A Hybrid Orbitrap-Nanoelectromechanical Systems Approach for the Analysis of Individual, Intact Proteins in Real Time.

Nanoelectromechanical systems (NEMS)-based mass spectrometry (MS) is an emerging technique that enables determination of the mass of individual adsorbed particles by driving nanomechanical devices at resonance and monitoring the real-time changes in their resonance frequencies induced by each single molecule adsorption event. We incorporate NEMS into an Orbitrap mass spectrometer and report our progress towards leveraging the single-molecule capabilities of the NEMS to enhance the dynamic range of conventional MS instrumentation and to offer new capabilities for performing deep proteomic analysis of clinically relevant samples. We use the hybrid instrument to deliver E. coli GroEL molecules (801 kDa) to the NEMS devices in their native, intact state. Custom ion optics are used to focus the beam down to 40 µm diameter with a maximum flux of 25 molecules/second. The mass spectrum obtained with NEMS-MS shows good agreement with the known mass of GroEL.

Nanomechanical mass measurements through feature-based time series clustering.

Recent years have seen explosive growth in miniaturized sensors that can continuously monitor a wide variety of processes, with applications in healthcare, manufacturing, and environmental sensing. The time series generated by these sensors often involves abrupt jumps in the detected signal. One such application uses nanoelectromechanical systems (NEMS) for mass spectrometry, where analyte adsorption produces a quick but finite-time jump in the resonance frequencies of the sensor eigenmodes. This finite-time response can lead to ambiguity in the detection of adsorption events, particularly in high event-rate mass adsorption. Here, we develop a computational algorithm that robustly eliminates this often-encountered ambiguity. A moving-window statistical test together with a feature-based clustering algorithm is proposed to automate the identification of single-event jumps. We validate the method using numerical simulations and demonstrate its application in practice using time-series data that are experimentally generated by molecules adsorbing onto NEMS sensors at a high event rate. This computational algorithm enables new applications, including high-throughput, single-molecule proteomics.

Comparison of the ADNEX and ROMA risk prediction models for the diagnosis of ovarian cancer: a multicentre external validation in patients who underwent surgery.

BACKGROUND: Several diagnostic prediction models to help clinicians discriminate between benign and malignant adnexal masses are available. This study is a head-to-head comparison of the performance of the Assessment of Different NEoplasias in the adneXa (ADNEX) model with that of the Risk of Ovarian Malignancy Algorithm (ROMA). METHODS: This is a retrospective study based on prospectively included consecutive women with an adnexal tumour scheduled for surgery at five oncology centres and one non-oncology centre in four countries between 2015 and 2019. The reference standard was histology. Model performance for ADNEX and ROMA was evaluated regarding discrimination, calibration, and clinical utility. RESULTS: The primary analysis included 894 patients, of whom 434 (49%) had a malignant tumour. The area under the receiver operating characteristic curve (AUC) was 0.92 (95% CI 0.88-0.95) for ADNEX with CA125, 0.90 (0.84-0.94) for ADNEX without CA125, and 0.85 (0.80-0.89) for ROMA. ROMA, and to a lesser extent ADNEX, underestimated the risk of malignancy. Clinical utility was highest for ADNEX. ROMA had no clinical utility at decision thresholds <27%. CONCLUSIONS: ADNEX had better ability to discriminate between benign and malignant adnexal tumours and higher clinical utility than ROMA. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov NCT01698632 and NCT02847832.

Cortical reactivation of spatial and non-spatial features coordinates with hippocampus to form a memory dialogue.

Episodic memories comprise diverse attributes of experience distributed across neocortical areas. The hippocampus is integral to rapidly binding these diffuse representations, as they occur, to be later reinstated. However, the nature of the information exchanged during this hippocampal-cortical dialogue remains poorly understood. A recent study has shown that the secondary motor cortex carries two types of representations: place cell-like activity, which were impaired by hippocampal lesions, and responses tied to visuo-tactile cues, which became more pronounced following hippocampal lesions. Using two-photon Ca2+ imaging to record neuronal activities in the secondary motor cortex of male Thy1-GCaMP6s mice, we assessed the cortical retrieval of spatial and non-spatial attributes from previous explorations in a virtual environment. We show that, following navigation, spontaneous resting state reactivations convey varying degrees of spatial (trajectory sequences) and non-spatial (visuo-tactile attributes) information, while reactivations of non-spatial attributes tend to precede reactivations of spatial representations surrounding hippocampal sharp-wave ripples.

A Multi-task Platform for Profiling Cognitive and Motivational Constructs in Humans and Nonhuman Primates.

