Multimodal exercise training to reduce frailty in people with multiple sclerosis: study protocol for a pilot randomized controlled trial.

BACKGROUND: Frailty, a syndrome characterized by decreased reserve and resistance to stressors across multiple physiologic systems, is highly prevalent in people living with multiple sclerosis (pwMS), independent of age or disability level. Frailty in MS is strongly associated with adverse clinical outcomes, such as falls, and may aggravate MS-related symptoms. Consequently, there is a pressing necessity to explore and evaluate strategies to reduce frailty levels in pwMS. The purpose of this pilot randomized controlled trial (RCT) will be to examine the feasibility and preliminary efficacy of a multimodal exercise training program to reduce frailty in pwMS. METHODS: A total of 24 participants will be randomly assigned to 6 weeks of multimodal exercise or to a waitlist control group with a 1:1 allocation. PwMS aged 40-65 years and living with frailty will be eligible. The multimodal exercise program will consist of cognitive-motor rehabilitation (i.e., virtual reality treadmill training) combined with progressive, evidence-based resistance training. At baseline and post-intervention, participants will complete the Evaluative Frailty Index for Physical Activity (EFIP), measures of fall risk, and quality of life. Frailty-related biomarkers will also be assessed. In addition, the feasibility of the multimodal exercise program will be systematically and multidimensionally evaluated. DISCUSSION: To date, no RCT has yet been conducted to evaluate whether targeted exercise interventions can minimize frailty in MS. The current study will provide novel data on the feasibility and preliminary efficacy of multimodal exercise training as a strategy for counteracting frailty in pwMS. TRIAL REGISTRATION: ClinicalTrials.gov, NCT06042244 (registered in September 2023).

CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization.

BACKGROUND: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. METHODS: Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. FINDINGS: CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. INTERPRETATION: CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Nuclear Factor I A and Nuclear Factor I B Are Jointly Required for Mouse Postnatal Neural Stem Cell Self-Renewal.

Mouse postnatal neural stem cells (pNSCs) can be expanded in vitro in the presence of epidermal growth factor and fibroblast growth factor 2 and upon removal of these factors cease proliferation and generate neurons, astrocytes, and oligodendrocytes. The genetic requirements for self-renewal and lineage-commitment of pNSCs are incompletely understood. In this study, we show that the transcription factors NFIA and NFIB, previously shown individually, to be essential for the normal commitment of pNSCs to the astrocytic lineage in vivo, are jointly required for normal self-renewal of pNSCs in vitro and in vivo. Using conditional knockout alleles of Nfia and Nfib, we show that the simultaneous loss of these two genes under self-renewal conditions in vitro reduces the expression of the proliferation markers PCNA and Ki67, eliminates clonogenicity of the cells, reduces the number of cells in S phase, and induces aberrant differentiation primarily into the neuroblast lineage. This phenotype requires the loss of both genes and is not seen upon loss of Nfia or Nfib alone, nor with combined loss of Nfia and Nfix or Nfib and Nfix. These data demonstrate a unique combined requirement for both Nfia and Nfib for pNSC self-renewal.

An immunoregulator nanomedicine approach for the treatment of tuberculosis.

