IL-10 suppresses T cell expansion while promoting tissue-resident memory cell formation during SARS-CoV-2 infection in rhesus macaques.

The regulation of inflammatory responses and pulmonary disease during SARS-CoV-2 infection is incompletely understood. Here we examine the roles of the prototypic pro- and anti-inflammatory cytokines IFNγ and IL-10 using the rhesus macaque model of mild COVID-19. We find that IFNγ drives the development of 18fluorodeoxyglucose (FDG)-avid lesions in the lungs as measured by PET/CT imaging but is not required for suppression of viral replication. In contrast, IL-10 limits the duration of acute pulmonary lesions, serum markers of inflammation and the magnitude of virus-specific T cell expansion but does not impair viral clearance. We also show that IL-10 induces the subsequent differentiation of virus-specific effector T cells into CD69+CD103+ tissue resident memory cells (Trm) in the airways and maintains Trm cells in nasal mucosal surfaces, highlighting an unexpected role for IL-10 in promoting airway memory T cells during SARS-CoV-2 infection of macaques.

Governance and legal considerations supporting prehospital emergency care in low and middle-income countries-For the Special Series on Prehospital Care in LMICs.

The global imperative to expand prehospital emergency care in low and middle-income countries to reduce health disparities and improve outcomes for time-sensitive health conditions is well established in academic literature and public policy discussions. However, the governance and legal frameworks essential for the strategic development of prehospital systems remain understudied and inadequately addressed. This paper delves into the critical role of governance in prehospital systems, emphasizing its impact on equity, human rights, and the provision of timely, quality emergency care. Health system governance, defined as a complex interplay of mechanisms, processes, and institutions, is a neglected yet pivotal component of prehospital care. By highlighting previously described barriers, we underscore the opportunity to strengthen prehospital care through improved governance, particularly in leadership and legislative standards. Drawing on the World Health Organization's Health System Building Blocks and the Emergency Care System Framework, we elucidate the multifaceted nature of governance in the prehospital context, including the coordination of diverse stakeholders, the establishment of standards, and the creation of accountability mechanisms. We emphasize the importance of applying a human rights perspective to governance, ensuring non-discriminatory and timely access to emergency care. Through the application of an established governance framework of 10 principles to assess prehospital system governance, we offer policymakers and stakeholders a structured approach to identify weaknesses, propose solutions, and evaluate progress in the prehospital system. To provide practical insights, we present a contemporary case study of Ghana's National Ambulance Service Act and the Health Institutions and Facilities Act of 2011, which establish a structured approach to governance and oversight while reflecting Ghana's commitment to advancing emergency care yet faces common challenges to operationalizing the laws. We advocate for a renewed focus on governance as an essential building block for effective prehospital emergency care. By providing a comprehensive framework and case study analysis, the paper offers actionable insights to guide policymakers and stakeholders in developing and evaluating governance initiatives that improve the availability, accessibility, acceptability, and quality of prehospital care globally.

Neuromodulatory subcortical nucleus integrity is associated with white matter microstructure, tauopathy and APOE status.

The neuromodulatory subcortical nuclei within the isodendritic core (IdC) are the earliest sites of tauopathy in Alzheimer's disease (AD). They project broadly throughout the brain's white matter. We investigated the relationship between IdC microstructure and whole-brain white matter microstructure to better understand early neuropathological changes in AD. Using multiparametric quantitative magnetic resonance imaging we observed two covariance patterns between IdC and white matter microstructure in 133 cognitively unimpaired older adults (age 67.9 ± 5.3 years) with familial risk for AD. IdC integrity related to 1) whole-brain neurite density, and 2) neurite orientation dispersion in white matter tracts known to be affected early in AD. Pattern 2 was associated with CSF concentration of phosphorylated-tau, indicating AD specificity. Apolipoprotein-E4 carriers expressed both patterns more strongly than non-carriers. IdC microstructure variation is reflected in white matter, particularly in AD-affected tracts, highlighting an early mechanism of pathological development.

p75 neurotrophin receptor modulation in mild to moderate Alzheimer disease: a randomized, placebo-controlled phase 2a trial.