Background: Understanding the neurobiological substrates of psychiatric disorders requires comprehensive evaluations of cognitive and motivational functions in preclinical research settings. The translational validity of such evaluations will be supported by (1) tasks with high construct validity that are engaging and easy to teach to human and nonhuman participants, (2) software that enables efficient switching between multiple tasks in single sessions, (3) software that supports tasks across a broad range of physical experimental setups, and (4) by platform architectures that are easily extendable and customizable to encourage future optimization and development. New Method: We describe the Multi-task Universal Suite for Experiments (M-USE), a software platform designed to meet these requirements. It leverages the Unity video game engine and C# programming language to (1) support immersive and engaging tasks for humans and nonhuman primates, (2) allow experimenters or participants to switch between multiple tasks within-session, (3) generate builds that function across computers, tablets, and websites, and (4) is freely available online with documentation and tutorials for users and developers. M-USE includes a task library with seven pre-existing tasks assessing cognitive and motivational constructs of perception, attention, working memory, cognitive flexibility, motivational and affective self-control, relational long-term memory, and visuo-spatial problem solving. Results: M-USE was used to test NHPs on up to six tasks per session, all available as part of the Task Library, and to extract performance metrics for all major cognitive and motivational constructs spanning the Research Domain Criteria (RDoC) of the National Institutes of Mental Health. Comparison with Existing Methods: Other experiment design and control systems exist, but do not provide the full range of features available in M-USE, including a pre-existing task library for cross-species assessments; the ability to switch seamlessly between tasks in individual sessions; cross-platform build capabilities; license-free availability; and its leveraging of video-engine capabilities used to gamify tasks. Conclusions: The new multi-task platform facilitates cross-species translational research for understanding the neurobiological substrates of higher cognitive and motivational functions.

M1-selective muscarinic allosteric modulation enhances cognitive flexibility and effective salience in nonhuman primates.

Acetylcholine (ACh) in cortical neural circuits mediates how selective attention is sustained in the presence of distractors and how flexible cognition adjusts to changing task demands. The cognitive domains of attention and cognitive flexibility might be differentially supported by the M1 muscarinic acetylcholine receptor (mAChR) subtype. Understanding how M1 mAChR mechanisms support these cognitive subdomains is of highest importance for advancing novel drug treatments for conditions with altered attention and reduced cognitive control including Alzheimer's disease or schizophrenia. Here, we tested this question by assessing how the subtype-selective M1 mAChR positive allosteric modulator (PAM) VU0453595 affects visual search and flexible reward learning in nonhuman primates. We found that allosteric potentiation of M1 mAChRs enhanced flexible learning performance by improving extradimensional set shifting, reducing latent inhibition from previously experienced distractors and reducing response perseveration in the absence of adverse side effects. These procognitive effects occurred in the absence of apparent changes of attentional performance during visual search. In contrast, nonselective ACh modulation using the acetylcholinesterase inhibitor (AChEI) donepezil improved attention during visual search at doses that did not alter cognitive flexibility and that already triggered gastrointestinal cholinergic side effects. These findings illustrate that M1 mAChR positive allosteric modulation enhances cognitive flexibility without affecting attentional filtering of distraction, consistent with M1 activity boosting the effective salience of relevant over irrelevant objects specifically during learning. These results suggest that M1 PAMs are versatile compounds for enhancing cognitive flexibility in disorders spanning schizophrenia and Alzheimer's diseases.

Consolidation of cellular memory representations in superficial neocortex.

Systems-level memory consolidation, a key concept in memory research, involves the conversion of memories that depend on the hippocampus for their formation into efficient hippocampus-independent forms, presumably encoded by cortico-cortical connections. Yet, little is understood about the nature of consolidated neural codes at the cellular ensemble level. Mice require an intact hippocampus for "virtual" spatial learning and to develop neocortical representations of the corresponding experiences. We find that, whereas a novel virtual environment is neither learned nor represented in superficial cortex following severe damage to hippocampus, pre-operatively learned memories and their corresponding sparse and widespread neural ensemble representations in cortical layers II-III are preserved, a sine qua non of memory consolidation. These findings provide a new window for future study of the cellular mechanisms of memory consolidation.

Dose-Dependent Dissociation of Pro-cognitive Effects of Donepezil on Attention and Cognitive Flexibility in Rhesus Monkeys.