Introduction: A nanoparticle composed of a poly (lactic-co-glycolic acid) (PLGA) core and a chitosan (CS) shell with surface-adsorbed 1,3 ß-glucan (ß-glucan) was synthesized. The exposure response of CS-PLGA nanoparticles (0.1 mg/mL) with surface-bound ß-glucan at 0, 5, 10, 15, 20, or 25 ng or free ß-glucan at 5, 10, 15, 20, or 25 ng/mL in macrophage in vitro and in vivo was investigated. Results: In vitro studies demonstrate that gene expression for IL-1ß, IL-6, and TNFα increased at 10 and 15 ng surface-bound ß-glucan on CS-PLGA nanoparticles (0.1 mg/mL) and at 20 and 25 ng/mL of free ß-glucan both at 24 h and 48 h. Secretion of TNFα protein and ROS production increased at 5, 10, 15, and 20 ng surface-bound ß-glucan on CS-PLGA nanoparticles and at 20 and 25 ng/mL of free ß-glucan at 24 h. Laminarin, a Dectin-1 antagonist, prevented the increase in cytokine gene expression induced by CS-PLGA nanoparticles with surface-bound ß-glucan at 10 and 15 ng, indicating a Dectin-1 receptor mechanism. Efficacy studies showed a significant reduction in intracellular accumulation of mycobacterium tuberculosis (Mtb) in monocyte-derived macrophages (MDM) incubated with on CS-PLGA (0.1 mg/ml) nanoparticles with 5, 10, and 15 ng surface-bound ß-glucan or with 10 and 15 ng/mL of free ß-glucan. ß-glucan-CS-PLGA nanoparticles inhibited intracellular Mtb growth more than free ß-glucan alone supporting the role of ß-glucan-CS-PLGA nanoparticles as stronger adjuvants than free ß-glucan. In vivo studies demonstrate that oropharyngeal aspiration (OPA) of CS-PLGA nanoparticles with nanogram concentrations of surface-bound ß-glucan or free ß-glucan increased TNFα gene expression in alveolar macrophages and TNFα protein secretion in bronchoalveolar lavage supernatants. Discussion: Data also demonstrate no damage to the alveolar epithelium or changes in the murine sepsis score following exposure to ß-glucan-CS-PLGA nanoparticles only, indicating safety and feasibility of this nanoparticle adjuvant platform to mice by OPA.

The first endovascular rat glioma model for pre-clinical evaluation of intra-arterial therapeutics.

BACKGROUND: Several translational animal models have been described assessing intra-arterial (IA) treatments for malignant gliomas. We describe the first endovascular animal model that allows testing of IA drug delivery as a first-line treatment, which is difficult to do in actual patients. We report a unique protocol for vascular access and IA delivery in the rat model that, unlike prior reports, does not require direct puncture and opening of proximal cerebrovasculature which carries risk of ischemia in the animal brain post-delivery. METHODS: Wistar rats underwent left femoral artery catherization with a Balt Magic 1.2F catheter or Marathon Flow directed 1.5F Microcatheter with an Asahi Chikai 0.008 micro-guidewire which was navigated to the left internal carotid artery under x-ray. 25% mannitol was administered to test blood brain barrier breakdown (BBBB). Additional rats were implanted with C6 glioma cells in the left frontal lobe. C6 Glioma-Implanted Rats (C6GRs) were monitored for overall survival and tumor growth. Tumor volumes from MRI images were calculated utilizing 3D slicer. Additional rats underwent femoral artery catheterization with Bevacizumab, carboplatin, or irinotecan injected into the left internal carotid artery to test feasibility and safety. RESULTS: A successful endovascular access and BBBB protocol was established. BBBB was confirmed with positive Evans blue staining. 10 rats were successfully implanted with C6 gliomas with confirmed growths on MRI. Overall survival was 19.75 ± 2.21 days. 5 rats were utilized for the development of our femoral catheterization protocol and BBBB testing. With regards to IA chemotherapy dosage testing, control rats tolerated targeted 10 mg/kg of bevascizumab, 2.4 mg/kg of carboplatin, and 15 mg/kg of irinotecan IA ICA injections without any complications. CONCLUSIONS: We present the first endovascular IA rat glioma model that allows selective catheterization of the intracranial vasculature and assessment of IA therapies for gliomas without need for access and sacrifice of proximal cerebrovasculature.

Defining the Intravital Renal Disposition of Fluorescence-Quenched Exenatide.

Despite the understanding that renal clearance is pivotal for driving the pharmacokinetics of numerous therapeutic proteins and peptides, the specific processes that occur following glomerular filtration remain poorly defined. For instance, sites of catabolism within the proximal tubule can occur at the brush border, within lysosomes following endocytosis, or even within the tubule lumen itself. The objective of the current study was to address these limitations and develop methodology to study the kidney disposition of a model therapeutic protein. Exenatide is a peptide used to treat type 2 diabetes mellitus. Glomerular filtration and ensuing renal catabolism have been shown to be its principal clearance pathway. Here, we designed and validated a Förster resonance energy transfer-quenched exenatide derivative to provide critical information on the renal handling of exenatide. A combination of in vitro techniques was used to confirm substantial fluorescence quenching of intact peptide that was released upon proteolytic cleavage. This evaluation was then followed by an assessment of the in vivo disposition of quenched exenatide directly within kidneys of living rats via intravital two-photon microscopy. Live imaging demonstrated rapid glomerular filtration and identified exenatide metabolism occurred within the subapical regions of the proximal tubule epithelia, with subsequent intracellular trafficking of cleaved fragments. These results provide a novel examination into the real-time, intravital disposition of a protein therapeutic within the kidney and offer a platform to build upon for future work.

