Large-Scale Screening of Pharmaceutical Compounds to Explore the Application Space of On-Tissue MALDI and MALDI-2 Mass Spectrometry.

The successful application of matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in pharmaceutical research is strongly dependent on the detection of the drug of interest at physiologically relevant concentrations. Here we explored how insufficient sensitivity due to low ionization efficiency and/or the interaction of the drug molecule with the local biochemical environment of the tissue can be mitigated for many compound classes using the recently introduced MALDI-MSI coupled with laser-induced postionization, known as MALDI-2-MSI. Leveraging a MALDI-MSI screen of about 1,200 medicines/drug-like compounds from a broad range of medicinal application areas, we demonstrate a significant improvement in drug detection and the degree of sensitivity uplift by using MALDI-2 versus traditional MALDI. Our evaluation was made under simulated imaging conditions using liver homogenate sections as substrate, onto which the compounds were spotted to mimic biological conditions to the first order. To enable an evaluable detection by both MALDI and MALDI-2 for the majority of employed compounds, we spotted 1 µL of a 10 mM solution using a spotting robot and performed our experiments with a Bruker timsTOF fleX MALDI-2 instrument in both positive and negative ion modes. Specifically, we demonstrate using a large cohort of drug-like compounds that ∼60% of the tested compounds showed a more than 10-fold increase in signal intensity and ∼16% showed a more than 100-fold increase upon use of MALDI-2 postionization. Such increases in sensitivity could help advance pharmaceutical MALDI-MSI applications toward the single-cell level.

Release of biogenic volatile organic compounds and physiological responses of two sub-tropical tree species to smoke derived from forest fire.

Forests emit a large amount of biogenic volatile organic compounds (BVOCs) in response to biotic and abiotic stress. Despite frequent occurrence of large forest fires in recent years, the impact of smoke stress derived from these forest fires on the emission of BVOCs is largely unexplored. Thus, the aims of the study were to quantify the amount and composition of BVOCs released by two sub-tropical tree species, Cunninghamia lanceolata and Schima superba, in response to exposure to smoke. Physiological responses and their relationship with BVOCs were also investigated. The results showed that smoke treatments significantly (p < 0.001) promoted short-term release of BVOCs by C. lanceolata leaves than S. superba; and alkanes, olefins and benzene homologs were identified as major classes of BVOCs. Both C. lanceolata and S. superba seedlings showed significant (p < 0.005) physiological responses after being smoke-stressed where photosynthetic rate remained unaffected, chlorophyll content greatly reduced and Activities of anti-oxidant enzymes and the malondialdehyde content generally increased with the increase in smoke concentration. Activities of anti-oxidant enzymes showed mainly positive correlations with the major BVOCs. In conclusion, the release of BVOCs following smoke stress is species-specific and there exists a link between activities of antioxidant enzymes and BVOCs released. The findings provide insight about management of forest fires in order to control excessive emission of smoke that would trigger increased release of BVOCs.

The order of attentional focus instructions affects how postural control processes compensate for multisensory mismatch: a crossover study.

Directing attention during balance training can have an immediate and lasting impact on a patient's balance and ultimately decrease the risk of future falls. However, it is unclear how attention can best be utilized to improve postural control. The current study uses a 2 × 2 crossover design to investigate the potential impact of receiving multiple verbal instructions during a single session of sensorimotor control testing for balance. Twenty-eight healthy adults were tasked to balance on a rocker board while immersed in virtual reality (VR). The VR created a multisensory mismatch between visual VR motion and body motion. The strength of the relationship between visual motion and body motion was measured to assess visual dependence. Alpha and theta frequency bands in electroencephalography (EEG) recordings were also analyzed to identify potential neural correlates of visual dependence and postural stability. Participants were randomized into two groups: one group was first instructed to keep the board leveled (external focus) and then instructed to keep both feet leveled (internal focus) to help maintain stability. The other group was given these two instructions in reverse order. Analyses focused on time, instruction, and group effects from receiving multiple instructions. Results revealed that when participants are given external focus first, and internal focus second, they are more likely to demonstrate lower visual dependence and better postural stability throughout the entire session than participants given internal focus first and external focus second. However, channel-level EEG analyses did not reveal differences between the groups. Current findings suggest that the order of attentional focus instructions may influence how the postural control system resolves sensory incongruence during a single testing session.

Anticipation across modalities in children and adults: Relating anticipatory alpha rhythm lateralization, reaction time, and executive function.

