Development of binaural temporal fine structure sensitivity in children.

The highest frequency for which the temporal fine structure (TFS) of a sinewave can be compared across ears varies between listeners with an upper limit of about 1400 Hz for young normal-hearing adults (YNHA). In this study, binaural TFS sensitivity was investigated for 63 typically developing children, aged 5 years, 6 months to 9 years, 4 months using the temporal fine structure-adaptive frequency (TFS-AF) test of Füllgrabe, Harland, Sek, and Moore [Int. J. Audiol. 56, 926-935 (2017)]. The test assesses the highest frequency at which an interaural phase difference (IPD) of ϕ° can be distinguished from an IPD of 0°. The values of ϕ were 30° and 180°. The starting frequency was 200 Hz. The thresholds for the children were significantly lower (worse) than the thresholds reported by Füllgrabe, Harland, Sek, and Moore [Int. J. Audiol. 56, 926-935 (2017)] for YNHA. For both values of ϕ, the median age at which children performed above chance level was significantly higher (p < 0.001) than for those who performed at chance. For the subgroup of 40 children who performed above chance for ϕ = 180°, the linear regression analyses showed that the thresholds for ϕ = 180° increased (improved) significantly with increasing age (p < 0.001) with adult-like thresholds predicted to be reached at 10 years, 2 months of age. The implications for spatial release from masking are discussed.

Luminex-based quantification of Alzheimer's disease neuropathologic change in formalin-fixed post-mortem human brain tissue.

The vast majority of archived research and clinical pathological specimens are stored in the form of formalin fixed, paraffin-embedded (FFPE) tissues, but, unlike fresh frozen tissue samples, highly quantitative measures in FFPE tissues are limited to immunohistochemical and immunofluorescence thresholding image analysis studies, cell counting, and ordinal ranking systems. This poses a significant obstacle for clinical investigations that aim to correlate diagnostic markers of neurodegenerative diseases like Alzheimer's disease (AD) with parameters like age, gender, drug exposures, genotype, disease stage, co-morbidities, or environmental factors. To overcome this limitation, we have developed Luminex-based techniques and protocols for the quantification of amyloid ß and hyperphosphorylated Tau in FFPE brain sections. We validated the Luminex assay in FFPE sections from prefrontal cortex, hippocampus, and neostriatum from 30 cases that underwent prior neuropathological diagnostic assessment of AD following the current NIA-AA recommendations for AD: 10 cases diagnosed as not or low, 10 cases as intermediate, and 10 cases as high AD neuropathologic change. Consistent with the neuropathologic assessment, Luminex assay detected high amounts of amyloid beta in the frontal cortex and striatum, and high amounts of hyperphosphorylated Tau in the frontal cortex and hippocampus, of cases with high AD neuropathologic change. This assay can be expanded to detect diverse antigenic targets of interest, as we show here with IBA1 and GFAP. This novel approach supports multiplexed highly quantitative, molecularly specific neuropathology measures to further explore mechanisms of neurodegeneration in AD.

A nonhuman primate model of early Alzheimer's disease pathologic change: Implications for disease pathogenesis.

INTRODUCTION: Nonhuman primates may serve as excellent models of sporadic age-associated brain ß-amyloid deposition and Alzheimer's disease pathologic changes. We examined whether a vervet nonhuman primate model recapitulated pathologic, physiologic, and behavioral features of early Alzheimer's disease. METHODS: Nine middle-aged (mean = 11.2 years) and nine aged (mean = 21.7 years) female vervet/African green monkeys underwent cerebrospinal fluid collection, gait speed measurement, and neuroimaging before neuropathologic assessment. RESULTS: ß-amyloid plaques were identified in all aged vervets and paired helical filament tau immunoreactivity was observed in all animals. Cerebrospinal fluid ß-amyloid42 and gait speed correlated negatively with age and plaque density. Greater plaque and paired helical filament tau burden predicted reduced volumes and CMRg in several brain regions. DISCUSSION: We observed a coordinated set of relationships among neuropathologic, cerebrospinal fluid, imaging, and behavioral modalities consistent with early Alzheimer's disease. Our results support future use of the vervet model to explore disease mechanisms, biomarkers, and novel therapeutic strategies.