Background: Donepezil exerts pro-cognitive effects by nonselectively enhancing acetylcholine (ACh) across multiple brain systems. Two brain systems that mediate pro-cognitive effects of attentional control and cognitive flexibility are the prefrontal cortex and the anterior striatum, which have different pharmacokinetic sensitivities to ACh modulation. We speculated that these area-specific ACh profiles lead to distinct optimal dose ranges for donepezil to enhance the cognitive domains of attention and flexible learning. Methods: To test for dose-specific effects of donepezil on different cognitive domains, we devised a multitask paradigm for nonhuman primates that assessed attention and cognitive flexibility. The nonhuman primates received either vehicle or variable doses of donepezil before task performance. We measured intracerebral donepezil and its strength in preventing the breakdown of ACh within the prefrontal cortex and anterior striatum using solid phase microextraction neurochemistry. Results: The highest administered donepezil dose improved attention and made the subjects more robust against distractor interference, but it did not improve flexible learning. In contrast, only a lower dose range of donepezil improved flexible learning and reduced perseveration, but without distractor-dependent attentional improvement. Neurochemical measurements confirmed a dose-dependent increase of extracellular donepezil and decreases in choline within the prefrontal cortex and the striatum. Conclusions: The donepezil dose for maximally improving attention differed from the dose range that enhanced cognitive flexibility despite the availability of the drug in two major brain systems supporting these functions. These results suggest that in our cohort of adult monkeys, donepezil traded improvements in attention for improvements in cognitive flexibility at a given dose range.

A Kiosk Station for the Assessment of Multiple Cognitive Domains and Cognitive Enrichment of Monkeys.

Nonhuman primates (NHP's) are self-motivated to perform cognitive tasks on touchscreens in their animal housing setting. To leverage this ability, fully integrated hardware and software solutions are needed that work within housing and husbandry routines while also spanning cognitive task constructs of the Research Domain Criteria (RDoC). Here, we detail such an integrated robust hardware and software solution for running cognitive tasks in cage-housed NHP's with a cage-mounted Kiosk Station (KS-1). KS-1 consists of a frame for mounting flexibly on housing cages, a touchscreen animal interface with mounts for receptables, reward pumps, and cameras, and a compact computer cabinet with an interface for controlling behavior. Behavioral control is achieved with a Unity3D program that is virtual-reality capable, allowing semi-naturalistic visual tasks to assess multiple cognitive domains.KS-1 is fully integrated into the regular housing routines of monkeys. A single person can operate multiple KS-1's. Monkeys engage with KS-1 at high motivation and cognitive performance levels at high intra-individual consistency. KS-1 is optimized for flexible mounting onto standard apartment cage systems and provides a new design variation complementing existing cage-mounted touchscreen systems. KS-1 has a robust animal interface with options for gaze/reach monitoring. It has an integrated user interface for controlling multiple cognitive tasks using a common naturalistic object space designed to enhance task engagement. All custom KS-1 components are open-sourced.In summary, KS-1 is a versatile new tool for cognitive profiling and cognitive enrichment of cage-housed monkeys. It reliably measures multiple cognitive domains which promises to advance our understanding of animal cognition, inter-individual differences, and underlying neurobiology in refined, ethologically meaningful behavioral foraging contexts.

Spatial Information Encoding across Multiple Neocortical Regions Depends on an Intact Hippocampus.

There has been considerable research showing populations of neurons encoding for different aspects of space in the brain. Recently, several studies using two-photon calcium imaging and virtual navigation have identified "spatially" modulated neurons in the posterior cortex. We enquire here whether the presence of such spatial representations may be a cortex-wide phenomenon and, if so, whether these representations can be organized in the absence of the hippocampus. To this end, we imaged the dorsal cortex of mice running on a treadmill populated with tactile cues. A high percentage (40-80%) of the detected neurons exhibited sparse, spatially localized activity, with activity fields uniformly localized over the track. The development of this location specificity was impaired by hippocampal damage. Thus, there is a substantial population of neurons distributed widely over the cortex that collectively form a continuous representation of the explored environment, and hippocampal outflow is necessary to organize this phenomenon.SIGNIFICANCE STATEMENT Increasing evidence points to the role of the neocortex in encoding spatial information. Whether this feature is linked to hippocampal functions is largely unknown. Here, we systematically surveyed multiple regions in the dorsal cortex of the same animal for the presence of signals encoding for spatial position. We described populations of cortical neurons expressing sequential patterns of activity localized in space in primary, secondary, and associational areas. Furthermore, we showed that the formation of these spatial representations was impacted by hippocampal lesion. Our results indicate that hippocampal inputs are necessary to maintain a precise cortical representation of space.

Coordinated activities of retrosplenial ensembles during resting-state encode spatial landmarks.