The development of the ability to anticipate-as manifested by preparatory actions and neural activation related to the expectation of an upcoming stimulus-may play a key role in the ontogeny of cognitive skills more broadly. This preregistered study examined anticipatory brain potentials and behavioral responses (reaction time; RT) to anticipated target stimuli in relation to individual differences in the ability to use goals to direct action (as indexed by measures of executive function; EF). A cross-sectional investigation was conducted in 40 adults (aged 18-25 years) and 40 children (aged 6-8 years) to examine the association of changes in the amplitude of modality-specific alpha-range rhythms in the electroencephalogram (EEG) during anticipation of lateralized visual, tactile, or auditory stimuli with inter- and intraindividual variation in RT and EF. Children and adults exhibited contralateral anticipatory reductions in the mu rhythm and the visual alpha rhythm for tactile and visual anticipation, respectively, indicating modality and spatially specific attention allocation. Variability in within-subject anticipatory alpha lateralization (the difference between contralateral and ipsilateral alpha power) was related to single-trial RT. This relation was more prominent in adults than in children, and was not apparent for auditory stimuli. Multilevel models indicated that interindividual differences in anticipatory mu rhythm lateralization contributed to the significant association with variability in EF, but this was not the case for visual or auditory alpha rhythms. Exploratory microstate analyses were undertaken to cluster global field power (GFP) into a distribution-free temporal analysis examining developmental differences across samples and in relation to RT and EF. Anticipation is suggested as a developmental bridge construct connecting neuroscience, behavior, and cognition, with anticipatory EEG oscillations being discussed as quantifiable and potentially malleable indicators of stimulus prediction.

Genes, genomes, and developmental process.

The view advanced by Madole & Harden falls back on the dogma of a gene as a DNA sequence that codes for a fixed product with an invariant function regardless of temporal and spatial contexts. This outdated perspective entrenches the metaphor of genes as static units of information and glosses over developmental complexities.

Mass spectrometry detection of inhaled drug in distal fibrotic lung.

BACKGROUND: Currently the only available therapies for fibrotic Interstitial Lung Disease are administered systemically, often causing significant side effects. Inhaled therapy could avoid these but to date there is no evidence that drug can be effectively delivered to distal, fibrosed lung. We set out to combine mass spectrometry and histopathology with rapid sample acquisition using transbronchial cryobiopsy to determine whether an inhaled drug can be delivered to fibrotic, distal lung parenchyma in participants with Interstitial Lung Disease. METHODS: Patients with radiologically and multidisciplinary team confirmed fibrotic Interstitial Lung Disease were eligible for this study. Transbronchial cryobiopsies and endobronchial biopsies were taken from five participants, with Interstitial Lung Disease, within 70 min of administration of a single dose of nebulised ipratropium bromide. Thin tissue cryosections were analysed by Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry imaging and correlated with histopathology. The remainder of the cryobiopsies were homogenised and analysed by Liquid Chromatography-tandem Mass Spectrometry. RESULTS: Drug was detected in proximal and distal lung samples from all participants. Fibrotic regions were identified in research samples of four of the five participants. Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry imaging showed co-location of ipratropium with fibrotic regions in samples from three participants. CONCLUSIONS: In this proof of concept study, using mass spectrometry, we demonstrate for the first-time that an inhaled drug can deposit in distal fibrotic lung parenchyma in patients with Interstitial Lung Disease. This suggests that drugs to treat pulmonary fibrosis could potentially be administered by the inhaled route. Trial registration A prospective clinical study approved by London Camden and Kings Cross Research Ethics Committee and registered on clinicaltrials.gov (NCT03136120).

The impact of external and internal focus of attention on visual dependence and EEG alpha oscillations during postural control.

BACKGROUND: The ability to maintain upright posture requires successful integration of multiple sensory inputs (visual, vestibular, and somatosensory). When one or more sensory systems become unreliable, the postural control system must "down-weight" (or reduce the influence of) those senses and rely on other senses to maintain postural stability. As individuals age, their ability to successfully reweight sensory inputs diminishes, leading to increased fall risk. The present study investigates whether manipulating attentional focus can improve the ability to prioritize different sensory inputs for postural control. METHODS: Forty-two healthy adults stood on a balance board while wearing a virtual reality (VR) head-mounted display. The VR environment created a multisensory conflict amongst the different sensory signals as participants were tasked with maintaining postural stability on the balance board. Postural sway and scalp electroencephalography (EEG) were measured to assess visual weighting and cortical activity changes. Participants were randomized into groups that received different instructions on where to focus their attention during the balance task. RESULTS: Following the instructions to direct attention toward the movement of the board (external focus group) was associated with lower visual weighting and better balance performance than when not given any instructions on attentional focus (control group). Following the instructions to direct attention towards movement of the feet (internal focus group) did not lead to any changes in visual weighting or balance performance. Both external and internal focus groups exhibited increased EEG alpha power (8-13 Hz) activity over the occipital cortex as compared to the control group. CONCLUSIONS: Current results suggest that directing one's attention externally, away from one's body, may optimize sensory integration for postural control when visual inputs are incongruent with somatosensory and vestibular inputs. Current findings may be helpful for clinicians and researchers in developing strategies to improve sensorimotor mechanisms for balance.