Neural responses to natural and enhanced speech edges in children with and without dyslexia.

Sensory-neural studies indicate that children with developmental dyslexia show impairments in processing acoustic speech envelope information. Prior studies suggest that this arises in part from reduced sensory sensitivity to amplitude rise times (ARTs or speech "edges") in the envelope, accompanied by less accurate neural encoding of low-frequency envelope information. Accordingly, enhancing these characteristics of the speech envelope may enhance neural speech processing in children with dyslexia. Here we applied an envelope modulation enhancement (EME) algorithm to a 10-min story read in child-directed speech (CDS), enhancing ARTs and also enhancing low-frequency envelope information. We compared neural speech processing (as measured using MEG) for the EME story with the same story read in natural CDS for 9-year-old children with and without dyslexia. The EME story affected neural processing in the power domain for children with dyslexia, particularly in the delta band (0.5-4 Hz) in the superior temporal gyrus. This may suggest that prolonged experience with EME speech could ameliorate some of the impairments shown in natural speech processing by children with dyslexia.

Protocol for the Systematic Fixation, Circuit-Based Sampling, and Qualitative and Quantitative Neuropathological Analysis of Human Brain Tissue.

Human brain tissue has long been a critical resource for neuroanatomy and neuropathology, but with the advent of advanced imaging and molecular sequencing techniques, it has become possible to use human brain tissue to study, in great detail, the structural, molecular, and even functional underpinnings of human brain disease. In the century following the first description of Alzheimer's disease (AD), numerous technological advances applied to human tissue have enabled novel diagnostic approaches using diverse physical and molecular biomarkers, and many drug therapies have been tested in clinical trials (Schachter and Davis, Dialogues Clin Neurosci 2:91-100, 2000). The methods for brain procurement and tissue stabilization have remained somewhat consistently focused on formalin fixation and freezing. Although these methods have enabled research protocols of multiple modalities, new, more advanced technologies demand improved methodologies for the procurement, characterization, stabilization, and preparation of both normal and diseased human brain tissues. Here, we describe our current protocols for the procurement and characterization of fixed brain tissue, to enable systematic and precisely targeted diagnoses, and describe the novel, quantitative molecular, and neuroanatomical studies that broadly expand the use of formalin-fixed, paraffin-embedded (FFPE) tissue that will further our understanding of the mechanisms underlying human neuropathologies.

Preferences of Dairy Cattle for Supplemental Light-Emitting Diode Lighting in the Resting Area.

Light from the environment is important for vision and regulating various biological processes. Providing supplemental lighting in the stall area could allow for individually targeted or group-level control of light. This study aimed to determine whether dairy cattle had preferences for short-term exposure to white (full-spectrum) light-emitting diode (LED) light or no LED light, yellow-green or white LED light, and blue or white LED light in the stall area. In total, 14 lactating cows were housed in a free-stall pen with unrestricted access to 28 stalls. LED light was controlled separately for each side of the stall platform. Two combinations of light were tested per week, and each week consisted of three adaptation days and four treatment days. Lying behaviour and video data were recorded continuously using leg-mounted pedometers and cameras, respectively. Preference was assessed by the amount of time spent lying and the number of bouts under each light treatment. No differences occurred between treatments within each week for daily lying time and number of bouts. Similarly, no differences occurred between treatments within each time period. Further controlled studies of long-term exposure to different LED wavelengths and intensities are required to determine potential benefits on metabolic processes.

The Delayed Neuropathological Consequences of Traumatic Brain Injury in a Community-Based Sample.

