Validation of a functional human AD model with four AD therapeutics utilizing patterned iPSC-derived cortical neurons integrated with microelectrode arrays.

Preclinical methods are needed for screening potential Alzheimer's disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. A potential functional parameter to be monitored is long-term potentiation (LTP), which is a correlate of learning and memory, that would be one of the first functions effected by AD onset. Mature human iPSC-derived cortical neurons and primary astrocytes were co-cultured on microelectrode arrays (MEA) where surface chemistry was utilized to create circuit patterns connecting two adjacent electrodes to model LTP function. LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aß42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aß42 induced LTP impairment. The results presented here illustrate the significance of the system as a validated platform that can be utilized to model and study MCI AD pathology, and potentially for the pre-MCI phase before the occurrence of significant cell death. It also has the potential to become an ideal platform for high content therapeutic screening for other neurodegenerative diseases.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's disease microglia.

Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aß induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's microglia.

Several genetic risk factors for Alzheimer's Disease (AD) implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells. However, the relationship between lipid metabolism in glia and AD pathology remains poorly understood. Through single-nucleus RNA-sequencing of AD brain tissue, we have identified a microglial state defined by the expression of the lipid droplet (LD) associated enzyme ACSL1 with ACSL1-positive microglia most abundant in AD patients with the APOE4/4 genotype. In human iPSC-derived microglia (iMG) fibrillar Aß (fAß) induces ACSL1 expression, triglyceride synthesis, and LD accumulation in an APOE-dependent manner. Additionally, conditioned media from LD-containing microglia leads to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for AD with microglial LD accumulation and neurotoxic microglial-derived factors, potentially providing novel therapeutic strategies for AD.

Delivering a toxic metal to the active site of urease.

Urease is a nickel (Ni) enzyme that is essential for the colonization of Helicobacter pylori in the human stomach. To solve the problem of delivering the toxic Ni ion to the active site without diffusing into the cytoplasm, cells have evolved metal carrier proteins, or metallochaperones, to deliver the toxic ions to specific protein complexes. Ni delivery requires urease to form an activation complex with the urease accessory proteins UreFD and UreG. Here, we determined the cryo-electron microscopy structures of H. pylori UreFD/urease and Klebsiella pneumoniae UreD/urease complexes at 2.3- and 2.7-angstrom resolutions, respectively. Combining structural, mutagenesis, and biochemical studies, we show that the formation of the activation complex opens a 100-angstrom-long tunnel, where the Ni ion is delivered through UreFD to the active site of urease.

Tuning Polymer Hydrophilicity to Regulate Gel Mechanics and Encapsulated Cell Morphology.

Mechanically tunable hydrogels are attractive platforms for 3D cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, an alternative strategy based upon tuning the hydrophilicity of an elastin-like protein (ELP) is presented. ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. It is hypothesized that increasing this transition temperature through bioconjugation with azide-containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide-modified ELPs are crosslinked into hydrogels with bicyclononyne-modified hyaluronic acid (HA-BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100-1000 Pa). Human mesenchymal stromal cells (hMSCs), human umbilical vein endothelial cells (HUVECs), and human neural progenitor cells (hNPCs) are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties is demonstrated. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness.

Inequities in Access to Medical Care Among Adults Diagnosed with Diabetes: Comparisons Between the US Population and a Sample of US-Residing Marshallese Islanders.

OBJECTIVE: We examined barriers to accessing medical care for migrant US-residing Marshallese Islanders. METHODS: Cross-sectional analyses were conducted to identify potential inequities. Surveys from largely migrant diabetic Marshallese Islanders (n = 255) were compared with nationally representative data. Two major outcomes were assessed including 1-whether or not one reported having forgone medical care in the past year because of cost-and 2-whether or not one reported not having a usual source of care. RESULTS: Overall, 74% and 77% of Marshallese Islanders reported forgone care and no usual source of care, respectively, versus 15% and 7% of the US diabetic population. In multivariable analyses, being younger; uninsured; unemployed; male; of lower education; Native American or Hispanic (versus White); and residing in the South were associated with forgone care nationwide, whereas only lacking insurance was associated with forgone care among Marshallese Islanders. Nationwide being younger; uninsured; unmarried; female; of lower education; Native American or Hispanic (versus White); and residing in the South were associated with not having a usual source of care, whereas only being younger and uninsured were associated with not having a usual source of care among Marshallese Islanders. CONCLUSION: The largest group of diabetic Marshallese Islanders in the continental US faces severe healthcare access inequities necessitating policies that increase access to health insurance options and associated resources.

Management of Vesicoureteral Reflux in a Patient With Recurrent Febrile Urinary Tract Infections, Neurogenic Bladder, and Ureteral Triplication.

