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Ischaemic brain infarction can occur without acute neurological symptoms (covert strokes) or with symptoms (overt strokes), both associated with poor health outcomes. We conducted a pilot study of the incidence of preoperative and postoperative (intraoperative or postoperative) covert strokes, and explored the relationship of postoperative ischaemic brain injury to blood levels of neurofilament light, a biomarker of neuronal damage.We analysed 101 preoperative (within 2 weeks of surgery) and 58 postoperative research MRIs on postoperative days 2-9 from two prospective cohorts collected at the University of Wisconsin (NCT01980511 and NCT03124303). Participants were aged65 yr and undergoing non-intracranial, non-carotid surgery.Preoperative covert stroke was identified in 2/101 participants (2%; Bayesian 95% confidence interval [CI], 0.2-5.4). This rate was statistically different from the postoperative ischaemic brain injury rate of 7/58 (12%, 4.9-21.3%; P=0.01) based on postoperative imaging. However, in a smaller group of participants with paired imaging (n=30), we did not identify the same effect (P=0.67). Patients with postoperative brain injury had elevated peak neurofilament light levels (median [inter-quartile range], 2.34 [2.24-2.64] logAlthough limited by a small sample size, these data suggest that preoperative covert stroke occurs more commonly than previously anticipated. Plasma neurofilament light is a potential screening biomarker for postoperative ischaemic brain injury.

Movement and posture depend on sensory feedback that is regulated by specialized GABAergic neurons (GAD2+) that form axo-axonic contacts onto myelinated proprioceptive sensory axons and are thought to be inhibitory. However, we report here that activating GAD2+ neurons, directly with optogenetics or indirectly by cutaneous stimulation, facilitates sensory feedback to motoneurons in awake rodents and humans. GABAA receptors and GAD2+ innervation at or near nodes of Ranvier of sensory axons cause this facilitation, preventing spike propagation failure at the many axon branch points, which is otherwise common without GABA. In contrast, GABAA receptors are generally lacking from axon terminals (unlike GABAB) and do not presynaptically inhibit transmitter release onto motoneurons. GABAergic innervation near nodes and branch points allows individual branches to function autonomously, with GAD2+ neurons regulating which branches conduct, adding a computational layer to the neuronal networks generating movement and likely generalizing to other CNS axons.

Case-control studies have associated delirium with blood-brain barrier (BBB) permeability. However, this approach cannot determine whether delirium is attributable to high pre-existing permeability or to perioperative changes. We tested whether perioperative changes in cerebrospinal fluid/plasma albumin ratio (CPAR) and plasma S100B were associated with delirium severity.Participants were recruited to two prospective cohort studies of non-intracranial surgery (NCT01980511, NCT03124303, and NCT02926417). Delirium severity was assessed using the Delirium Rating Scale-98. Delirium incidence was diagnosed with the 3D-Confusion Assessment Method (3D-CAM) or CAM-ICU (CAM for the ICU). CSF samples from 25 patients and plasma from 78 patients were analysed for albumin and S100B. We tested associations between change in CPAR (n=11) and S100B (n=61) and delirium, blood loss, CSF interleukin-6 (IL-6), and CSF lactate.The perioperative increase in CPAR and S100B correlated with delirium severity (CPAR ρ=0.78, P=0.01; S100B ρ=0.41, P0.001), delirium incidence (CPAR P=0.012; S100B P0.001) and CSF IL-6 (CPAR ρ=0.66 P=0.04; S100B ρ=0.75, P=0.025). Linear mixed-effect analysis also showed that decreased levels of S100B predicted recovery from delirium symptoms (P=0.001). Linear regression demonstrated that change in plasma S100B was independently associated with surgical risk, cardiovascular surgery, blood loss, and hypotension. Blood loss also correlated with CPAR (ρ=0.64, P=0.04), S100B (ρ=0.70, P0.001), CSF lactate (R=0.81, P=0.01), and peak delirium severity (ρ=0.36, P=0.01).Postoperative delirium is associated with a breakdown in the BBB. This increased permeability is dynamic and associated with a neuroinflammatory and lactate response. Strategies to mitigate blood loss may protect the BBB.

γ-Aminobutyric acid type A receptors that incorporate α5 subunits (α5-GABAARs) are highly enriched in the hippocampus and are strongly implicated in control of learning and memory. Receptors located on pyramidal neuron dendrites have long been considered responsible, but here we report that mice in which α5-GABAARs have been eliminated from pyramidal neurons (α5-pyr-KO) continue to form strong spatial engrams and that they remain as sensitive as their pseudo-wild-type (p-WT) littermates to etomidate-induced suppression of place cells and spatial engrams. By contrast, mice with selective knockout in interneurons (α5-i-KO) no longer exhibit etomidate-induced suppression of place cells. In addition, the strength of spatial engrams is lower in α5-i-KO mice than p-WT littermates under control conditions. Consistent with the established role of the hippocampus in contextual fear conditioning, α5-i-KO mice resisted etomidate’s suppression of freezing to context, but so too did α5-pyr-KO mice, supporting a role for extra-hippocampal regions in the development of contextual fear memory. Overall, our results indicate that interneuronal α5-GABAARs serve a physiological role in promoting spatial learning and that they mediate suppression of hippocampus-dependent contextual memory by etomidate.

Etomidate exerts its anesthetic actions through GABAA receptors. However, the mechanism remains unknown. Here, using a hippocampal brain slice model, we show that β2-GABAARs are essential to this effect. We also show that these receptors contribute to long-lasting dendritic inhibition in feedback but not feedforward inhibition of pyramidal neurons. These findings hold implications for understanding how anesthetics block memory formation and, more generally, how inhibitory circuits control learning and memory.