The late neuropathological effects of traumatic brain injury have yet to be fully elucidated, particularly with respect to community-based cohorts. To contribute to this critical gap in knowledge, we designed a multimodal neuropathological study, integrating traditional and quantitative approaches to detect pathologic changes in 532 consecutive brain autopsies from participants in the Adult Changes in Thought (ACT) study. Diagnostic evaluation including assessment for chronic traumatic encephalopathy (CTE) and quantitative immunoassay-based methods were deployed to examine levels of pathological (hyperphosphorylated) tau (pTau) and amyloid (A) ß in brains from ACT participants with (n = 107) and without (n = 425) history of remote TBI with loss of consciousness (w/LOC). Further neuropathological assessments included immunohistochemistry for α-synuclein and phospho-TDP-43 pathology and astro- (GFAP) and micro- (Iba1) gliosis, mass spectrometry analysis of free radical injury, and gene expression evaluation (RNA sequencing) in a smaller sub-cohort of matched samples (49 cases with TBI and 49 non-exposed matched controls). Out of 532 cases, only 3 (0.6%-none with TBI w/LOC history) showed evidence of the neuropathologic signature of chronic traumatic encephalopathy (CTE). Across the entire cohort, the levels of pTau and Aß showed expected differences for brain region (higher levels in temporal cortex), neuropathological diagnosis (higher in participants with Alzheimer's disease), and APOE genotype (higher in participants with one or more APOE ε4 allele). However, no differences in PHF-tau or Aß1-42 were identified by Histelide with respect to the history of TBI w/LOC. In a subset of TBI cases with more carefully matched control samples and more extensive analysis, those with TBI w/LOC history had higher levels of hippocampal pTau but no significant differences in Aß, α-synuclein, pTDP-43, GFAP, Iba1, or free radical injury. RNA-sequencing also did not reveal significant gene expression associated with any measure of TBI exposure. Combined, these findings suggest long term neuropathological changes associated with TBI w/LOC may be subtle, involve non-traditional pathways of neurotoxicity and neurodegeneration, and/or differ from those in autopsy cohorts specifically selected for neurotrauma exposure.

Attenuated progression of diet-induced steatohepatitis in glutathione-deficient mice.

In nonalcoholic fatty liver disease (NAFLD), depletion of hepatic antioxidants may contribute to the progression of steatosis to nonalcoholic steatohepatitis (NASH) by increasing oxidative stress that produces lipid peroxidation, inflammation, and fibrosis. We investigated whether depletion of glutathione (GSH) increases NASH-associated hepatic pathology in mice fed a diet deficient in methionine and choline (MCD diet). Wild-type (wt) mice and genetically GSH-deficient mice lacking the modifier subunit of glutamate cysteine ligase (Gclm null mice), the rate-limiting enzyme for de novo synthesis of GSH, were fed the MCD diet, a methionine/choline-sufficient diet, or standard chow for 21 days. We assessed NASH-associated hepatic pathology, including steatosis, fibrosis, inflammation, and hepatocyte ballooning, and used the NAFLD Scoring System to evaluate the extent of changes. We measured triglyceride levels, determined the level of lipid peroxidation products, and measured by qPCR the expression of mRNAs for several proteins associated with lipid metabolism, oxidative stress, and fibrosis. MCD-fed GSH-deficient Gclm null mice were to a large extent protected from MCD diet-induced excessive fat accumulation, hepatocyte injury, inflammation, and fibrosis. Compared with wt animals, MCD-fed Gclm null mice had much lower levels of F2-isoprostanes, lower expression of acyl-CoA oxidase, carnitine palmitoyltransferase 1a, uncoupling protein-2, stearoyl-coenzyme A desaturase-1, transforming growth factor-ß, and plasminogen activator inhibitor-1 mRNAs, and higher activity of catalase, indicative of low oxidative stress, inhibition of triglyceride synthesis, and lower expression of profibrotic proteins. Global gene analysis of hepatic RNA showed that compared with wt mice, the livers of Gclm null mice have a high capacity to metabolize endogenous and exogenous compounds, have lower levels of lipogenic proteins, and increased antioxidant activity. Thus, metabolic adaptations resulting from severe GSH deficiency seem to protect against the development of steatohepatitis.

Identification of glutathione S-transferase pi as a protein involved in Parkinson disease progression.

Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD.

Perception of Filtered Speech by Children with Developmental Dyslexia and Children with Specific Language Impairments.