Profiling of Circulating Gene Expression Reveals Molecular Signatures Associated with Intracranial Aneurysm Rupture Risk.

BACKGROUND: Following detection, rupture risk assessment for intracranial aneurysms (IAs) is critical. Towards molecular prognostics, we hypothesized that circulating blood RNA expression profiles are associated with IA risk. METHODS: We performed RNA sequencing on 68 blood samples from IA patients. Here, patients were categorized as either high or low risk by assessment of aneurysm size (≥ 5 mm = high risk) and Population, Hypertension, Age, Size, Earlier subarachnoid hemorrhage, Site (PHASES) score (≥ 1 = high risk). Modified F-statistics and Benjamini-Hochberg false discovery rate correction was performed on transcripts per million-normalized gene counts. Protein-coding genes expressed in ≥ 50% of samples with a q value < 0.05 and an absolute fold-change ≥ 2 were considered significantly differentially expressed. Bioinformatics in Ingenuity Pathway Analysis was performed to understand the biology of risk-associated expression profiles. Association was assessed between gene expression and risk via Pearson correlation analysis. Linear discriminant analysis models using significant genes were created and validated for classification of high-risk cases. RESULTS: We analyzed transcriptomes of 68 IA patients. In these cases, 31 IAs were large (≥ 5 mm), while 26 IAs had a high PHASES score. Based on size, 36 genes associated with high-risk IAs, and two were correlated with the size measurement. Alternatively, based on PHASES score, 76 genes associated with high-risk cases, and nine of them showed significant correlation to the score. Similar ontological terms were associated with both gene profiles, which reflected inflammatory signaling and vascular remodeling. Prediction models based on size and PHASES stratification were able to correctly predict IA risk status, with > 80% testing accuracy for both. CONCLUSIONS: Here, we identified genes associated with IA risk, as quantified by common clinical metrics. Preliminary classification models demonstrated feasibility of assessing IA risk using whole blood expression.

Gene expression profiles of ischemic stroke clots retrieved by mechanical thrombectomy are associated with disease etiology.

BACKGROUND: Determining stroke etiology is crucial for secondary prevention, but intensive workups fail to classify ~30% of strokes that are cryptogenic. OBJECTIVE: To examine the hypothesis that the transcriptomic profiles of clots retrieved during mechanical thrombectomy are unique to strokes of different subtypes. METHODS: We isolated RNA from the clots of 73 patients undergoing mechanical thrombectomy. Samples of sufficient quality were subjected to 100-cycle, paired-end RNAseq, and transcriptomes with less than 10 million unique reads were excluded from analysis. Significant differentially expressed genes (DEGs) between subtypes (defined by the Trial of Org 10 172 in Acute Stroke Treatment) were identified by expression analysis in edgeR. Gene ontology enrichment analysis was used to study the biologic differences between stroke etiologies. RESULTS: In all, 38 clot transcriptomes were analyzed; 6 from large artery atherosclerosis (LAA), 21 from cardioembolism (CE), 5 from strokes of other determined origin, and 6 from cryptogenic strokes. Among all comparisons, there were 816 unique DEGs, 174 of which were shared by at least two comparisons, and 20 of which were shared by all three. Gene ontology analysis showed that CE clots reflected high levels of inflammation, LAA clots had greater oxidoreduction and T-cell processes, and clots of other determined origin were enriched for aberrant platelet and hemoglobin-related processes. Principal component analysis indicated separation between these subtypes and showed cryptogenic samples clustered among several different groups. CONCLUSIONS: Expression profiles of stroke clots were identified between stroke etiologies and reflected different biologic responses. Cryptogenic thrombi may be related to multiple etiologies.