BackgroundPostoperative neurocognitive disorder (poNCD), previously also referred to as postoperative cognitive dysfunction (POCD) following surgery and anesthesia, is a significant public health problem in patients over 60 years. Propofol is the most commonly used intravenous anesthetic, acting primarily via GABAAreceptors. However, it has also been shown to have apparent neuroprotective effects in aged mice and mouse models of Alzheimer’s disease. Therefore, we postulated that chronic intermittent propofol would attenuate the development of postoperative cognitive deficits in aged mice.MethodsAbdominal surgery was performed under isoflurane anesthesia in 21-24 months-old mice. Animals received either chronic intermittent propofol (CIP, 75 mg/kg i.p.) or vehicle (IntralipidR) every 5thday throughout the experiment, starting 17 days before surgery. The levels of α5-GABAAreceptors on cell surface membranes, as well as behavioral or biochemical manifestations of poNCD were studied.ResultsCIP led to a sustained redistribution of the GABAAreceptor α5 subunit to the cell surface membranes. Laparotomy impaired learning and memory functions, as determined using a behavioral test battery that included Y maze alternation, novel object recognition, water maze learning and reversal learning, and cued and contextual fear conditioning, compared to no surgery controls. Strikingly, CIP strongly attenuated the surgery-induced memory impairment. Western blots on hippocampal tissues showed increased expression of the pro-apoptotic markers cleaved caspase-3, cleaved caspase-9, and Bax, indicating that abdominal surgery promoted apoptosis. Moreover, surgery increased expression of Iba-1, a marker of microglial activation. Chronic intermittent propofol prevented this proapoptotic and microglial activation.ConclusionsOur results suggest that propofol – via mechanisms not fully understood, potentially via the sustained increased availability of α5-GABAAreceptors on cell surface membranes – improves cognitive function by attenuating surgery-induced neuroinflammation and caspase activation and/or independently of this by rebalancing neuronal inhibition in aged mice. These findings support a therapeutic potential of perioperative propofol or of other compounds leading to a sustained redistribution of α5-GABAAreceptors to the cell surface membranes in the perioperative period for reducing the risk of surgery-induced cognitive decline in aged individuals.

Recent trials are conflicting as to whether titration of anaesthetic dose using electroencephalography monitoring reduces postoperative delirium. Titration to anaesthetic dose itself might yield clearer conclusions. We analysed our observational cohort to clarify both dose ranges for trials of anaesthetic dose and biological plausibility of anaesthetic dose influencing delirium.We analysed the use of sevoflurane in an ongoing prospective cohort of non-intracranial surgery. Of 167 participants, 118 received sevoflurane and were aged65 yr. We tested associations between age-adjusted median sevoflurane (AMS) minimum alveolar concentration fraction or area under the sevoflurane time×dose curve (AUC-S) and delirium severity (Delirium Rating Scale-98). Delirium incidence was measured with 3-minute Diagnostic Confusion Assessment Method (3D-CAM) or CAM-ICU. Associations with previously identified delirium biomarkers (interleukin-8, neurofilament light, total tau, or S100B) were tested.Delirium severity did not correlate with AMS (Spearman's ρ=-0.014, P=0.89) or AUC-S (ρ=0.093, P=0.35), nor did delirium incidence (AMS Wilcoxon P=0.86, AUC-S P=0.78). Further sensitivity analyses including propofol dose also demonstrated no relationship. Linear regression confirmed no association for AMS in unadjusted (log (IRR)=-0.06 P=0.645) or adjusted models (log (IRR)=-0.0454, P=0.735). No association was observed for AUC-S in unadjusted (log (IRR)=0.00, P=0.054) or adjusted models (log (IRR)=0.00, P=0.832). No association of anaesthetic dose with delirium biomarkers was identified (P0.05).Sevoflurane dose was not associated with delirium severity or incidence. Other biological mechanisms of delirium, such as inflammation and neuronal injury, appear more plausible than dose of sevoflurane.NCT03124303, NCT01980511.

BACKGROUND: Myocardial and neuronal injury occur commonly after noncardiac surgery. We examined whether patients who had perioperative myocardial injury (PMI) also incurred neuronal injury, and whether myocardial and neuronal injury were associated with similar changes in inflammatory markers or overlapping clinical predictors. METHODS: A total of 114 individuals >65 yr old were recruited from two ongoing perioperative cohort studies (NCT02926417; NCT03124303). Plasma samples were collected before and daily after surgery to process assays for troponin I (PMI), neurofilament light (NfL; neuronal injury) and multiplexed plasma cytokines (inflammation). The primary outcome was the change in NfL in individuals with PMI (>40 pg ml(−1) increase in troponin above preoperative values). We conducted logistic regression to identify if there were shared clinical predictors for myocardial and neuronal injury. RESULTS: Ninety-six patients had paired NfL and troponin data. Twenty-three of 94 subjects (24%) with PMI had greater increases in NfL (median [inter-quartile range, IQR]: 29 pg ml(−1) [3–95 pg ml(−1)]; 2.8-fold increase) compared with subjects with no troponin increase (8 pg ml(−1) [3–20]; 1.3-fold increase; P=0.008). PMI was associated with increased interleukin (IL)-1ra (P=0.005), IL-2 (P=0.045), IL-8 (P=0.002), and IL-10 (P

BACKGROUND: Postoperative delirium is associated with increases in the neuronal injury biomarker, neurofilament light (NfL). Here we tested whether two other biomarkers, glial fibrillary acidic protein (GFAP) and tau, are associated with postoperative delirium. METHODS: A total of 114 surgical patients were recruited into two prospective biomarker cohort studies with assessment of delirium severity and incidence. Plasma samples were sent for biomarker analysis including tau, NfL, and GFAP, and a panel of 10 cytokines. We determined a priori to adjust for interleukin-8 (IL-8), a marker of inflammation, when assessing associations between biomarkers and delirium incidence and severity. RESULTS: GFAP concentrations showed no relationship to delirium. The change in tau from preoperative concentrations to postoperative Day 1 was greater in patients with postoperative delirium (P