Ureteral triplication is a rare anomaly with about 100 total cases reported in the literature. In this case presentation, we present a case of ureteral triplication in a young female with a history of neurogenic bladder secondary to L5 lipomeningocele who presented with recurrent febrile urinary tract infections (UTIs) and vesicoureteral reflux despite antibiotic prophylaxis. Given her high grade reflux, she underwent ureteral reimplantation which ultimately led to resolution of her UTIs and reflux. Later in her clinical course, toilet training unmasked additional bladder dysfunction and she was successfully managed with clean intermittent catheterization and anticholinergics. To the best of our knowledge, this is the first case report to describe ureteral triplication, recurrent febrile UTIs and vesicoureteral reflux in the setting of a concomitant neurogenic bladder with a successful outcome.

The Influence of Combat Experience on Psychologically Healthy Soldiers' Attentiveness to Environmental Threats.

INTRODUCTION: In contrast to previous research that has primarily examined how psychological disorders (e.g., post-traumatic stress disorder [PTSD], anxiety) are affected by and affect individuals' threat perceptions, this study examines the relationship between combat experience and threat-monitoring in psychologically healthy Soldiers. Existing research has established how prolonged or intense experiences with war-related stressors can lead individuals to undergo an unconscious fear-conditioning process that affects the circuitry of the prefrontal cortex, hippocampus, amygdala, and anterior cingulate cortex regions of the brain. We predict that the intensity of one's combat experience positively influences Soldiers' attention to environmental threats. MATERIALS AND METHODS: Participants included U. S. Army Soldiers with a score of 50 or below on the PTSD Checklist-Military Version. Participants completed the Combat Exposure Scale and the State-Trait Anxiety Inventory. The experimental prediction task we employed assesses the expectation of an intrusively loud white noise sound that occurred on three variable patterns in a pseudorandomized order. Each tone pattern was used 20 times over a total of 60 trials. The experimental prediction task included two neutral tones (700 and 1,300 Hz) that were presented in a repeated pattern along with a 100-dB burst of white noise (0.5-second duration). In each trial, one of three possible tone combinations was presented. To assess their attentiveness to threats, participants were asked to continuously rate their expectancy of the burst of white noise using a visual analogue scale (VAS) ranging from 0 to 100. VAS ratings were collected at controlled points throughout the task. RESULTS: None of the participants reported scores on any of the diagnostic surveys that met standards for clinical significance. A repeated-measures analysis of variance was conducted to assess the overall effect of the three prediction conditions on participants' VAS ratings. There was a significant main effect for Combat Exposure Scale scores on VAS ratings [F(1, 27) = 5.19, p = 0.031], with high scorers demonstrating a generally higher expectancy of the white noise burst throughout the entire experimental sequence. Results suggest that within subclinical populations of Soldiers, the intensity of one's combat experience is positively associated with their attention to threats. CONCLUSION: These findings suggest that Soldiers who experience combat should be observed for signs of increased threat-attention bias, as this may indicate that their capacities for information processing, decision-making, and emotion regulation could be compromised. The positive relationship we observe between a level of combat experience and attentional biases toward threatening stimuli may also help to explain why these veterans engage in "externalizing" behaviors that are risky, aggressive, or violent as well as relational problems and antisocial behaviors that are reported in higher-than-average rates among these populations of Soldiers. Acknowledging that increased threat attention may be a preclinical indication of developing PTSD or other related psychological conditions (e.g., depression, anxiety) should motivate clinicians to more actively diagnose and treat this condition.

Strategic improvements for gross anatomy web-based teaching.

Current generations of graduate students have been immersed in technology from their early school years and have high expectations regarding digital resources. To better meet the expectations of Gross Anatomy students at our institution, electronic radiology teaching files for first-year coursework were organized into a web site. The web site was custom designed to provide material that directly correlated to the Gross Anatomy dissection and lectures. Quick links provided sets of images grouped by anatomic location. Additionally, Lab and Study Companions provided specific material for the students to review prior to and after lectures and gross dissections. Student opinions of this education resource were compared to student opinions of the prior year's digital teaching files. The new content was ranked as more user friendly (3.1 points versus 2.3 points) and more useful for learning anatomy (3.3 points versus 2.6 points). Many students reported that using the web portal was critical in helping them to better understand relationships of anatomical structures. These findings suggest that a well-organized web portal can provide a user-friendly, valuable educational resource for medical students who are studying Gross Anatomy.

In vivo multicolor molecular MR imaging using diamagnetic chemical exchange saturation transfer liposomes.

