myocardial infarction) underwent four CMR scans each. Short-axis cine stacks were acquired and used for functional cardiac analysis. End-systolic and end-diastolic images were labelled manually by two observers and inter- and intra-observer variability were assessed. Aiming to make the functional analysis faster and more reproducible, an established deep learning (DL) model for myocardial segmentation in humans was re-trained using our preclinical 7 T data (n = 772 images and labels). We then tested the model on n = 288 images. Excellent agreement in parameters of cardiac function was found between manual and DL segmentation: For ejection fraction (EF) we achieved a Pearson's r of 0.95, an Intraclass correlation coefficient (ICC) of 0.97, and a Coefficient of variability (CoV) of 6.6%. Dice scores were 0.88 for the left ventricle and 0.84 for the myocardium.

Magnetic Modulation of Biochemical Synthesis in Synthetic Cells.

Synthetic cells can be constructed from diverse molecular components, without the design constraints associated with modifying 'living' biological systems. This can be exploited to generate cells with abiotic components, creating functionalities absent in biology. One example is magnetic responsiveness, the activation and modulation of encapsulated biochemical processes using a magnetic field, which is absent from existing synthetic cell designs. This is a critical oversight, as magnetic fields are uniquely bio-orthogonal, noninvasive, and highly penetrative. Here, we address this by producing artificial magneto-responsive organelles by coupling thermoresponsive membranes with hyperthermic Fe3O4 nanoparticles and embedding them in synthetic cells. Combining these systems enables synthetic cell microreactors to be built using a nested vesicle architecture, which can respond to alternating magnetic fields through in situ enzymatic catalysis. We also demonstrate the modulation of biochemical reactions by using different magnetic field strengths and the potential to tune the system using different lipid compositions. This platform could unlock a wide range of applications for synthetic cells as programmable micromachines in biomedicine and biotechnology.

Assessment of socioeconomic and racial differences in patients undergoing concurrent gynecologic oncology and urogynecology surgeries: a National Inpatient Sample (NIS) database study.

OBJECTIVE: To assess social determinants of health impacting patients undergoing gynecologic oncology versus combined gynecologic oncology and urogynecology surgeries. METHODS: We identified patients who underwent gynecologic oncology surgeries from 2016 to 2019 in the National Inpatient Sample using the International Classification of Diseases-10 codes. Demographics, including race and insurance status, were compared for patients who underwent gynecologic oncology procedures only (Oncologic) and those who underwent concurrent incontinence or pelvic organ prolapse procedures (Urogynecologic-Oncologic). A logistic regression model assessed variables of interest after adjustment for other relevant variables. RESULTS: From 2016 to 2019 the National Inpatient Sample database contained 389 (1.14%) Urogynecologic-Oncologic cases and 33 796 (98.9%) Oncologic cases. Urogynecologic-Oncologic patients were less likely to be white (62.1% vs 68.8%, p=0.02) and were older (median 67 vs 62 years, p<0.001) than Oncologic patients. The Urogynecologic-Oncologic cohort was less likely to have private insurance as their primary insurance (31.9% vs 38.9%, p=0.01) and was more likely to have Medicare (52.2% vs 42.8%, p=0.01). After multivariable analysis, black (adjusted odds ratio (aOR) 1.41, 95% CI 1.05 to 1.89, p=0.02) and Hispanic patients (aOR 1.53, 95% CI 1.11 to 2.10, p=0.02) remained more likely to undergo Urogynecologic-Oncologic surgeries but the primary expected payer no longer differed significantly between the two groups (p=0.95). Age at admission, patient residence, and teaching location remained significantly different between the groups. CONCLUSIONS: In this analysis of a large inpatient database we identified notable racial and geographical differences between the cohorts of patients who underwent Urogynecologic-Oncologic and Oncologic procedures.

CYRI-B mediated macropinocytosis drives metastasis via lysophosphatidic acid receptor uptake.

Pancreatic ductal adenocarcinoma carries a dismal prognosis, with high rates of metastasis and few treatment options. Hyperactivation of KRAS in almost all tumours drives RAC1 activation, conferring enhanced migratory and proliferative capacity as well as macropinocytosis. Macropinocytosis is well understood as a nutrient scavenging mechanism, but little is known about its functions in trafficking of signaling receptors. We find that CYRI-B is highly expressed in pancreatic tumours in a mouse model of KRAS and p53-driven pancreatic cancer. Deletion of Cyrib (the gene encoding CYRI-B protein) accelerates tumourigenesis, leading to enhanced ERK and JNK-induced proliferation in precancerous lesions, indicating a potential role as a buffer of RAC1 hyperactivation in early stages. However, as disease progresses, loss of CYRI-B inhibits metastasis. CYRI-B depleted tumour cells show reduced chemotactic responses to lysophosphatidic acid, a major driver of tumour spread, due to impaired macropinocytic uptake of the lysophosphatidic acid receptor-1. Overall, we implicate CYRI-B as a mediator of growth and signaling in pancreatic cancer, providing new insights into pathways controlling metastasis.