Field trials were conducted in Princeton, Murray, and Lexington, KY in 2016–2018 to determine response of dark and burley tobacco to potassium source (potassium sulfate or potassium chloride) and potassium rate (0, 93, 186, or 279 kg K ha−1). Field sites that showed higher potential for potassium yield response were selected based on low soil test potassium levels from soil samples collected in early spring each year. All potassium applications were made between 1 and 10 days before transplanting. Significant yield responses to potassium were seen in 5 of 12 trials at sites that had initial soil test potassium levels of ≤150 kg K ha−1. Although cured leaf chloride levels were >1% on average where potassium chloride was used, negative effects on cured leaf moisture were only seen in 1 of 12 trials, and negative effects on quality grade index were not seen in any trial. The most consistent effect of potassium chloride application seen in this research was a 28% reduction in average total tobacco-specific nitrosamines (TSNA) compared to potassium sulfate application. These results showing lower TSNA from potassium chloride applications, along with minimal effects on moisture and quality grade index, may cause the tobacco industry to reconsider the long-standing preference for potassium sulfate as the potassium source for tobacco production.

Chemical risk assessment considers potentially susceptible populations including pregnant women and developing fetuses. Humans encounter thousands of chemicals in their environments, few of which have been fully characterized. Toxicokinetic (TK) information is needed to relate chemical exposure to potentially bioactive tissue concentrations. Observational data describing human gestational exposures are unavailable for most chemicals, but physiologically based TK (PBTK) models estimate such exposures. Development of chemical-specific PBTK models requires considerable time and resources. As an alternative, generic PBTK approaches describe a standardized physiology and characterize chemicals with a set of standard physical and TK descriptors - primarily plasma protein binding and hepatic clearance. Here we report and evaluate a generic PBTK model of a human mother and developing fetus. We used a published set of formulas describing the major anatomical and physiological changes that occur during pregnancy to augment the High-Throughput Toxicokinetics (httk) software package. We simulated the ratio of concentrations in maternal and fetal plasma and compared to literature in vivo measurements. We evaluated the model with literature in vivo time-course measurements of maternal plasma concentrations in pregnant and non-pregnant women. Finally, we prioritized chemicals measured in maternal serum based on predicted fetal brain concentrations. This new model can be used for TK simulations of 859 chemicals with existing human-specific in vitro TK data as well as any new chemicals for which such data become available. This gestational model may allow for in vitro to in vivo extrapolation of point of departure doses relevant to reproductive and developmental toxicity.

γ-aminobutyric acid type A receptors that incorporate α5 subunits (α5-GABAARs) are highly enriched in the hippocampus and are strongly implicated in control of learning and memory. Receptors located on pyramidal neuron dendrites have long been considered responsible, but here we report that their selective knockout from either interneurons (α5-i-KO) or pyramidal neurons (α5-pyr-KO) interferes with the ability of the general anesthetic etomidate to suppress contextual conditioning. Using Ca2+imaging of CA1 pyramidal neuron activity in freely exploring mice to assess hippocampal function directly, we found that etomidate blocked the development of place cells and spatial engrams in wild type (WT) and α5-pyr-KO mice, but not in α5-i-KO mice. In addition, α5-i-KO mice developed weaker spatial engrams than WT mice under control conditions. These findings show that interneuronal α5-GABAARs serve a physiological role in promoting spatial learning, and that they mediate the suppression of hippocampus-dependent memory by etomidate.

Currently it is difficult to prospectively estimate human toxicokinetics (particularly for novel chemicals) in a high-throughput manner. The R software package httk has been developed, in part, to address this deficiency, and the aim of this investigation was to develop a generalized inhalation model for httk. The structure of the inhalation model was developed from two previously published physiologically based models from Jongeneelen and Berge (Ann Occup Hyg 55:841–864, 2011) and Clewell et al. (Toxicol Sci 63:160–172, 2001), while calculated physicochemical data was obtained from EPA’s CompTox Chemicals Dashboard. In total, 142 exposure scenarios across 41 volatile organic chemicals were modeled and compared to published data. The slope of the regression line of best fit between log-transformed simulated and observed blood and exhaled breath concentrations was 0.46 with an r2 = 0.45 and a root mean square error (RMSE) of direct comparison between the log-transformed simulated and observed values of 1.11. Approximately 5.1% (n = 108) of the data points analyzed were >2 orders of magnitude different than expected. The volatile organic chemicals examined in this investigation represent small, generally lipophilic molecules. Ultimately this paper details a generalized inhalation component that integrates with the httk physiologically based toxicokinetic model to provide high-throughput estimates of inhalation chemical exposures.

Azoxystrobin is the only synthetic, systemic fungicide labeled in the United States for management of frogeye leaf spot (FLS) of tobacco (Nicotiana tabacum L.), caused by Cercospora nicotianae. Though traditionally considered a minor disease in the United States, FLS has recently become yield and quality limiting. In 2016 and 2017, 100 C. nicotianae isolates were collected from symptomatic tobacco from eight counties in Kentucky, United States. Prior to azoxystrobin sensitivity testing, some C. nicotianae isolates were found to utilize the alternative oxidase pathway and, after assay comparisons, conidial germination was utilized to evaluate sensitivity in C. nicotianae as opposed to mycelial growth. Azoxystrobin sensitivity was determined by establishing the effective concentration to inhibit 50% conidial germination (EC50) for 47 (in 2016) and 53 (in 2017) C. nicotianae isolates. Distributions of C. nicotianae EC50 values indicated three qualitative levels of sensitivity to azoxystrobin. Partial cytochrome b sequence, encompassing the F129L and G143A mutation sites, indicated single-nucleotide polymorphisms (SNPs) conferring the F129L mutation in C. nicotianae of moderate resistance (azoxystrobin at 0.177 ≤ EC50 ≤ 0.535 µg/ml) and the G143A mutation in isolates with an azoxystrobin-resistant phenotype (azoxystrobin EC50 > 1.15 µg/ml). Higher frequencies of resistant isolates were identified from greenhouse transplant (4 of 17) and conventionally produced (58 of 62) tobacco samples, as compared with field-grown tobacco (