The brain likely uses offline periods to consolidate recent memories. One hypothesis holds that the hippocampal output provides a unique, global linking or 'index' code for each memory, and that this code is stored in the cortex in association with locally encoded attributes of each memory. Activation of the index code is hypothesized to evoke coordinated memory trace reactivation thus facilitating consolidation. Retrosplenial cortex (RSC) is a major recipient of hippocampal outflow and we have described populations of neurons there with sparse and orthogonal coding characteristics that resemble hippocampal 'place' cells, and whose expression depends on an intact hippocampus. Using two-photon Ca2+ imaging, we recorded ensembles of neurons in the RSC during periods of immobility before and after active running on a familiar linear treadmill track. Synchronous bursting of distinct groups of neurons occurred during rest both prior to and after running. In the second rest epoch, these patterns were associated with the locations of tactile landmarks and reward. Complementing established views on the functions of the RSC, our findings indicate that the structure is involved with processing landmark information during rest. This article is part of the Theo Murphy meeting issue 'Memory reactivation: replaying events past, present and future'.

Hippocampus-dependent emergence of spatial sequence coding in retrosplenial cortex.

Retrosplenial cortex (RSC) is involved in visuospatial integration and spatial learning, and RSC neurons exhibit discrete, place cell-like sequential activity that resembles the population code of space in hippocampus. To investigate the origins and population dynamics of this activity, we combined longitudinal cellular calcium imaging of dysgranular RSC neurons in mice with excitotoxic hippocampal lesions. We tracked the emergence and stability of RSC spatial activity over consecutive imaging sessions. Overall, spatial activity in RSC was experience-dependent, emerging gradually over time, but, as seen in the hippocampus, the spatial code changed dynamically across days. Bilateral but not unilateral hippocampal lesions impeded the development of spatial activity in RSC. Thus, the emergence of spatial activity in RSC, a major recipient of hippocampal information, depends critically on an intact hippocampus; the indirect connections between the dysgranular RSC and the hippocampus further indicate that hippocampus may exert such influences polysynaptically within neocortex.

Mass Spectrometry Using Nanomechanical Systems: Beyond the Point-Mass Approximation.

The mass measurement of single molecules, in real time, is performed routinely using resonant nanomechanical devices. This approach models the molecules as point particles. A recent development now allows the spatial extent (and, indeed, image) of the adsorbate to be characterized using multimode measurements ( Hanay , M. S. , Nature Nanotechnol. , 10 , 2015 , pp 339 - 344 ). This "inertial imaging" capability is achieved through virtual re-engineering of the resonator's vibrating modes, by linear superposition of their measured frequency shifts. Here, we present a complementary and simplified methodology for the analysis of these inertial imaging measurements that exhibits similar performance while streamlining implementation. This development, together with the software that we provide, enables the broad implementation of inertial imaging that opens the door to a range of novel characterization studies of nanoscale adsorbates.

Involvement of fast-spiking cells in ictal sequences during spontaneous seizures in rats with chronic temporal lobe epilepsy.

See Lenck-Santini (doi:10.1093/awx205) for a scientific commentary on this article. Epileptic seizures represent altered neuronal network dynamics, but the temporal evolution and cellular substrates of the neuronal activity patterns associated with spontaneous seizures are not fully understood. We used simultaneous recordings from multiple neurons in the hippocampus and neocortex of rats with chronic temporal lobe epilepsy to demonstrate that subsets of cells discharge in a highly stereotypical sequential pattern during ictal events, and that these stereotypical patterns were reproducible across consecutive seizures. In contrast to the canonical view that principal cell discharges dominate ictal events, the ictal sequences were predominantly composed of fast-spiking, putative inhibitory neurons, which displayed unusually strong coupling to local field potential even before seizures. The temporal evolution of activity was characterized by unique dynamics where the most correlated neuronal pairs before seizure onset displayed the largest increases in correlation strength during the seizures. These results demonstrate the selective involvement of fast spiking interneurons in structured temporal sequences during spontaneous ictal events in hippocampal and neocortical circuits in experimental models of chronic temporal lobe epilepsy.

A two years longitudinal study of a transgenic Huntington disease monkey.