Here we use two filtered speech tasks to investigate children's processing of slow (<4 Hz) versus faster (∼33 Hz) temporal modulations in speech. We compare groups of children with either developmental dyslexia (Experiment 1) or speech and language impairments (SLIs, Experiment 2) to groups of typically-developing (TD) children age-matched to each disorder group. Ten nursery rhymes were filtered so that their modulation frequencies were either low-pass filtered (<4 Hz) or band-pass filtered (22 - 40 Hz). Recognition of the filtered nursery rhymes was tested in a picture recognition multiple choice paradigm. Children with dyslexia aged 10 years showed equivalent recognition overall to TD controls for both the low-pass and band-pass filtered stimuli, but showed significantly impaired acoustic learning during the experiment from low-pass filtered targets. Children with oral SLIs aged 9 years showed significantly poorer recognition of band pass filtered targets compared to their TD controls, and showed comparable acoustic learning effects to TD children during the experiment. The SLI samples were also divided into children with and without phonological difficulties. The children with both SLI and phonological difficulties were impaired in recognizing both kinds of filtered speech. These data are suggestive of impaired temporal sampling of the speech signal at different modulation rates by children with different kinds of developmental language disorder. Both SLI and dyslexic samples showed impaired discrimination of amplitude rise times. Implications of these findings for a temporal sampling framework for understanding developmental language disorders are discussed.

Cerebrospinal Fluid Particles in Alzheimer Disease and Parkinson Disease.

Human cerebrospinal fluid (CSF) contains diverse lipid particles, including lipoproteins that are distinct from their plasma counterparts and contain apolipoprotein (apo) E isoforms, apoJ, and apoAI, and extracellular vesicles, which can be detected by annexin V binding. The aim of this study was to develop a method to quantify CSF particles and evaluate their relationship to aging and neurodegenerative diseases. We used a flow cytometric assay to detect annexin V-, apoE-, apoAI-, apoJ-, and amyloid (A) ß42-positive particles in CSF from 131 research volunteers who were neurologically normal or had mild cognitive impairment (MCI), Alzheimer disease (AD) dementia, or Parkinson disease. APOE ε4/ε4 participants had CSF apoE-positive particles that were more frequently larger but at an 88% lower level versus those in APOE ε3/ε3 or APOE ε3/ε4 patients; this finding was reproduced in conditioned medium from mouse primary glial cell cultures with targeted replacement of apoE. Cerebrospinal fluid apoE-positive and ß-amyloid (Aß42)-positive particle concentrations were persistently reduced one-third to one-half in middle and older age subjects; apoAI-positive particle concentration progressively increased approximately 2-fold with age. Both apoAI-positive and annexin V-positive CSF particle levels were reduced one-third to one-half in CSF of MCI and/or AD dementia patients versus age-matched controls. Our approach provides new methods to investigate CNS lipid biology in relation to neurodegeneration and perhaps develop new biomarkers for diagnosis or treatment monitoring.

Ablation of the chemokine monocyte chemoattractant protein-1 delays retrograde neuronal degeneration, attenuates microglial activation, and alters expression of cell death molecules.

The mechanisms regulating retrograde neuronal degeneration and subsequent death of thalamic neurons following cortical injury are not well understood. However, the delay in the onset of retrograde cell death and observed morphological changes are consistent with apoptosis. Our previous studies demonstrated that monocyte chemoattractant protein-1 (MCP-1), a beta-chemokine that attracts cells of monocytic origin to sites of injury, is rapidly and specifically expressed in the lateral geniculate nucleus following visual cortical lesions. To determine the potential role of MCP-1 in retrograde degeneration, the present study examined the effect of genetic deletion of MCP-1 (MCP-1 KO or -/-) or its high affinity receptor CCR2 (CCR2 KO or -/-) on thalamic microglial activation and neuronal cell death following aspiration lesions of the visual cortex in adult mice. Deletion of the MCP-1 gene delayed microglial activation and transiently improved the survival of thalamic neurons. Deletion of the CCR2 receptor resulted in a significant increase in apoptosis as measured by nucleosomal fragmentation after injury compared to wild-type mice, but did not alter neuron survival, suggesting that glial apoptosis is increased in the receptor knockout mice. Investigation of Bcl-2, Bax, Fas, Fas ligand (FasL) and activated caspase-3, key regulators of apoptosis that can be modulated by cytokines, revealed complex alterations of mRNA and protein levels in MCP-1(-/-) and CCR2(-/-) mice. As examples, Bcl-2 protein was detected in wild-type, but not in MCP-1(-/-) mice. Caspase-3 activity was higher in MCP-1(-/-) mice compared to wild-type and CCR2(-/-) mice at 5 days after injury. High levels of activated caspase-3 correlate with the beginning of a period of delayed, but rapid cell death in the thalami of MCP-1(-/-) mice. In summary, our data strongly suggest that MCP-1 is involved in early microglial response to axotomy and that modulation of this chemokine could provide a novel strategy for improved neuronal survival following injury to the central nervous system.