Experiments were initiated in 2015 to evaluate the efficacy of chemical topping for burley tobacco (Nicotiana tabacum L.). The major objectives for this study were to determine the optimum timing of suckercide application and appropriate cultivar maturity for effective chemical topping. Burley tobacco cultivars TN 90 (medium maturity), KT 210, and KT 215 (late maturity) were chemically topped at the 10% button, 50% button, and 10% bloom growth stages. The 10% button and 50% button application timings were best suited for chemical topping practices. Treatments that targeted the 10% bloom stage did not completely halt inflorescence growth; however, all application timings resulted in excellent sucker control. Both medium and late maturing burley cultivars proved to be acceptable for chemical topping methods; however, timing the suckercide application may be less difficult with later maturing cultivars. Chemically topped treatments generally resulted in shorter, narrower tip leaves than manually topped treatments. There were no significant differences in total yield of TN 90 when comparing tobacco that was manually topped at 10% bloom to tobacco that was chemically topped at 10% button, 50% button, or 10% bloom across all environments. In 4 out of 6 environments, total yield was not significantly different between manual topping and any chemically topped application timing in the late maturing burley cultivars; however, at least 1 chemically topped application timing had equivalent yield to manually topped tobacco in all environments.

The act of topping tobacco (Nicotiana tabacum L.) involves the removal of the terminal bud or inflorescence of the tobacco plant. This practice ordinarily is accomplished by manually removing the top of each tobacco plant in an entire field, which is labor intensive and costly. The major objectives for this research were to determine which labeled suckercides could be used effectively for chemical topping of burley tobacco and the effect of suckercide rate on sucker control, yield, leaf quality, maleic hydrazide (MH) residues, and leaf chemistry. A study was initiated at Murray, Princeton, and Lexington, KY that investigated the efficacy of suckercide applications using combinations of MH, butralin, and fatty alcohols (FA). The terminal bud was not well controlled with FA or butralin alone, nor was adequate sucker control or total yield achieved. A significant reduction in total yield and sucker control were observed when plants were chemically topped with MH alone compared to manually topped or chemically topped with a tank mixture of MH and butralin at Princeton only. At the other locations, all chemically topped plants had similar yield to manually topped plants. Our data suggested that chemical topping of burley tobacco with a tank mixture of MH and a local systemic can be an acceptable alternative to manual topping as total yield and leaf quality grade index were not significantly different at any location. Total tobacco-specific nitrosamine (TSNA) content and MH residues were significantly lower with chemical topping treatments in some years and locations.

Tobacco-specific nitrosamines (TSNAs) are known carcinogens in cured tobacco. They are produced primarily during the curing process, but agronomic practices occurring in the field as well as handling practices after curing may also influence TSNA levels, particularly if cured leaf is stored at high moisture. After curing and during market preparation, the cured leaf must be supple to avoid breakage. Ideally, this is after a period of wet weather during which the leaf absorbs moisture and comes into order or case. Often the weather remains dry for long periods after curing, and growers resort to artificial ordering to take down a sufficient amount of their crop to work on for several days, during which time the tobacco is bulked. The effect of this artificial ordering on TSNAs during short-term storage is not known. Field experiments were conducted in each of 3 years at two locations in Kentucky to evaluate TSNA accumulation following several ordering methods in dark air-cured and burley tobacco types. Treatments included natural ordering and variants of steaming and misting, which are both commonly used artificial ordering methods. At the Princeton location, samples were taken within 24 hr after the ordering treatments were done. In Lexington, samples were taken sequentially at takedown, after ordering, and after 14 d in the bulk. There were limited and inconsistent differences in total TSNAs between methods of ordering, and the TSNA levels were not affected by the moisture content of the leaf during bulking. There was a significant increase in TSNAs in the 24-hr period between takedown and bulking, which cannot be explained. We conclude that, in Kentucky, growers should use ordering methods that are best suited for their production system, but this may not be the case in warmer climates.

N ε-lysine acetylation within the lumen of the endoplasmic reticulum is a recently characterized protein quality control system that positively selects properly folded glycoproteins in the early secretory pathway. Overexpression of the endoplasmic reticulum acetyl-CoA transporter AT-1 in mouse forebrain neurons results in increased dendritic branching, spine formation and an autistic-like phenotype that is attributed to altered glycoprotein flux through the secretory pathway. AT-1 overexpressing neurons maintain the cytosolic pool of acetyl-CoA by upregulation of SLC25A1, the mitochondrial citrate/malate antiporter and ATP citrate lyase, which converts cytosolic citrate into acetyl-CoA. All three genes have been associated with autism spectrum disorder, suggesting that aberrant cytosolic-to-endoplasmic reticulum flux of acetyl-CoA can be a mechanistic driver for the development of autism spectrum disorder. We therefore generated a SLC25A1 neuron transgenic mouse with overexpression specifically in the forebrain neurons. The mice displayed autistic-like behaviours with a jumping stereotypy. They exhibited increased steady-state levels of citrate and acetyl-CoA, disrupted white matter integrity with activated microglia and altered synaptic plasticity and morphology. Finally, quantitative proteomic and acetyl-proteomic analyses revealed differential adaptations in the hippocampus and cortex. Overall, our study reinforces the connection between aberrant cytosolic-to-endoplasmic reticulum acetyl-CoA flux and the development of an autistic-like phenotype.