p75 neurotrophin receptor (p75NTR) signaling pathways substantially overlap with degenerative networks active in Alzheimer disease (AD). Modulation of p75NTR with the first-in-class small molecule LM11A-31 mitigates amyloid-induced and pathological tau-induced synaptic loss in preclinical models. Here we conducted a 26-week randomized, placebo-controlled, double-blinded phase 2a safety and exploratory endpoint trial of LM11A-31 in 242 participants with mild to moderate AD with three arms: placebo, 200 mg LM11A-31 and 400 mg LM11A-31, administered twice daily by oral capsules. This trial met its primary endpoint of safety and tolerability. Within the prespecified secondary and exploratory outcome domains (structural magnetic resonance imaging, fluorodeoxyglucose positron-emission tomography and cerebrospinal fluid biomarkers), significant drug-placebo differences were found, consistent with the hypothesis that LM11A-31 slows progression of pathophysiological features of AD; no significant effect of active treatment was observed on cognitive tests. Together, these results suggest that targeting p75NTR with LM11A-31 warrants further investigation in larger-scale clinical trials of longer duration. EU Clinical Trials registration: 2015-005263-16 ; ClinicalTrials.gov registration: NCT03069014 .

The relational bottleneck as an inductive bias for efficient abstraction.

A central challenge for cognitive science is to explain how abstract concepts are acquired from limited experience. This has often been framed in terms of a dichotomy between connectionist and symbolic cognitive models. Here, we highlight a recently emerging line of work that suggests a novel reconciliation of these approaches, by exploiting an inductive bias that we term the relational bottleneck. In that approach, neural networks are constrained via their architecture to focus on relations between perceptual inputs, rather than the attributes of individual inputs. We review a family of models that employ this approach to induce abstractions in a data-efficient manner, emphasizing their potential as candidate models for the acquisition of abstract concepts in the human mind and brain.

Mental Health across the Early Life Course at the Intersection of Race, Skin Tone, and School Racial Context.

Prior research documents higher levels of depressive symptoms among Black Americans relative to Whites. Yet, we know less about the role of other dimensions of stratification (e.g., skin tone) in shaping mental health inequality between Black and White adults, and whether mental health trajectories by race and skin tone among Black adults are contingent upon social contexts in childhood and adolescence. To address these gaps, this study asks: 1) to what extent do self-identified race and interviewer-rated skin tone among Black respondents shape inequalities in depressive symptoms between Black and White Americans across ages 12-42? 2) Are trajectories of depressive symptoms by race and skin tone among Black respondents contingent on school racial contexts (e.g., school racial composition)? Using five waves of data from the National Longitudinal Study of Adolescent to Adult Health and growth curve models, results suggest trajectories of depressive symptoms across ages 12-42 vary by race, school racial context, and skin tone among Black respondents. Specifically, Black students rated as having very dark, dark, and medium brown skin who attended high proportion Black schools in adolescence experienced lower levels of depressive symptoms than their White and light-skinned Black counterparts, particularly across the teen years and early 20s. Conversely, attending higher proportion White schools led to increases in depressive symptoms across earlier ages for Black students, particularly those who fell within the middle of the skin color continuum. Findings highlight competing advantages and disadvantages of navigating racialized spaces in childhood/adolescence for Black Americans of different skin tones.

Level One Trauma Center Proliferation May Worsen Patient Outcomes.