Exploring developmental changes in infant anticipation and perceptual processing: EEG responses to tactile stimulation.

There is an increasing interest in alpha-range rhythms in the electroencephalogram (EEG) in relation to perceptual and attentional processes. The infant mu rhythm has been extensively studied in the context of linkages between action observation and action production in infancy, but less is known about the mu rhythm in relation to cross-modal processes involving somatosensation. We investigated differences in mu responses to cued vibrotactile stimulation of the hand in two age groups of infants: From 6 to 7 months and 13 to 14 months. We were also interested in anticipatory neural responses in the alpha frequency range prior to tactile stimulation. Tactile stimulation of infants' left or right hand was preceded by an audiovisual cue signaling which hand would be stimulated. In response to the tactile stimulus, infants demonstrated significant mu desynchronization over the central areas contralateral to the hand stimulated, with higher mu peak frequency and greater contralateral mu desynchronization for older infants. Prior to the tactile stimulus, both age groups showed significant bilateral alpha desynchronization over frontocentral sites, which may be indicative of generalized anticipation of an upcoming stimulus. The findings highlight the potential of examining the sensorimotor mu rhythm in the context of infant attentional development.

Monitoring the three-dimensional distribution of endogenous species in the lungs by matrix-assisted laser desorption/ionization mass spectrometry imaging.

RATIONALE: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is routinely employed to monitor the distribution of compounds in tissue sections and generate two-dimensional (2D) images. Whilst informative the images do not represent the distribution of the analyte of interest through the entire organ. The generation of 3D images is an exciting field that can provide a deeper view of the analyte of interest throughout an entire organ. METHODS: Serial sections of mouse and rat lung tissue were obtained at 120 µm depth intervals and imaged individually. Homogenate registration markers were incorporated in order to aid the final 3D image construction. Using freely available software packages, the images were stacked together to generate a 3D image that showed the distribution of endogenous species throughout the lungs. RESULTS: Preliminary tests were performed on 16 serial tissue sections of mouse lungs. A 3D model showing the distribution of phosphocholine at m/z 184.09 was constructed, which defined the external structure of the lungs and trachea. Later, a second experiment was performed using 24 serial tissue sections of the left lung of a rat. Two molecular markers, identified as [PC (32:1) + K]+ at m/z 770.51 and [PC (36:4) + K]+ at m/z 820.52, were used to generate 3D models of the parenchyma and airways, respectively. CONCLUSIONS: A straightforward method to generate 3D MALDI-MS images of selected molecules in lung tissue has been presented. Using freely available imaging software, the 3D distributions of molecules related to different anatomical features were determined.

Multimodal imaging of drug and excipients in rat lungs following an inhaled administration of controlled-release drug laden PLGA microparticles.

Controlled-release formulations, in the form of micro- or nanoparticles, are increasingly attractive to the pharmaceutical industry for drug delivery. For respiratory illnesses, controlled-release microparticle formulations provide an opportunity to deliver a higher percentage of an inhaled medicament dose to the lung, thus potentially reducing the therapeutic dose, frequency of dosing, and minimising side-effects. We describe the use of a multimodal approach consisting of MALDI MS imaging, 3D depth profiling TOF-SIMS analysis, and histopathology to monitor the distribution of drug and excipients in sections taken from excised rat lungs following an inhaled administration of drug-laden microparticles. Following a single dose, the administered drug was detected in the lung via both MALDI MS and TOF-SIMS over a range of time points. Both imaging techniques enabled the characterisation of the distribution and retention of drug particles and identified differences in the capabilities of both imaging modalities. Histochemical staining of consecutive sections was used to provide biological context to the findings and will also be discussed in this presentation. We demonstrate how this multimodal approach could be used to help increase our understanding of the use of controlled release microparticles.