Delirium is associated with electroencephalogram (EEG) slowing and impairments in connectivity. We hypothesized that delirium would be accompanied by a reduction in the available cortical information (ie, there is less information processing occurring), as measured by a surrogate, Lempil-Ziv Complexity (LZC), a measure of time-domain complexity. Two ongoing perioperative cohort studies (NCT03124303, NCT02926417) contributed EEG data from 91 patients before and after surgery; 89 participants were used in the analyses. After cleaning and filtering (0.1–50Hz), the perioperative change in LZC and LZC normalized (LZCn) to a phase-shuffled distribution were calculated. The primary outcome was the correlation of within-patient paired changes in delirium severity (Delirium Rating Scale-98 [DRS]) and LZC. Scalp-wide threshold-free cluster enhancement was employed for multiple comparison correction. LZC negatively correlated with DRS in a scalp-wide manner (peak channel r2 = .199, p < .001). This whole brain effect remained for LZCn, though the correlations were weaker (peak channel r2 = .076, p = .010). Delirium diagnosis was similarly associated with decreases in LZC (peak channel p < .001). For LZCn, the topological significance was constrained to the midline posterior regions (peak channel p = .006). We found a negative correlation of LZC in the posterior and temporal regions with monocyte chemoattractant protein-1 (peak channel r2 = .264, p < .001, n = 47) but not for LZCn. Complexity of the EEG signal fades proportionately to delirium severity implying reduced cortical information. Peripheral inflammation, as assessed by monocyte chemoattractant protein-1, does not entirely account for this effect, suggesting that additional pathogenic mechanisms are involved.

Altered predictive coding may underlie the reduced auditory mismatch negativity amplitude observed in patients with dementia. We hypothesized that accumulating dementia-associated pathologies, including amyloid and tau, lead to disturbed predictions of our sensory environment. This would manifest as increased reliance on ‘observed’ sensory information with an associated increase in feedforward, and decrease in feedback, signalling. To test this hypothesis, we studied a cross-sectional cohort of participants who underwent PET imaging and high-density EEG during an oddball paradigm, and used dynamic casual modelling and Bayesian statistics to make inferences about the neuronal architectures (generators) and mechanisms (effective connectivity) underlying the observed auditory-evoked responses. Amyloid-β imaging with [C-11] Pittsburgh Compound-B PET was qualitatively rated using established criteria. Tau-positive PET scans, with [F-18]MK-6240, were defined by an MK-6240 standardized uptake value ratio positivity threshold at 2 standard deviations above the mean of the Amyloid(–) group in the entorhinal cortex (entorhinal MK-6240 standardized uptake value ratio > 1.27). The cross-sectional cohort included a total of 56 participants [9 and 13 participants in the Tau(+) and Amyloid(+) subgroups, respectively: age interquartile range of (73.50–75.34) and (70.5–75.34) years, 56 and 69% females, respectively; 46 and 43 participants in the Tau(−) and Amyloid(−) subgroups, respectively: age interquartile range of (62.72–72.5) and (62.64–72.48) years, 67 and 65% females, respectively]. Mismatch negativity amplitudes were significantly smaller in Tau+ subgroup than Tau− subgroup (cluster statistics corrected for multiple comparisons: P = 0.028). Dynamic causal modelling showed that tau pathology was associated with increased feedforward connectivity and decreased feedback connectivity, with increased excitability of superior temporal gyrus but not inferior frontal regions. This effect on superior temporal gyrus was consistent with the distribution of tau disease on PET in these participants, indicating that the observed differences in mismatch negativity reflect pathological changes evolving in preclinical dementia. Exclusion of participants with diagnosed mild cognitive impairment or dementia did not affect the results. These observational data provide proof of concept that abnormalities in predictive coding may be detected in the preclinical phase of Alzheimer’s disease. This framework also provides a construct to understand how progressive impairments lead to loss of orientation to the sensory world in dementia. Based on our modelling results, plus animal models indicating that Alzheimer’s disease pathologies produce hyperexcitability of higher cortical regions through local disinhibition, mismatch negativity might be a useful monitor to deploy as strategies that target interneuron dysfunction are developed.

BACKGROUND: How conscious experience becomes disconnected from the environment, or disappears, across arousal states is unknown. We sought to identify the neural correlates of sensory disconnection and unconsciousness using a novel serial awakening paradigm. METHODS: Volunteers were recruited for sedation with dexmedetomidine i.v., propofol i.v., or natural sleep with high-density EEG monitoring and serial awakenings to establish whether subjects were in states of disconnected consciousness or unconsciousness in the preceding 20 s. The primary outcome was classification of conscious states by occipital delta power (0.5–4 Hz). Secondary analyses included derivation (dexmedetomidine) and validation (sleep/propofol) studies of EEG signatures of conscious states. RESULTS: Occipital delta power differentiated disconnected and unconscious states for dexmedetomidine (area under the curve [AUC] for receiver operating characteristic 0.605 [95% confidence interval {CI}: 0.516; 0.694]) but not for sleep/propofol (AUC 0.512 [95% CI: 0.380; 0.645]). Distinct source localised signatures of sensory disconnection (AUC 0.999 [95% CI: 0.9954; 1.0000]) and unconsciousness (AUC 0.972 [95% CI: 0.9507; 0.9879]) were identified using support vector machine classification of dexmedetomidine data. These findings generalised to sleep/propofol (validation data set: sensory disconnection [AUC 0.743 {95% CI: 0.6784; 0.8050}]) and unconsciousness (AUC 0.622 [95% CI: 0.5176; 0.7238]). We identified that sensory disconnection was associated with broad spatial and spectral changes. In contrast, unconsciousness was associated with focal decreases in activity in anterior and posterior cingulate cortices. CONCLUSIONS: These findings may enable novel monitors of the anaesthetic state that can distinguish sensory disconnection and unconsciousness, and these may provide novel insights into the biology of arousal. CLINICAL TRIAL REGISTRATION: NCT03284307.