Metabolism of the soil and groundwater contaminants, ethylene dibromide and trichloroethylene, by the tropical leguminous tree, Leuceana leucocephala.

Ethylene dibromide (EDB; dibromoethane) and trichloroethylene (TCE) are hazardous environmental pollutants. The use of plants to treat polluted sites and groundwater, termed phytoremediation, requires plants that can both effectively remove the pollutant as well as grow in the climatic region of the site. In this paper, we report that the tropical leguminous tree, Leuceana leucocephala var. K636, is able to take up and metabolize EDB and TCE. The plants were grown in sterile hydroponic solution without its symbiont, Rhizobium. EDB and TCE were both metabolized by the plant, as indicated by the formation of bromide ion from EDB and trichloroethanol from TCE. Each plant organ was independently capable of debromination of EDB. L. leucocephala is being used to treat perched groundwater as part of a remedial alternative to address an accidental EDB spill in Hawaii. Bromide levels of plant tissues from the trees grown in the phytoremediation treatment cells at the Hawaii Site were elevated, indicating uptake and degradation of brominated compounds in the trees. This report is the first evidence of a tropical tree effectively metabolizing these common organic pollutants.

Inflammation and oxidative stress markers and esophageal adenocarcinoma incidence in a Barrett's esophagus cohort.

BACKGROUND: Persons with Barrett's esophagus experience increased risk of esophageal adenocarcinoma. Prediagnostic inflammation markers predict several cancers, but their role in predicting esophageal adenocarcinoma is unknown. METHODS: We investigated whether biomarkers of inflammation [C-reactive protein (CRP), interleukin-6 (IL6), soluble tumor necrosis factor (sTNF) receptors I and II], and of oxidative stress (F2-isoprostanes) predicted progression to esophageal adenocarcinoma in a prospective cohort of 397 patients with Barrett's esophagus, 45 of whom developed esophageal adenocarcinoma. Biomarkers were measured in stored plasma samples from two time points during follow-up, the mean of which served as the primary predictor. Adjusted hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression. RESULTS: CRP level above the median was associated with an 80% increased risk of esophageal adenocarcinoma. The HR and 95% CI adjusted for age, gender, and further adjusted for waist-hip ratio and smoking were 1.98 (1.05-3.73) and 1.77 (0.93-3.37), respectively, with Ptrend for continuous CRP = 0.04. Persons with IL6 levels above the median also had almost 2-fold increased risk [HR and 95% CI adjusted for age and gender, and further adjusted for waist-hip ratio and smoking were 1.95 (1.03-3.72) and 1.79 (0.93-3.43), respectively, but no evidence of a trend was observed]. Concentrations of TNF receptors and F2-isoprostanes were not associated with esophageal adenocarcinoma risk. CONCLUSIONS: Further research is needed to evaluate the role of inflammation and associated markers in esophageal adenocarcinoma development in persons with Barrett's esophagus. IMPACT: This prospective study suggests that inflammation markers, particularly CRP and IL6, may help identify persons at higher risk of progression to esophageal adenocarcinoma.

Increased cerebrospinal fluid F2-isoprostanes are associated with aging and latent Alzheimer's disease as identified by biomarkers.