BACKGROUND: From 2013 to 2020, Arizona state trauma system expanded from seven to thirteen level 1 trauma centers (L1TCs). This study utilized the state trauma registry to analyze the effect of L1TC proliferation on patient outcomes. METHODS: Adult patients age≥15 in the state trauma registry from 2007-2020 were queried for demographic, injury, and outcome variables. These variables were compared across the 2 time periods: 2007-2012 as pre-proliferation (PRE) and 2013-2020 as post-proliferation (POST). Multivariate logistic regression was performed to assess independent predictors of mortality. Subgroup analyses were done for Injury Severity Score (ISS)≥15, age≥65, and trauma mechanisms. RESULTS: A total of 482,896 trauma patients were included in this study. 40% were female, 29% were geriatric patients, and 8.6% sustained penetrating trauma. The median ISS was 4. Inpatient mortality overall was 2.7%. POST consisted of more female, geriatric, and blunt trauma patients (P < .001). Both periods had similar median ISS. POST had more interfacility transfers (14.5% vs 10.3%, P < .001). Inpatient, unadjusted mortality decreased by .5% in POST (P < .001). After adjusting for age, gender, ISS, and trauma mechanism, being in POST was predictive of death (OR: 1.4, CI:1.3-1.5, P < .001). This was consistent across all subgroups except for geriatric subgroup, which there was no significant correlation. DISCUSSION: Despite advances in trauma care and almost doubling of L1TCs, POST had minimal reduction of unadjusted mortality and was an independent predictor of death. Results suggest increasing number of L1TCs alone may not improve mortality. Alternative approaches should be sought with future regional trauma system design and implementation.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Impact of lower level trauma center proliferation on patient outcomes.

Background: In attempt to increase trauma system coverage, our state added 21 level 3 (L3TC) and level 4 trauma centers (L4TC) to the existing 7 level 1 trauma centers from 2008 to 2012. This study examined the impact of adding these lower-level trauma centers (LLTC) on patient outcomes. Methods: Patients in the state trauma registry age ≥ 15 from 2007 to 2012 were queried for demographic, injury, and outcome variables. These were compared between 2007 (PRE) and 2008-2012 (POST) cohorts. Multivariate logistic regression was performed to assess independent predictors of mortality. Subgroup analyses were performed for Injury Severity Score (ISS) ≥15, age ≥ 65, and trauma mechanisms. Results: 143,919 adults were evaluated. POST had significantly more female, geriatric, and blunt traumas (all p < 0.001). ISS was similar. Interfacility transfers increased by 10.2 %. Overall mortality decreased by 0.6 % (p < 0.001). Multivariate logistic regression analysis showed that being in POST was not associated with survival (OR: 1.07, CI: 0.96-1.18, p = 0.227). Subgroup analyses showed small reductions in mortality, except for geriatric patients. After adjusting for covariates, POST was not associated with survival in any subgroup, and trended toward being a predictor for death in penetrating traumas (OR: 1.23; 1.00-1.53, p = 0.059). Conclusions: Unregulated proliferation of LLTCs was associated with increased interfacility transfers without significant increase in trauma patients treated. LLTC proliferation was not an independent protector against mortality in the overall cohort and may worsen mortality for penetrating trauma patients. Rather than simply increasing the number of LLTCs within a region, perhaps more planned approaches are needed. Key message: This is, to our knowledge, the first work to study the effect of rapid lower level trauma center proliferation on patient outcomes. The findings of our analysis have implications for strategic planning of future trauma systems.

Need for Gastrostomy Tube in Periviable Infants.

OBJECTIVE: We sought to identify clinical and demographic factors associated with gastrostomy tube (g-tube) placement in periviable infants. STUDY DESIGN: We conducted a single-center retrospective cohort study of live-born infants between 22 and 25 weeks' gestation. Infants not actively resuscitated and those with congenital anomalies were excluded from analysis. RESULTS: Of the 243 infants included, 158 survived until discharge. Of those that survived to discharge, 35 required g-tube prior to discharge. Maternal race/ethnicity (p = 0.006), intraventricular hemorrhage (p = 0.013), periventricular leukomalacia (p = 0.003), bronchopulmonary dysplasia (BPD; p ≤ 0.001), and singleton gestation (p = 0.009) were associated with need for gastrostomy. In a multivariable logistic regression, maternal Black race (Odds Ratio [OR] 2.88; 95% confidence interval [CI] 1.11-7.47; p = 0.029), singleton gestation (OR 3.99; 95% CI 1.28-12.4; p = 0.017) and BPD (zero g-tube placement in the no BPD arm; p ≤ 0.001) were associated with need for g-tube. CONCLUSION: A high percentage of periviable infants surviving until discharge require g-tube at our institution. In this single-center retrospective study, we noted that maternal Black race, singleton gestation, and BPD were associated with increased risk for g-tube placement in infants born between 22 and 25 weeks' gestation. The finding of increased risk with maternal Black race is consistent with previous reports of racial/ethnic disparities in preterm morbidities. Additional studies examining factors associated with successful achievement of oral feedings in preterm infants are necessary and will inform future efforts to advance equity in newborn health. KEY POINTS: · BPD, singleton birth, and Black race are associated with need for g-tube in periviable infants.. · Severe intraventricular hemorrhage is associated with increased mortality or g-tube placement in periviable infants.. · Further investigation into the relationship between maternal race and g-tube placement is warranted..