Mitochondrial Dynamics in SARS-COV2 Spike Protein Treated Human Microglia: Implications for Neuro-COVID.

Emerging clinical data from the current COVID-19 pandemic suggests that ~ 40% of COVID-19 patients develop neurological symptoms attributed to viral encephalitis while in COVID long haulers chronic neuro-inflammation and neuronal damage result in a syndrome described as Neuro-COVID. We hypothesize that SAR-COV2 induces mitochondrial dysfunction and activation of the mitochondrial-dependent intrinsic apoptotic pathway, resulting in microglial and neuronal apoptosis. The goal of our study was to determine the effect of SARS-COV2 on mitochondrial biogenesis and to monitor cell apoptosis in human microglia non-invasively in real time using Raman spectroscopy, providing a unique spatio-temporal information on mitochondrial function in live cells. We treated human microglia with SARS-COV2 spike protein and examined the levels of cytokines and reactive oxygen species (ROS) production, determined the effect of SARS-COV2 on mitochondrial biogenesis and examined the changes in molecular composition of phospholipids. Our results show that SARS- COV2 spike protein increases the levels of pro-inflammatory cytokines and ROS production, increases apoptosis and increases the oxygen consumption rate (OCR) in microglial cells. Increases in OCR are indicative of increased ROS production and oxidative stress suggesting that SARS-COV2 induced cell death. Raman spectroscopy yielded significant differences in phospholipids such as Phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which account for ~ 80% of mitochondrial membrane lipids between SARS-COV2 treated and untreated microglial cells. These data provide important mechanistic insights into SARS-COV2 induced mitochondrial dysfunction which underlies neuropathology associated with Neuro-COVID.

Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy.

Mechanical thrombectomy (MT) for large vessel acute ischemic stroke (AIS) has enabled biologic analyses of resected clots. While clot histology has been well-studied, little is known about gene expression within the tissue, which could shed light on stroke pathophysiology. In this methodological study, we develop a pipeline for obtaining useful RNA from AIS clots. A total of 73 clot samples retrieved by MT were collected and stored in RNALater and in 10% phosphate-buffered formalin. RNA was extracted from all samples using a modified Chemagen magnetic bead extraction protocol on the PerkinElmer Chemagic 360. RNA was interrogated by UV-Vis absorption and electrophoretic quality control analysis. All samples with sufficient volume underwent traditional qPCR analysis and samples with sufficient RNA quality were subjected to next-generation RNA sequencing on the Illumina NovaSeq platform. Whole blood RNA samples from three patients were used as controls, and H&E-stained histological sections of the clots were used to assess clot cellular makeup. Isolated mRNA was eluted into a volume of 140 µL and had a concentration ranging from 0.01 ng/µL to 46 ng/µL. Most mRNA samples were partially degraded, with RNA integrity numbers ranging from 0 to 9.5. The majority of samples (71/73) underwent qPCR analysis, which showed linear relationships between the expression of three housekeeping genes (GAPDH, GPI, and HPRT1) across all samples. Of these, 48 samples were used for RNA sequencing, which had moderate quality based on MultiQC evaluation (on average, ~35 M reads were sequenced). Analysis of clot histology showed that more acellular samples yielded RNA of lower quantity and quality. We obtained useful mRNA from AIS clot samples stored in RNALater. qPCR analysis could be performed in almost all cases, while sequencing data could only be performed in approximately two-thirds of the samples. Acellular clots tended to have lower RNA quantity and quality.

RNA Sequencing Data from Human Intracranial Aneurysm Tissue Reveals a Complex Inflammatory Environment Associated with Rupture.