Endoplasmic reticulum-based Nɛ-lysine acetylation serves as an important protein quality control system for the secretory pathway. Dysfunctional endoplasmic reticulum-based acetylation, as caused by overexpression of the acetyl coenzyme A transporter AT-1 in the mouse, results in altered glycoprotein flux through the secretory pathway and an autistic-like phenotype. AT-1 works in concert with SLC25A1, the citrate/malate antiporter in the mitochondria, SLC13A5, the plasma membrane sodium/citrate symporter and ATP citrate lyase, the cytosolic enzyme that converts citrate into acetyl coenzyme A. Here, we report that mice with neuron-specific overexpression of SLC13A5 exhibit autistic-like behaviours with a jumping stereotypy. The mice displayed disrupted white matter integrity and altered synaptic structure and function. Analysis of both the proteome and acetyl-proteome revealed unique adaptations in the hippocampus and cortex, highlighting a metabolic response that likely plays an important role in the SLC13A5 neuron transgenic phenotype. Overall, our results support a mechanistic link between aberrant intracellular citrate/acetyl coenzyme A flux and the development of an autistic-like phenotype.

A common way to study the functional importance of N-methyl-D-aspartate receptors (NMDARs) in hippocampal memory-encoding circuits is by administering NMDAR antagonists. We recently compared the effects of (R,S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), a competitive NMDAR antagonist, on suppression of memory in vivo versus suppression of NMDAR-mediated field EPSPs (fEPSPNMDA) and long-term potentiation (LTP) in vitro. Surprisingly, we found that concentrations that block contextual conditioning in vivo are ineffective at blocking the fEPSPNMDA or LTP in vitro. Here we tested one possible explanation for the mismatch – that the hippocampus is relatively resistant to CPP compared to other brain structures engaged in contextual fear conditioning. We used the context pre-exposure facilitation effect (CPFE) paradigm to isolate the hippocampal component of contextual learning, and in-vivo calcium imaging of place cells and spatial engrams to directly assess hippocampal spatial coding. We found that, by both measures, the active enantiomer (R)-CPP did interfere with hippocampal function at concentrations below those that block fEPSPs or LTP. We conclude that the alternative – that CPP interferes with memory by targeting NMDARs in interneurons rather than pyramidal neurons – is the more likely explanation.

The general anesthetic etomidate, which acts through γ-aminobutyric acid type A (GABA(A)) receptors, impairs the formation of new memories under anesthesia. This study addresses the molecular and cellular mechanisms by which this occurs. Here, using a new line of genetically engineered mice carrying the GABA(A) receptor (GABA(A)R) β2-N265M mutation, we tested the roles of receptors that incorporate GABA(A) receptor β2 versus β3 subunits to suppression of long-term potentiation (LTP), a cellular model of learning and memory. We found that brain slices from β2-N265M mice resisted etomidate suppression of LTP, indicating that the β2-GABA(A)Rs are an essential target in this model. As these receptors are most heavily expressed by interneurons in the hippocampus, this finding supports a role for interneuron modulation in etomidate control of synaptic plasticity. Nevertheless, β2 subunits are also expressed by pyramidal neurons, so they might also contribute. Therefore, using a previously established line of β3-N265M mice, we also examined the contributions of β2- versus β3-GABA(A)Rs to GABA(A,slow) dendritic inhibition, because dendritic inhibition is particularly well suited to controlling synaptic plasticity. We also examined their roles in long-lasting suppression of population activity through feedforward and feedback inhibition. We found that both β2- and β3-GABA(A)Rs contribute to GABA(A,slow) inhibition and that both β2- and β3-GABA(A)Rs contribute to feedback inhibition, whereas only β3-GABA(A)Rs contribute to feedforward inhibition. We conclude that modulation of β2-GABA(A)Rs is essential to etomidate suppression of LTP. Furthermore, to the extent that this occurs through GABA(A)Rs on pyramidal neurons, it is through modulation of feedback inhibition. NEW & NOTEWORTHY Etomidate exerts its anesthetic actions through GABA(A) receptors. However, the mechanism remains unknown. Here, using a hippocampal brain slice model, we show that β2-GABA(A)Rs are essential to this effect. We also show that these receptors contribute to long-lasting dendritic inhibition in feedback but not feedforward inhibition of pyramidal neurons. These findings hold implications for understanding how anesthetics block memory formation and, more generally, how inhibitory circuits control learning and memory.

Drugs that block N-methyl- d -aspartate receptors (NMDARs) suppress hippocampus-dependent memory formation; they also block long-term potentiation (LTP), a cellular model of learning and memory. However, the fractional block that is required to achieve these effects is unknown. Here, we measured the dose-dependent suppression of contextual memory in vivo by systemic administration of the competitive antagonist (R,S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP); in parallel, we measured the concentration-dependent block by CPP of NMDAR-mediated synapses and LTP of excitatory synapses in hippocampal brain slices in vitro. We found that the dose of CPP that suppresses contextual memory in vivo (EC50 = 2.3 mg/kg) corresponds to a free concentration of 53 nM. Surprisingly, applying this concentration of CPP to hippocampal brain slices had no effect on the NMDAR component of evoked field excitatory postsynaptic potentials (fEPSPNMDA), or on LTP. Rather, the IC50 for blocking the fEPSPNMDA was 434 nM, and for blocking LTP was 361 nM – both nearly an order of magnitude higher. We conclude that memory impairment produced by systemically administered CPP is not due primarily to its blockade of NMDARs on hippocampal pyramidal neurons. Rather, systemic CPP suppresses memory formation by actions elsewhere in the memory-encoding circuitry.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play a key role in the regulation of reproductive function. In this study, we sought an efficient method for generating GnRH neurons from human embryonic and induced pluripotent stem cells (hESC and hiPSC, respectively). First, we found that exposure of primitive neuroepithelial cells, rather than neuroprogenitor cells, to fibroblast growth factor 8 (FGF8), was more effective in generating GnRH neurons. Second, addition of kisspeptin to FGF8 further increased the efficiency rates of GnRH neurogeneration. Third, we generated a fluorescent marker mCherry labeled human embryonic GnRH cell line (mCh-hESC) using a CRISPR-Cas9 targeting approach. Fourth, we examined physiological characteristics of GnRH (mCh-hESC) neurons: similar to GnRH neurons in vivo, they released the GnRH peptide in a pulsatile manner at ~60 min intervals; GnRH release increased in response to high potassium, kisspeptin, estradiol, and neurokinin B challenges; and injection of depolarizing current induced action potentials. Finally, we characterized developmental changes in transcriptomes of GnRH neurons using hESC, hiPSC, and mCh-hESC. The developmental pattern of transcriptomes was remarkably similar among the 3 cell lines. Collectively, human stem cell–derived GnRH neurons will be an important tool for establishing disease models to understand diseases, such as idiopathic hypothalamic hypogonadism, and testing contraceptive drugs.