Parkinson's disease CA2-CA3 hippocampal atrophy is accompanied by increased cholinergic innervation in patients with normal cognition but not in patients with mild cognitive impairment.

Although brain cholinergic denervation has been largely associated with cognitive decline in patients with Parkinson's disease (PD), new evidence suggests that cholinergic upregulation occurs in the hippocampus of PD patients without cognitive deficits. The specific hippocampal sectors and potential mechanisms of this cholinergic compensatory process have been further studied here, using MRI volumetry and morphometry coupled with molecular imaging using the PET radiotracer [18F]-Fluoroethoxybenzovesamicol ([18F]-FEOBV). Following a thorough screening procedure, 18 participants were selected and evenly distributed in three groups, including cognitively normal PD patients (PD-CN), PD patients with mild cognitive impairment (PD-MCI), and healthy volunteers (HV). Participants underwent a detailed neuropsychological assessment, structural MRI, and PET imaging with [18F]-FEOBV. Basal forebrain Ch1-Ch2 volumes were measured using stereotaxic mapping. Hippocampal subfields were automatically defined using the MAGeT-Brain segmentation algorithm. Cholinergic innervation density was quantified using [18F]-FEOBV uptake. Compared with HV, both PD-CN and PD-MCI displayed significantly reduced volumes in CA2-CA3 bilaterally. We found no other hippocampal subfield nor Ch1-Ch2 volume differences between the three groups. PET imaging revealed higher [18F]-FEOBV uptake in CA2-CA3 of the PD-CN compared with HV or PD-MCI. A positive correlation was observed between cognitive performances and [18F]-FEOBV uptake in the right CA2-CA3 subfield. Reduced volume, together with increased [18F]-FEOBV uptake, were observed specifically in the CA2-CA3 hippocampal subfields. However, while the volume change was observed in both PD-CN and PD-MCI, increased [18F]-FEOBV uptake was present only in the PD-CN group. This suggests that a cholinergic compensatory process takes place in the atrophied CA2-CA3 hippocampal subfields and might underlie normal cognition in PD.

Comparing the effects of different circular vection stimuli on upright stance.

BACKGROUND: Upright quiet stance is maintained through the complex integration of sensory information from the visual, vestibular, and somatosensory systems [1]. Virtual reality (VR) is a well-established tool that has been used to study sensory contributions to balance and induce visual perturbations. Previous assessments of virtual environments have suggested that VR can be used to create various visual stimuli that affect balance [2]; however, there is limited work examining which dynamic visual stimulus, in the form of circular vection (CV), is the most effective at inducing whole body lean. RESEARCH QUESTION: Therefore, this study assessed the effects of two visual stimuli using VR to better understand their effects on postural control. METHODS: 33 healthy young adults between the ages of 18-40, free of neurological impairments, stood quietly on a force plate for 30 s while wearing a head-mounted display. Participants were exposed to a field of random white dots (DOTS) or a black and white striped tunnel (TUNNEL) that rotated in the roll plane at 60°/s clockwise or counterclockwise. Amplitude was calculated from head orientation data recorded from a head-mounted display, and centre of pressure (COP). RESULTS: Independent of visual stimuli, postural lean was in the same direction as the stimulus. The DOTS stimulus increased Head orientation and COP position compared to the TUNNEL stimulus. There was no significant main effect or interaction with direction for Head or COP data. SIGNIFICANCE: When comparing the effect of stimulus design on postural sway, a DOTS stimulus was most effective at inducing direction-modulated postural sway This study builds on our understanding of the VR-related destabilizing effects on postural control and shows evidence that a DOTS stimulus has a stronger effect than a TUNNEL stimulus. Overall, it is important to consider the design of visual stimuli when examining VR effects on upright stance.