BACKGROUND: Intracranial aneurysm (IA) rupture leads to deadly subarachnoid hemorrhages. However, the mechanisms leading to rupture remain poorly understood. Altered gene expression within IA tissue is linked to the pathobiology of aneurysm development and progression. Here, we analyzed expression patterns of control tissue samples and compared them to those of unruptured and ruptured IA tissue samples using data from the Gene Expression Omnibus (GEO). METHODS: FASTQ files for 21 ruptured IAs, 21 unruptured IAs, and 16 control tissue samples were accessed from the GEO database. DESeq2 was used for differential expression analysis in three comparisons: unruptured IA versus control, ruptured IA versus control, and ruptured versus unruptured IA. Genes that were differentially expressed in multiple comparisons were evaluated to find those progressively increasing/decreasing from control to unruptured to ruptured. Significance was tested by either analysis of variance/Gabriel or Brown-Forsythe/Games Howell (p < 0.05 was considered significant). We used additional RNA sequencing and proteomics datasets to evaluate if our differentially expressed genes (DEGs) were present in other studies. Bioinformatics analyses were performed with g:Profiler and Ingenuity Pathway Analysis. RESULTS: In total, we identified 1768 DEGs, of which 318 were found in multiple comparisons. Unruptured versus control reflected vascular remodeling processes, while ruptured versus control reflected inflammatory responses and cell activation/signaling. When comparing ruptured to unruptured IAs, we found massive activation of inflammation, inflammatory responses, and leukocyte responses. Of the 318 genes in multiple comparisons, 127 were found to be significant in the multi-cohort correlation analysis. Those that progressively increased (70 genes) were associated with immune system processes, while those that progressively decreased (38 genes) did not return any gene ontology terms. Many of our DEGs were also found in the other IA tissue sequencing studies. CONCLUSIONS: We found unruptured IAs relate more to remodeling processes, while ruptured IAs reflect more inflammatory and immune responses.

Unconjugated p-cresol activates macrophage macropinocytosis leading to increased LDL uptake.

Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated that a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first-pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, not only did we measure the effect of altering the intestinal microbiota on lipid accumulation in coronary arteries, but we also examined macrophage lipid uptake and handling pathways in response to uPC. We found that atherosclerosis-prone mice fed a high-fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, and hepatic and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated that uPC exposure increased apoptosis whereas PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate LDL uptake and only uPC induced endocytosis- and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate that uPC itself had a distinct effect on macrophage biology that might have contributed to increased cardiovascular risk in patients with CKD.

Immunoglobulin G Is a Novel Substrate for the Endocytic Protein Megalin.

Therapeutic immunoglobulin G (IgG) antibodies comprise the largest class of protein therapeutics. Several factors that influence their overall disposition have been well-characterized, including target-mediated mechanics and convective flow. What remains poorly defined is the potential for non-targeted entry into various tissues or cell types by means of uptake via cell surface receptors at those sites. Megalin and cubilin are large endocytic receptors whose cooperative function plays important physiological roles at the tissues in which they are expressed. One such example is the kidney, where loss of either results in significant declines in proximal tubule protein reabsorption. Due to their diverse ligand profile and broad tissue expression, megalin and cubilin represent potential candidates for receptor-mediated uptake of IgG into various epithelia. Therefore, the objective of the current work was to determine if IgG was a novel ligand of megalin and/or cubilin. Direct binding was measured for human IgG with both megalin and the cubilin/amnionless complex. Additional work focusing on the megalin-IgG interaction was then conducted to build upon these findings. Cell uptake studies using megalin ligands for competitive inhibition or proximal tubule cells stably transduced with megalin-targeted shRNA constructs supported a role for megalin in the endocytosis of human IgG. Furthermore, a pharmacokinetic study using transgenic mice with a kidney-specific mosaic knockout of megalin demonstrated increased urinary excretion of human IgG in megalin knockout mice when compared to wild-type controls. These findings indicate that megalin is capable of binding and internalizing IgG via a high affinity interaction.

Hybrid Curdlan Poly(γ -Glutamic Acid) Nanoassembly for Immune Modulation in Macrophage.