Purpose The purpose of this paper is to evaluate multinational enterprises’ (MNEs) performance and impact in contexts beyond their own internal objectives. Design/methodology/approach Based on an “eclectic” paradigm and the range of motivations, the framework is designed around three layers of evaluation: “efficiency” as static optimisation, “growth and development” as the dynamics of change on a purely economic view; political/economic sphere in terms of the outcomes of “distribution”, and “sovereignty” on the more purely political concern of how MNEs may undermine countries’ policy independence. Findings MNE and national economies have to use current sources of competitiveness efficiently while addressing the necessity to reinforce and refocus them through time. Within these broad agendas, significant interactions and outcomes reflect a range of contingencies conditioned by both MNE objectives and hosts’ competitive status. Originality/value The paper concerns economics and MNE role in globalisation. The paper defines a framework of four generic evaluative issues of MNE performance, which subsume a wider range of important but more niche concerns.

Winter cover crops (CCs) provide soil conservation benefits for strip-tillage tobacco producers, but soil-residual herbicides may interfere with their establishment and growth. Tobacco is planted later than many agronomic crops, but growers often terminate CCs early to minimize CC residue at planting, and this may reduce weed suppression potential. We examined residual herbicide effects on CCs across two seasons and the potential for CC-based weed suppression within strip-tilled tobacco. Mixtures of wheat plus crimson clover and cereal rye plus crimson clover were examined, with a no-CC control. Herbicides included two rates of PRE sulfentrazone (177 or 354 g ai ha–1) plus carfentrazone (20 or 40 g ai ha–1); the higher rate was also followed by POST clomazone (840 g ai ha–1) or mixed with PRE pendimethalin (1,400 g ai ha–1). Controls with no weed management and hand weeding were also included. CC density and biomass were not reduced by weed management (WM) treatments with residual herbicides. However, CCs did not reduce density of annual grasses, small-seeded broadleaves, or perennials in the tilled in-row or untilled between-row zones. Cereal rye plus crimson clover resulted in lower weed biomass at tobacco harvest in the untilled between-row zone in 2017. WM effects were variable between the years, weed groups, and zones. Adding clomazone or pendimethalin was more consistent for reducing weed density and biomass compared to the low rate of sulfentrazone plus carfentrazone. Tobacco yield was unaffected by CCs in 2017 but lower in some WM treatments in 2018. In this study, tobacco herbicides did not interfere with wheat, cereal rye, or crimson clover establishment, but additional research should determine if these results apply to other environments and soil types. However, when these CC species were terminated 5 to 6 wk before transplanting, they did not consistently contribute to weed control.

High(er) throughput toxicokinetics (HTTK) encompasses in vitro measures of key determinants of chemical toxicokinetics and reverse dosimetry approaches for in vitro-in vivo extrapolation (IVIVE). With HTTK, the bioactivity identified by any in vitro assay can be converted to human equivalent doses and compared with chemical intake estimates. Biological variability in HTTK has been previously considered, but the relative impact of measurement uncertainty has not. Bayesian methods were developed to provide chemical-specific uncertainty estimates for 2 in vitro toxicokinetic parameters: unbound fraction in plasma (fup) and intrinsic hepatic clearance (Clint). New experimental measurements of fup and Clint are reported for 418 and 467 chemicals, respectively. These data raise the HTTK chemical coverage of the ToxCast Phase I and II libraries to 57%. Although the standard protocol for Clint was followed, a revised protocol for fup measured unbound chemical at 10%, 30%, and 100% of physiologic plasma protein concentrations, allowing estimation of protein binding affinity. This protocol reduced the occurrence of chemicals with fup too low to measure from 44% to 9.1%. Uncertainty in fup was also reduced, with the median coefficient of variation dropping from 0.4 to 0.1. Monte Carlo simulation was used to propagate both measurement uncertainty and biological variability into IVIVE. The uncertainty propagation techniques used here also allow incorporation of other sources of uncertainty such as in silico predictors of HTTK parameters. These methods have the potential to inform risk-based prioritization based on the relationship between in vitro bioactivities and exposures.

Weed management in tobacco (Nicotiana tabacumL.) is accomplished primarily with soil-residual herbicides, cultivation, and hand removal. Management practices that reduce weed emergence, like reduced tillage and cover crop mulches, may improve weed management efficacy. Depending on cover-cropping goals, growers face trade-offs in species selection and management priorities—producing weed-suppressive mulches may lead to transplanting difficulties and soil-residual herbicide interception. Managing more complex cover crop mixtures may result in different challenges. We established on-farm trials across 4 site-years to study impacts of cover crop composition [wheat (Triticum aestivumL.) monoculture or mixture], termination treatment (early or late chemical termination or removing aboveground biomass), and soil-residual herbicides on weed density and biomass. The cover crop mixture contained cereal rye (Secale cerealeL.), crimson clover (Trifolium incarnatumL.), and hairy vetch (Vicia villosaRoth.), with canola (Brassica napusL.) at 1 site-year. The mixture typically produced more biomass than monoculture wheat, although composition had few impacts on weed density or biomass. With residual herbicides, termination treatment had few impacts on weed density, suggesting that residues did not adversely affect herbicide efficacy. Without residual herbicides, early-season weed density was often higher following the late-terminated cover crop compared with other termination treatments, though midseason weed density was typically lower. When termination treatment affected final weed biomass, it was lower following late termination, with one exception—crop establishment was reduced at 1 site-year, leading to reduced weed–crop competition and greater weed biomass. Our results suggest that growers can use mixtures and, if well-timed to a rainfall event for incorporation, still effectively use soil-residual herbicides to maintain adequate weed control in tobacco regardless of how the cover crop is managed. Later termination, resulting in more residue, may lead to less weed biomass accumulation in the absence of herbicide use.