Alzheimer's disease (AD) is a common age-related chronic illness with latent, prodrome, and fully symptomatic dementia stages. Increased free radical injury to regions of brain is one feature of prodrome and dementia stages of AD; however, it also is associated with advancing age. This raises the possibility that age-related free radical injury to brain might be caused in part or in full by latent AD. We quantified free radical injury in the central nervous system with cerebrospinal fluid (CSF) F(2)-isoprostanes (IsoPs) in 421 clinically normal individuals and observed a significant increase over the adult human lifespan (P < 0.001). Using CSF amyloid (A) ß(42) and tau, we defined normality using results from 28 clinically normal individuals <50 years old, and then stratified 74 clinically normal subjects ≥60 years into those with CSF that had normal CSF Aß(42) and tau (n = 37); abnormal CSF Aß(42) and tau, the biomarker signature of AD (n = 24); decreased Aß(42) only (n = 4); or increased tau only (n = 9). Increased CSF F(2)-IsoPs were present in clinically normal subjects with the biomarker signature of AD (P < 0.05) and those subjects with increased CSF tau (P < 0.001). Finally, we analyzed the relationship between age and CSF F(2)-IsoPs for those clinically normal adults with normal CSF (n = 37) and those with abnormal CSF Aß(42) and/or tau (n = 37); only those with normal CSF demonstrated a significant increase with age (P < 0.01). These results show that CSF F(2)-IsoPs increased across the human lifespan and that this age-related increase in free radical injury to brain persisted after culling those with laboratory evidence of latent AD.

A randomized, placebo-controlled clinical trial of bupropion for the prevention of smoking in children and adolescents with attention-deficit/hyperactivity disorder.

OBJECTIVE: Since attention-deficit/hyperactivity disorder (ADHD) is a well-documented risk factor for smoking and bupropion has been shown to be effective for smoking cessation, we tested the efficacy of bupropion as a prophylactic agent for the prevention of smoking in children and adolescents with ADHD. METHOD: We conducted a longitudinal, randomized, double-blind, placebo-controlled, parallel-group study of bupropion at a large, urban, outpatient medical center. Recruitment began in April 1999, and the last subject was followed until September 2004. Patients were nonsmoking youth, of both sexes, between 9 and 18 years of age, with DSM-IV ADHD. After random assignment to either bupropion or placebo, subjects were assessed weekly for 8 weeks, biweekly for 4 weeks, and monthly thereafter for up to 6.5 years (mean 12 months). Also, patients received treatment with psychostimulants for ADHD symptoms as needed. To assess smoking, we used an assay of cotinine in urine. RESULTS: Fifty-seven subjects (28 receiving bupropion and 29 receiving placebo) were randomly assigned and included in the analysis. No differences were found between the bupropion and placebo groups on demographic factors. About half of each group was treated with stimulants for ADHD. Statistical separation between bupropion and placebo in the rate of smoking initiation or continued smoking was not demonstrated. However, secondary post hoc analyses revealed that concurrent stimulant treatment was significantly associated with a lower rate of smoking onset (hazard ratio [HR] = 0.2, 95% CI = 0.08 to 0.89; z = -2.2, p = .03) and a lower rate of continued smoking (HR = 0.3, 95% CI = 0.11 to 0.85; z = -2.3, p = .02). CONCLUSION: While bupropion was not associated with a lower rate of smoking in youth with ADHD, post hoc analyses suggest that stimulant treatment was. Future controlled studies should investigate the role of stimulants in the prevention of smoking in children and adolescents with ADHD.

Expression of functional mammalian P450 2E1 in hairy root cultures.

P450 2E1 is an important mammalian liver enzyme known to metabolize a wide range of compounds including several common environmental pollutants. The medicinal plant, Atropa belladonna, was transformed with Agrobacterium rhizogenes containing a binary vector with rabbit P450 2E1 in either the sense or antisense orientation. The resulting "hairy roots" were isolated and grown in liquid medium. Production of P450 2E1 protein was verified in the roots containing the 2E1 gene in the sense orientation. Transgenic and control root cultures were dosed with the environmental pollutant, trichloroethylene (TCE), and were analyzed for the TCE metabolites, chloral and trichloroethanol. The root cultures expressing the mammalian P450 2E1 had increased levels of the metabolites compared to the levels in the control roots. This method represents a quick way to screen transformants for expression of foreign genes before regeneration of whole plants, and also as a possible source of foreign protein for purification.