A variety of (super)paramagnetic contrast agents are available for enhanced MR visualization of specific tissues, cells, or molecules. To develop alternative contrast agents without the presence of metal ions, liposomes were developed containing simple bioorganic and biodegradable compounds that produce diamagnetic chemical exchange saturation transfer MR contrast. This diamagnetic chemical exchange saturation transfer contrast is frequency-dependent, allowing the unique generation of "multicolor" images. The contrast can be turned on and off at will, and standard images do not show the presence of these agents. As an example, glycogen, L-arginine, and poly-L-lysine were encapsulated inside liposomes and injected intradermally into mice to image the lymphatic uptake of these liposomes. Using a frequency-dependent acquisition scheme, it is demonstrated that multicolor MRI can differentiate between different contrast particles in vivo following their homing to draining lymph nodes. Being nonmetallic and bioorganic, these diamagnetic chemical exchange saturation transfer liposomes form an attractive novel platform for multicolor imaging in vivo.

Spatiotemporal wavelet analysis for functional MRI.

Characterizing the spatiotemporal behavior of the BOLD signal in functional Magnetic Resonance Imaging (fMRI) is a central issue in understanding brain function. While the nature of functional activation clusters is fundamentally heterogeneous, many current analysis approaches use spatially invariant models that can degrade anatomic boundaries and distort the underlying spatiotemporal signal. Furthermore, few analysis approaches use true spatiotemporal continuity in their statistical formulations. To address these issues, we present a novel spatiotemporal wavelet procedure that uses a stimulus-convolved hemodynamic signal plus correlated noise model. The wavelet fits, computed by spatially constrained maximum-likelihood estimation, provide efficient multiscale representations of heterogeneous brain structures and give well-identified, parsimonious spatial activation estimates that are modulated by the temporal fMRI dynamics. In a study of both simulated data and actual fMRI memory task experiments, our new method gave lower mean-squared error and seemed to result in more localized fMRI activation maps compared to models using standard wavelet or smoothing techniques. Our spatiotemporal wavelet framework suggests a useful tool for the analysis of fMRI studies.

Cross-beta order and diversity in nanocrystals of an amyloid-forming peptide.

The seven-residue peptide GNNQQNY from the N-terminal region of the yeast prion protein Sup35, which forms amyloid fibers, colloidal aggregates and highly ordered nanocrystals, provides a model system for characterizing the elusively protean cross-beta conformation. Depending on preparative conditions, orthorhombic and monoclinic crystals with similar lath-shaped morphology have been obtained. Ultra high-resolution (<0.5A spacing) electron diffraction patterns from single nanocrystals show that the peptide chains pack in parallel cross-beta columns with approximately 4.86A axial spacing. Mosaic striations 20-50 nm wide observed by electron microscopy indicate lateral size-limiting crystal growth related to amyloid fiber formation. Frequently obtained orthorhombic forms, with apparent space group symmetry P2(1)2(1)2(1), have cell dimensions ranging from /a/=22.7-21.2A, /b/=39.9-39.3A, /c/=4.89-4.86A for wet to dried states. Electron diffraction data from single nanocrystals, recorded in tilt series of still frames, have been mapped in reciprocal space. However, reliable integrated intensities cannot be obtained from these series, and dynamical electron diffraction effects present problems in data analysis. The diversity of ordered structures formed under similar conditions has made it difficult to obtain reproducible X-ray diffraction data from powder specimens; and overlapping Bragg reflections in the powder patterns preclude separated structure factor measurements for these data. Model protofilaments, consisting of tightly paired, half-staggered beta strands related by a screw axis, can be fit in the crystal lattices, but model refinement will require accurate structure factor measurements. Nearly anhydrous packing of this hydrophilic peptide can account for the insolubility of the crystals, since the activation energy for rehydration may be extremely high. Water-excluding packing of paired cross-beta peptide segments in thin protofilaments may be characteristic of the wide variety of anomalously stable amyloid aggregates.

Practice and difficulty evoke anatomically and pharmacologically dissociable brain activation dynamics.

Brain activation is adaptive to task difficulty and practice. We used functional MRI to map brain systems activated by an object-location learning task in 24 healthy elderly volunteers each scanned following placebo and two of four active drugs studied. We distinguished a fronto-striatal system adaptive to difficulty from a posterior system adaptive to practice. Fronto-striatal response to increased cognitive load was significantly attenuated by scopolamine, sulpiride and methylphenidate; practice effects were not modulated by these drugs but were enhanced by diazepam. We also found enhancement by methylphenidate, and attenuation by sulpiride, of load response in premotor, cingulate and parietal regions comprising a spatial attention network. Difficulty and practice evoke anatomically and pharmacologically dissociable brain activation dynamics, which are probably mediated by different neurotransmitter systems in humans.