BACKGROUND: It is unclear how preoperative neurodegeneration and postoperative changes in EEG delta power relate to postoperative delirium severity. We sought to understand the relative relationships between neurodegeneration and delta power as predictors of delirium severity. METHODS: We undertook a prospective cohort study of high-risk surgical patients (>65 yr old) to identify predictors of peak delirium severity (Delirium Rating Scale-98) with twice-daily delirium assessments (NCT 03124303). Participants (n=86) underwent preoperative MRI; 54 had both an MRI and a postoperative EEG. Cortical thickness was calculated from the MRI and delta power from the EEG. RESULTS: In a linear regression model, the interaction between delirium status and preoperative mean cortical thickness (suggesting neurodegeneration) across the entire cortex was a significant predictor of delirium severity (P

Delirium is associated with cognitive decline and subsequent dementia, and rises in plasma total tau (tTau) and neurofilament light (NfL), providing links to Amyloid-Tau-Neurodegeneration (ATN) pathophysiology. We investigated whether changes in delirium severity after surgery correlated with changes in cerebrospinal fluid (CSF) ATN biomarkers. Thirty-two thoracic vascular surgical patients were recruited into a prospective biomarker cohort study with assessment of delirium severity and incidence (NCT02926417). CSF (n = 54) and plasma (n = 118) samples were sent for biomarker analysis for tTau, phosphorylated tau-181 (pTau) (plasma n = 53), NfL, and amyloid-β 42/40 ratio (Ab42/40-ratio). The primary outcome was the correlation of preoperative to postoperative change in ATN biomarkers with the highest postoperative Delirium Rating Scale-98 score. CSF and plasma biomarkers all increased postoperatively (all p < .05, n = 13 paired preoperative–postoperative samples). Delirium severity was associated with peak changes in CSF tTau (p = .007, r = .710) and pTau (p = .01, r = .667) but not NfL (p = .09, ρ = .491) or Ab42/40-ratio (p = .18, ρ = .394). Sensitivity analysis with exclusion of participants with putative spinal cord ischemia shifted the NfL result to significance (p < .001, ρ = .847). Our data show that changes in tau and biomarkers of neurodegeneration in the CSF are associated with delirium severity. These data should be considered hypothesis-generating and future studies should identify if these changes are robust to confounding.

Background: Mice carrying the GABAA receptor &beta, 3(N265M) point mutation, which renders receptors incorporating &beta, 3-subunits insensitive to many general anesthetics, have been used experimentally to link modulation of different receptor subtypes to distinct behavioral endpoints. Remarkably, however, the effect of the mutation on the susceptibility to modulation by isoflurane (a standard reference agent for inhalational vapors) has never been tested directly. Therefore, we compared the modulation by isoflurane of expressed &alpha, 5&beta, 3(N265M)&gamma, 2L receptors with their wild type counterparts. Methods: Using whole-cell electrophysiological recording and rapid solution exchange techniques, we tested the effects of isoflurane at concentrations ranging from 80 &mu, M to 320 &mu, M on currents activated by 1 &mu, M GABA. We measured drug modulation of wild-type &alpha, 3&gamma, 2L GABAA receptors and their counterparts harboring the &beta, 3(N265M) mutation. Results: Currents elicited by GABA were enhanced two- to four-fold by isoflurane, in a concentration-dependent manner. Under the same conditions, receptors incorporating the &beta, 3(N265M) mutation were enhanced by approximately 1.5- to two-fold, i.e., modulation by isoflurane was attenuated by approximately one-half. Direct activation by isoflurane was also present in mutant receptors but also attenuated. Conclusions: In contrast to the complete insensitivity of &beta, 3(N265M) mutant receptors to etomidate and propofol, the mutation has only a partial effect on receptor modulation by isoflurane. Therefore, the persistence of isoflurane effects in mutant mice does not exclude a possible contribution of &beta, 3-GABAA receptors.

The toxicokinetic (TK) parameters fraction of the chemical unbound to plasma proteins and metabolic clearance are critical for relating exposure and internal dose when building in vitro-based risk assessment models. However, experimental toxicokinetic studies have only been carried out on limited chemicals of environmental interest (~1000 chemicals with TK data relative to tens of thousands of chemicals of interest). This work evaluated the utility of chemical structure information to predict TK parameters in silico; development of cluster-based read-across and quantitative structure-activity relationship models of fraction unbound or fub (regression) and intrinsic clearance or Cl(int) (classification and regression) using a dataset of 1487 chemicals; utilization of predicted TK parameters to estimate uncertainty in steady-state plasma concentration (C(ss)); and subsequent in vitro–in vivo extrapolation analyses to derive bioactivity-exposure ratio (BER) plot to compare human oral equivalent doses and exposure predictions using androgen and estrogen receptor activity data for 233 chemicals as an example dataset. The results demonstrate that fub is structurally more predictable than Cl(int). The model with the highest observed performance for fub had an external test set RMSE/σ=0.62 and R(2)=0.61, for Cl(int) classification had an external test set accuracy = 65.9%, and for intrinsic clearance regression had an external test set RMSE/σ=0.90 and R(2)=0.20. This relatively low performance is in part due to the large uncertainty in the underlying Cl(int) data. We show that C(ss) is relatively insensitive to uncertainty in Cl(int). The models were benchmarked against the ADMET Predictor software. Finally, the BER analysis allowed identification of 14 out of 136 chemicals for further risk assessment demonstrating the utility of these models in aiding risk-based chemical prioritization.