Dual-layered micromesh stent technology for embolic prevention in carotid revascularization: technical experience and clinical outcomes from a high-volume interventional radiology center.

BACKGROUND: Carotid artery stenting (CAS) has become a cornerstone of carotid revascularization for stroke prevention. Despite the advantages of CAS, large-scale randomized trials involving prior (single-layer) generation of carotid stents demonstrated its higher risk of periprocedural cerebrovascular events compared to carotid endarterectomy (CEA). Dual-layer mesh-covered stents (DLSs) showed promising results in terms of 30-day embolic events. This study aims to evaluate 30-day clinical efficacy of DLS against a closed-cell single-layer stent, based on large-volume data. METHODS: The study center is part of the Italian National Outcomes Evaluation Program (PNE). CAS procedures performed between November 2017 and September 2023 were retrospectively analyzed. Our primary endpoint was 30-day survival free of death, stroke, and myocardial infarction (MI). Periprocedural stroke rate, technical success and restenosis rate of CAS procedures performed with DLSs and first-generation stents (FGSs) were also evaluated. RESULTS: Over a total of 1101 CAS procedures (55 men; 745 males; mean age of 79±7.8 years), 80.2% were treated with DLS and 48.6% were symptomatic. The cumulative stroke-, MI- and death-free 30-day survival was 98.9%, Technical success was achieved in 98.9% of cases. The DLS group showed significantly lower 30-day death, stroke and death+stroke and periprocedural minor stroke rates compared to FGS group (P=0.04; P=0.04; P=0.003 and P=0.0002, respectively). CONCLUSIONS: The use of DLS in patients undergoing CAS in our large-volume center showed a high technical success rate and minimal cerebral embolic complications by 30 days. High volumes and an experienced interventional team may contribute to these favorable outcomes.

Ectomycorrhizal fungi alter soil food webs and the functional potential of bacterial communities.

Most of Earth's trees rely on critical soil nutrients that ectomycorrhizal fungi (EcMF) liberate and provide, and all of Earth's land plants associate with bacteria that help them survive in nature. Yet, our understanding of how the presence of EcMF modifies soil bacterial communities, soil food webs, and root chemistry requires direct experimental evidence to comprehend the effects that EcMF may generate in the belowground plant microbiome. To this end, we grew Pinus muricata plants in soils that were either inoculated with EcMF and native forest bacterial communities or only native bacterial communities. We then profiled the soil bacterial communities, applied metabolomics and lipidomics, and linked omics data sets to understand how the presence of EcMF modifies belowground biogeochemistry, bacterial community structure, and their functional potential. We found that the presence of EcMF (i) enriches soil bacteria linked to enhanced plant growth in nature, (ii) alters the quantity and composition of lipid and non-lipid soil metabolites, and (iii) modifies plant root chemistry toward pathogen suppression, enzymatic conservation, and reactive oxygen species scavenging. Using this multi-omic approach, we therefore show that this widespread fungal symbiosis may be a common factor for structuring soil food webs.IMPORTANCEUnderstanding how soil microbes interact with one another and their host plant will help us combat the negative effects that climate change has on terrestrial ecosystems. Unfortunately, we lack a clear understanding of how the presence of ectomycorrhizal fungi (EcMF)-one of the most dominant soil microbial groups on Earth-shapes belowground organic resources and the composition of bacterial communities. To address this knowledge gap, we profiled lipid and non-lipid metabolites in soils and plant roots, characterized soil bacterial communities, and compared soils amended either with or without EcMF. Our results show that the presence of EcMF changes soil organic resource availability, impacts the proliferation of different bacterial communities (in terms of both type and potential function), and primes plant root chemistry for pathogen suppression and energy conservation. Our findings therefore provide much-needed insight into how two of the most dominant soil microbial groups interact with one another and with their host plant.

Robust and heritable knockdown of gene expression using a self-cleaving ribozyme in Drosophila.

The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111 bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into four independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3' fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes.

Insecure attachment and support-seeking during COVID-19: a sequential mixed methods investigation.

The COVID-19 pandemic led many couples to stay at home together with minimal contact with others. As social distancing measures reduced contact with outside households, many partners could seek support only from one another. In two studies using a sequential mixed methods design, we investigated support-seeking behaviors in romantic relationships during COVID-19. In the qualitative study, semi-structured interviews (n = 48) showed differences in how and why partners seek support with an initial consideration of the role of attachment. We identified the following themes: direct support-seeking, indirect support-seeking, (in)dependence, and gender dynamics. In the quantitative study (n = 588), high COVID-19 worry, high attachment anxiety, and low attachment avoidance were associated with more support-seeking. Interestingly, we also found that when COVID-19 worry was high, individuals high in attachment avoidance were more likely to report seeking support. The paper provides a unique insight into the impact of COVID-19 on support-seeking behaviors in romantic relationships.