Retrospective Analysis of Circumstances of Falls and Related Injuries across Levels of Care in Older Adult Retirement Home Facilities.

Towards developing more effective interventions for fall-related injuries, this study analysed a novel database from six retirement home facilities over a 4-year period comprising 1,877 fallers and 12,445 falls. Falls were characterized based on location, activity, injury site, and type, and the database was stratified across four levels of care: Independent Living, Retirement Care, Assisted Care, and Memory care. Falls most occurred within the bedroom (62.8%), and during unknown (38.1%), walking (20.2%), and transfer tasks (14.6%). Approximately one in three (37%) of all falls resulted in an injury, most commonly involving the upper limb (31.8%), head (26.3%), and lower limb (22.2%), resulting in skin tears (35.3%), aches/pains (29.1%), or bruises (28.0%). While fall location, activity, and injury site were different across levels of care, injury type was not. The data from this study can assist in targeting fall-related injury prevention strategies across levels of care within retirement facilities.

Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths.

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M-1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M-1. In the plot of ΔH-T*ΔS, the of Enthalpy-Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

Focal acetylcholinergic modulation of the human midcingulo-insular network during attention: Meta-analytic neuroimaging and behavioral evidence.

The basal forebrain cholinergic neurons provide acetylcholine to the cortex via large projections. Recent molecular imaging work in humans indicates that the cortical cholinergic innervation is not uniformly distributed, but rather may disproportionately innervate cortical areas relevant to supervisory attention. In this study, we therefore reexamined the spatial relationship between acetylcholinergic modulation and attention in the human cortex using meta-analytic strategies targeting both pharmacological and non-pharmacological neuroimaging studies. We found that pharmaco-modulation of acetylcholine evoked both increased activity in the anterior cingulate and decreased activity in the opercular and insular cortex. In large independent meta-analyses of non-pharmacological neuroimaging research, we demonstrate that during attentional engagement these cortical areas exhibit (1) task-related co-activation with the basal forebrain, (2) task-related co-activation with one another, and (3) spatial overlap with dense cholinergic innervations originating from the basal forebrain, as estimated by multimodal positron emission tomography and magnetic resonance imaging. Finally, we provide meta-analytic evidence that pharmaco-modulation of acetylcholine also induces a speeding of responses to targets with no apparent tradeoff in accuracy. In sum, we demonstrate in humans that acetylcholinergic modulation of midcingulo-insular hubs of the ventral attention/salience network via basal forebrain afferents may coordinate selection of task relevant information, thereby facilitating cognition and behavior.

Sex-dependent cholinergic effects on amyloid pathology: A translational study.

INTRODUCTION: About two-thirds of Alzheimer's Disease (AD) patients are women, who exhibit more severe pathology and cognitive decline than men. Whether biological sex causally modulates the relationship between cholinergic signaling and amyloid pathology remains unknown. METHODS: We quantified amyloid beta (Aß) in male and female App-mutant mice with either decreased or increased cholinergic tone and examined the impact of ovariectomy and estradiol replacement in this relationship. We also investigated longitudinal changes in basal forebrain (cholinergic function) and Aß in elderly individuals. RESULTS: We show a causal relationship between cholinergic tone and amyloid pathology in males and ovariectomized female mice, which is decoupled in ovary-intact and ovariectomized females receiving estradiol. In elderly humans, cholinergic loss exacerbates Aß. DISCUSSION: Our findings emphasize the importance of reflecting human menopause in mouse models. They also support a role for therapies targeting estradiol and cholinergic signaling to reduce Aß. HIGHLIGHTS: Cholinergic tone regulates amyloid beta (Aß) pathology in males and ovariectomized female mice. Estradiol uncouples the relationship between cholinergic tone and Aß. In elderly humans, cholinergic loss correlates with increased Aß in both sexes.

Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples and in normal urine.

We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.

Multimodal gradients of human basal forebrain connectivity.

The cholinergic innervation of the cortex originates almost entirely from populations of neurons in the basal forebrain (BF). Structurally, the ascending BF cholinergic projections are highly branched, with individual cells targeting multiple different cortical regions. However, it is not known whether the structural organization of basal forebrain projections reflects their functional integration with the cortex. We therefore used high-resolution 7T diffusion and resting state functional MRI in humans to examine multimodal gradients of BF cholinergic connectivity with the cortex. Moving from anteromedial to posterolateral BF, we observed reduced tethering between structural and functional connectivity gradients, with the most pronounced dissimilarity localized in the nucleus basalis of Meynert (NbM). The cortical expression of this structure-function gradient revealed progressively weaker tethering moving from unimodal to transmodal cortex, with the lowest tethering in midcingulo-insular cortex. We used human [18F] fluoroethoxy-benzovesamicol (FEOBV) PET to demonstrate that cortical areas with higher concentrations of cholinergic innervation tend to exhibit lower tethering between BF structural and functional connectivity, suggesting a pattern of increasingly diffuse axonal arborization. Optogenetic tracing of cholinergic projections and [18F] FEOBV PET in mice confirmed a gradient of axonal arborization across individual BF cholinergic neurons. Like humans, cholinergic neurons with the highest arborization project to cingulo-insular areas of the mouse isocortex. Altogether, our findings reveal that BF cholinergic neurons vary in their branch complexity, with certain subpopulations exhibiting greater modularity and others greater diffusivity in the functional integration of their cortical targets.

Harnessing axonal transport to map reward circuitry: Differing brain-wide projections from medial prefrontal cortical domains.

Neurons project long axons that contact other distant neurons. Neurons in the medial prefrontal cortex project into the limbic system to regulate responses to reward or threat. Diminished neural activity in prefrontal cortex is associated with loss of executive function leading to drug use, yet the specific circuitry that mediate these effects is unknown. Different regions within the medial prefrontal cortex may project to differing limbic system nuclei. Here, we exploited the cell biology of intracellular membrane trafficking, fast axonal transport, to map projections from two adjacent medial prefrontal cortical regions. We used Mn(II), a calcium analog, to trace medial prefrontal cortical projections in the living animal by magnetic resonance imaging (MRI). Mn(II), a contrast agent for MRI, enters neurons through voltage-activated calcium channels and relies on kinesin-1 and amyloid-precursor protein to transport out axons to distal destinations. Aqueous MnCl2 together with fluorescent dextran (3--5 nL) was stereotactically injected precisely into two adjacent regions of the medial prefrontal cortex: anterior cingulate area (ACA) or infralimbic/prelimbic (IL/PL) region. Projections were traced, first live by manganese-enhanced MRI (MEMRI) at four time points in 3D, and then after fixation by microscopy. Data-driven unbiased voxel-wise statistical maps of aligned normalized MR images after either ACA or IL/PL injections revealed statistically significant progression of Mn(II) over time into deeper brain regions: dorsal striatum, globus pallidus, amygdala, hypothalamus, substantia nigra, dorsal raphe and locus coeruleus. Quantitative comparisons of these distal accumulations at 24 h revealed dramatic differences between ACA and IL/PL injection groups throughout the limbic system, and most particularly in subdomains of the hypothalamus. ACA projections targeted dorsomedial nucleus of the hypothalamus, posterior part of the periventricular region and mammillary body nuclei as well as periaqueductal gray, while IL/PL projections accumulated in anterior hypothalamic areas and lateral hypothalamic nuclei as well as amygdala. As hypothalamic subsegments relay CNS activity to the body, our results suggest new concepts about mind-body relationships and specific roles of distinct yet adjacent medial prefrontal cortical segments. Our MR imaging strategy, when applied to follow other cell biological processes in the living organism, will undoubtedly lead to an expanded perspective on how minute details of cellular processes influence whole body health and wellbeing.