BACKGROUND: A two-year longitudinal study composed of morphometric MRI measures and cognitive behavioral evaluation was performed on a transgenic Huntington's disease (HD) monkey. rHD1, a transgenic HD monkey expressing exon 1 of the human gene encoding huntingtin (HTT) with 29 CAG repeats regulated by a human polyubiquitin C promoter was used together with four age-matched wild-type control monkeys. This is the first study on a primate model of human HD based on longitudinal clinical measurements. RESULTS: Changes in striatal and hippocampal volumes in rHD1 were observed with progressive impairment in motor functions and cognitive decline, including deficits in learning stimulus-reward associations, recognition memory and spatial memory. The results demonstrate a progressive cognitive decline and morphometric changes in the striatum and hippocampus in a transgenic HD monkey. CONCLUSIONS: This is the first study on a primate model of human HD based on longitudinal clinical measurements. While this study is based a single HD monkey, an ongoing longitudinal study with additional HD monkeys will be important for the confirmation of our findings. A nonhuman primate model of HD could complement other animal models of HD to better understand the pathogenesis of HD and future development of diagnostics and therapeutics through longitudinal assessment.

Longitudinal transcriptomic dysregulation in the peripheral blood of transgenic Huntington's disease monkeys.

BACKGROUND: Huntington's Disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the polyglutamine (polyQ) region of the Huntingtin (HTT) gene. The clinical features of HD are characterized by cognitive, psychological, and motor deficits. Molecular instability, a core component in neurological disease progression, can be comprehensively evaluated through longitudinal transcriptomic profiling. Development of animal models amenable to longitudinal examination enables distinct disease-associated mechanisms to be identified. RESULTS: Here we report the first longitudinal study of transgenic monkeys with genomic integration of various lengths of the human HTT gene and a range of polyQ repeats. With this unique group of transgenic HD nonhuman primates (HD monkeys), we profiled over 47,000 transcripts from peripheral blood collected over a 2 year timespan from HD monkeys and age-matched wild-type control monkeys. CONCLUSIONS: Messenger RNAs with expression patterns which diverged with disease progression in the HD monkeys considerably facilitated our search for transcripts with diagnostic or therapeutic potential in the blood of human HD patients, opening up a new avenue for clinical investigation.

Reprogramming Huntington monkey skin cells into pluripotent stem cells.

Induced pluripotent Huntington's disease monkey stem cells (rHD-iPSCs) were established by the overexpression of rhesus macaque transcription factors (Oct4, Sox2, and Klf4) in transgenic Huntington's monkey skin fibroblasts. The rHD-iPSCs were pluripotent and capable of differentiating into neuronal cell types in vitro and developed teratoma in immune compromised mice. We also demonstrated the upregulation of endogenous Oct4 and Sox2 after successful reprogramming to pluripotency in rHD-iPSCs, which was not expressed in skin fibroblasts. rHD-iPSCs also developed cellular features comparable to Huntington's disease (HD), including the accumulation of mutant huntingtin (htt) aggregate and the formation of intranuclear inclusions (NIs) paralleling neural differentiation in vitro. Induced pluripotent stem cells from transgenic HD monkeys open a new era of nonhuman primate modeling of human diseases. rHD-iPSCs that develop key HD cellular features and parallel neural differentiation can be a powerful platform for investigating the developmental impact on HD pathogenesis and developing new therapies, which can be evaluated in HD monkeys from whom the rHD-iPSCs were derived.

Circadian clock and output genes are rhythmically expressed in extratesticular ducts and accessory organs of mice.

Circadian clocks regulate multiple rhythms in mammalian tissues. In most organs core clock gene expression is oscillatory, with negative components Per and Cry peaking in antiphase to Bmal1. A notable exception is the testis, where clock genes seem nonrhythmic. Earlier mammalian studies, however, did not examine clock expression patterns in accessory ductal tissue required for sperm maturation and transport. Previous studies in insects demonstrated control of sperm maturation in vas deferens by a local circadian system. Sperm ducts express clock genes and display circadian pH changes controlled by vacuolar-type H(+)-ATPase and carbonic anhydrase (CA-II). It is unknown whether sperm-processing rhythms are conserved beyond insects. To address this question in mice housed in a light-dark environment, we examined temporal patterns of mPer1 and Bmal1 gene expression and protein abundance in epididymis, vas deferens, seminal vesicles, and prostate. Results demonstrate variable tissue-specific patterns of expression of the two genes, with variations in levels of clock proteins and their nucleo-cytoplasmic cycling observed among examined tissues. Strikingly, mPer1 and Bmal1 mRNA and proteins oscillate in antiphase in the prostate, with similar peak-trough patterns as observed in the suprachiasmatic nuclei, the brain's central clock. Genes encoding CA and a V-ATPase subunit, which are rhythmically expressed in sperm ducts of moths, are also rhythmic in some segments of murine sperm ducts. Our data suggest that some sperm duct segments may contain peripheral circadian systems whereas others may express clock genes in a pleiotropic manner.