A nanoformulation composed of curdlan, a linear polysaccharide of 1,3-ß-linked d-glucose units, hydrogen bonded to poly(γ -glutamic acid) (PGA), was developed to stimulate macrophage. Curdlan/PGA nanoparticles (C-NP) are formulated by physically blending curdlan (0.2 mg mL-1 in 0.4 m NaOH) with PGA (0.8 mg mL-1 ). Forster resonance energy transfer (FRET) analysis demonstrates a heterospecies interpolymer complex formed between curdlan and PGA. The 1 H-NMR spectra display significant peak broadening as well as downfield chemical shifts of the hydroxyl proton resonances of curdlan, indicating potential intermolecular hydrogen bonding interactions. In addition, the cross peaks in 1 H-1 H 2D-NOESY suggest intermolecular associations between the OH-2/OH-4 hydroxyl groups of curdlan and the carboxylic-/amide-groups of PGA via hydrogen bonding. Intracellular uptake of C-NP occurs over time in human monocyte-derived macrophage (MDM). Furthermore, C-NP nanoparticles dose-dependently increase gene expression for TNF-α, IL-6, and IL-8 at 24 h in MDM. C-NP nanoparticles also stimulate the release of IL-lß, MCP-1, TNF-α, IL-8, IL-12p70, IL-17, IL-18, and IL-23 from MDM. Overall, this is the first demonstration of a simplistic nanoformulation formed by hydrogen bonding between curdlan and PGA that modulates cytokine gene expression and release of cytokines from MDM.

Classification models using circulating neutrophil transcripts can detect unruptured intracranial aneurysm.

BACKGROUND: Intracranial aneurysms (IAs) are dangerous because of their potential to rupture. We previously found significant RNA expression differences in circulating neutrophils between patients with and without unruptured IAs and trained machine learning models to predict presence of IA using 40 neutrophil transcriptomes. Here, we aim to develop a predictive model for unruptured IA using neutrophil transcriptomes from a larger population and more robust machine learning methods. METHODS: Neutrophil RNA extracted from the blood of 134 patients (55 with IA, 79 IA-free controls) was subjected to next-generation RNA sequencing. In a randomly-selected training cohort (n = 94), the Least Absolute Shrinkage and Selection Operator (LASSO) selected transcripts, from which we constructed prediction models via 4 well-established supervised machine-learning algorithms (K-Nearest Neighbors, Random Forest, and Support Vector Machines with Gaussian and cubic kernels). We tested the models in the remaining samples (n = 40) and assessed model performance by receiver-operating-characteristic (ROC) curves. Real-time quantitative polymerase chain reaction (RT-qPCR) of 9 IA-associated genes was used to verify gene expression in a subset of 49 neutrophil RNA samples. We also examined the potential influence of demographics and comorbidities on model prediction. RESULTS: Feature selection using LASSO in the training cohort identified 37 IA-associated transcripts. Models trained using these transcripts had a maximum accuracy of 90% in the testing cohort. The testing performance across all methods had an average area under ROC curve (AUC) = 0.97, an improvement over our previous models. The Random Forest model performed best across both training and testing cohorts. RT-qPCR confirmed expression differences in 7 of 9 genes tested. Gene ontology and IPA network analyses performed on the 37 model genes reflected dysregulated inflammation, cell signaling, and apoptosis processes. In our data, demographics and comorbidities did not affect model performance. CONCLUSIONS: We improved upon our previous IA prediction models based on circulating neutrophil transcriptomes by increasing sample size and by implementing LASSO and more robust machine learning methods. Future studies are needed to validate these models in larger cohorts and further investigate effect of covariates.

CHRFAM7A: A human specific fusion gene, accounts for the translational gap for cholinergic strategies in Alzheimer's disease.

BACKGROUND: Cholinergic neuronal loss is one of the hallmarks of AD related neurodegeneration; however, preclinical promise of α7 nAChR drugs failed to translate into humans. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of α7 nAChR and was unaccounted for in preclinical models. METHODS: Molecular methods: Function of CHRFAM7A alleles was studied in vitro in two disease relevant phenotypic readouts: electrophysiology and Aß uptake. Genome edited human induced pluripotent stem cells (iPSC) were used as a model system with the human context. Double blind pharmacogenetic study: We performed double-blind pharmacogenetic analysis on the effect of AChEI therapy based on CHRFAM7A carrier status in two paradigms: response to drug initiation and DMT effect. Mini Mental Status Examination (MMSE) was used as outcome measure. Change in MMSE score from baseline was compared by 2-tailed T-test. Longitudinal analysis of clinical outcome (MMSE) was performed using a fitted general linear model, based on an assumed autoregressive covariance structure. Model independent variables included age, sex, and medication regimen at the time of the first utilized outcome measure (AChEI alone or AChEI plus memantine), APOE4 carrier status (0, 1 or 2 alleles as categorical variables) and CHRFAM7A genotype. FINDINGS: The direct and inverted alleles have distinct phenotypes. Functional CHRFAM7A allele classifies the population as 25% non-carriers and 75% carriers. Induced pluripotent stem cell (iPSC) models α7 nAChR mediated Aß neurotoxicity. Pharmacological readout translates into both first exposure (p = 0.037) and disease modifying effect (p = 0.0048) in two double blind pharmacogenetic studies. INTERPRETATION: CHRFAM7A accounts for the translational gap in cholinergic strategies in AD. Clinical trials not accounting for this uniquely human genetic factor may have rejected drug candidates that would benefit 25% of AD. Reanalyses of the completed trials using this pharmacogenetic paradigm may identify effective therapy.

Megalin-mediated albumin endocytosis in cultured murine mesangial cells.

Endocytosis by podocytes is gaining increased attention as a biologic means of removing large proteins such as serum albumin from the glomerular barrier. Some of this function has been attributed to the megalin/cubilin (Lrp2/Cubn) receptor complex and the albumin recycling protein FcRn (Fcgrt). However, whether other glomerular cells possess the potential to perform this same phenomenon or express these proteins remains uncharacterized. Mesangial cells are uniquely positioned in glomeruli and represent a cell type capable of performing several diverse functions. Here, the expression of megalin and FcRn in murine mesangial cells along with the megalin adaptor protein Dab-2 (Dab2) was shown for the first time. Cubilin mRNA expression was detected, but the absence of the cubilin partner amnionless (Amn) suggested that cubilin is minimally functional, if at all, in these cells. Mesangial cell endocytosis of albumin was characterized and shown to involve a receptor-mediated process. Albumin endocytosis was significantly impaired (p < 0.01) under inducible megalin knockdown conditions in stably transduced mesangial cells. The current work provides both the novel identification of megalin and FcRn in mesangial cells and the functional demonstration of megalin-mediated albumin endocytosis.

iPSC model of CHRFAM7A effect on α7 nicotinic acetylcholine receptor function in the human context.

The α7 nicotinic acetylcholine receptor (α7nAChR) has been a promising target for diseases affecting cognition and higher cortical functions; however, the effect observed in animal models failed to translate into human clinical trials identifying a translational gap. CHRFAM7A is a human-specific fusion gene with properties that enable incorporation into the α7nAChR and, being human specific, CHRFAM7A effect was not accounted for in preclinical studies. We hypothesized that CHRFAM7A may account for this translational gap and understanding its function may offer novel insights when exploring α7nAChR as a drug target. CHRFAM7A is present in different copy number variations (CNV) in the human genome with high frequency. To study the functional consequences of the presence of the CHRFAM7A, two induced pluripotent stem cell (iPSC) lines (0 copy and 1 copy direct) were developed. The 0 copy line was rescued with CHRFAM7A transfection to control for genetic heterogeneity. As readouts for genotype-phenotype correlation, α7nAChR synaptic transmission and amyloid beta 1-42 (Aß1-42) uptake were tested. Synaptic transmission in the presence of CHRFAM7A demonstrated that PNU-modulated desensitization of α7nAChR currents increased as a function of CHRFAM7A dosage. CHRFAM7A mitigated the dose response of Aß1-42 uptake suggesting a protective effect beyond physiological concentrations. Furthermore, in the presence of CHRFAM7A Aß1-42 uptake activated neuronal interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) without activating the canonical inflammasome pathway. Lead optimization may identify more potent molecules when the screen has a model harboring CHRFAM7A. Incorporating pharmacogenetics into clinical trials may enhance signals in efficacy measures.

Fecal microbiota analysis of polycystic kidney disease patients according to renal function: A pilot study.

IMPACT STATEMENT: The heterogeneity of the renal disease, therapeutic interventions, and the original cause of the renal failure, all directly affect the microbiota. We delineate in this report the direct effect of decreased renal function on the bacterial composition following stringent criteria to eliminate the possibilities of other confounding factors and dissect the direct effects of the uremic milieu. We analyzed the microbiome following three different approaches to further evaluate the effects of mild, moderate and advanced renal insufficiency on the microbiome. We also present here a detailed functional analysis of the projected altered pathways secondary to changes in the microbiome composition.