Individual differences in anticipatory mu rhythm modulation are associated with executive function and processing speed.

There is increasing interest in the role of brain oscillations in the regulation and control of behavior. The current study examined the relations between specific cognitive abilities and changes in brain oscillatory activity during anticipation of, and in response to, tactile stimulation of the hand. The oscillation of interest was the sensorimotor mu rhythm (8-14 Hz) at central electrode sites. The electroencephalogram (EEG) was recorded during a task in which a visuospatial cue directed adults (N = 40) that a tactile stimulus would be delivered to their left or right hand. Lateralized changes in mu power following tactile stimulation were associated with reaction time to the tactile stimulus. The extent of a contralateral anticipatory reduction in mu power during the 500 ms before the tactile stimulus was associated with performance on a separate processing speed task. Changes in ipsilateral mu power during anticipation of the tactile stimulus were associated with performance on a flanker task and were marginally correlated with performance on a card sort task. Regression analyses further indicated the specificity of these relations to anticipatory changes in mu power. In summary, mu rhythm modulation during anticipation of tactile stimulation to a specific bodily location was related to a broad measure of processing speed and to variability in the broader ability to regulate behavior in a goal-directed manner. Implications are discussed in terms of the foundational role of anticipatory attention in cognitive processes and the utility of selective attention to the body as an index of attentional control more broadly.

The 3D OrbiSIMS-label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power.

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 µm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters-gamma-aminobutyric acid, dopamine and serotonin-with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

Body maps in the infant brain: implications for neurodevelopmental disabilities.

This review and synthesis discusses recent work that has utilized brain imaging methods, such as the electroencephalogram (EEG) and magnetoencephalogram, to provide insights into the ways that the body is represented in the infant brain. One aspect of body representation concerns somatotopic maps of the body surface in somatosensory cortex. A good deal is known about the properties of these maps in adults, but there has been relatively little developmental work. Recent studies have provided new insights into the organization of infant neural body maps and have laid the foundations for examining their plasticity in relation to behavioral development. Other work has suggested that neural body maps may be involved in the registration of correspondences between self and other, with implications for early social development. Here, body representations are discussed in the context of preterm birth and autism spectrum disorder, providing novel perspectives relevant to developmental medicine and child neurology. WHAT THIS PAPER ADDS: ●Somatotopic body maps develop prenatally through intrinsic and activity-dependent mechanisms. ●There is increasing interest in understanding postnatal plasticity in body maps. ●Body representations may be involved in the registration of preverbal, interpersonal relationships.

Neural representations of the body in 60-day-old human infants.

The organization of body representations in the adult brain has been well documented. Little is understood about this aspect of brain organization in human infancy. The current study employed electroencephalography (EEG) with 60-day-old infants to test the distribution of brain responses to tactile stimulation of three different body parts: hand, foot, and lip. Analyses focused on a prominent positive response occurring at 150-200 ms in the somatosensory evoked potential at central and parietal electrode sites. The results show differential electrophysiological signatures for touch of these three body parts. Stimulation of the left hand was associated with greater positive amplitude over the lateral central region contralateral to the side stimulated. Left foot stimulation was associated with greater positivity over the midline parietal site. Stimulation of the midline of the upper lip was associated with a strong bilateral response over the central region. These findings provide new insights into the neural representation of the body in infancy and shed light on research and theories about the involvement of somatosensory cortex in infant imitation and social perception.

Determining a Threshold to Delimit the Amazonian Forests from the Tree Canopy Cover 2000 GFC Data.

Open global forest cover data can be a critical component for Reducing Emissions from Deforestation and Forest Degradation (REDD+) policies. In this work, we determine the best threshold, compatible with the official Brazilian dataset, for establishing a forest mask cover within the Amazon basin for the year 2000 using the Tree Canopy Cover 2000 GFC product. We compared forest cover maps produced using several thresholds (10%, 30%, 50%, 80%, 85%, 90%, and 95%) with a forest cover map for the same year from the Brazilian Amazon Deforestation Monitoring Project (PRODES) data, produced by the National Institute for Space Research (INPE). We also compared the forest cover classifications indicated by each of these maps to 2550 independently assessed Landsat pixels for the year 2000, providing an accuracy assessment for each of these map products. We found that thresholds of 80% and 85% best matched with the PRODES data. Consequently, we recommend using an 80% threshold for the Tree Canopy Cover 2000 data for assessing forest cover in the Amazon basin.

Neuropsychology of Human Body Parts: Exploring Categorical Boundaries of Tactile Perception Using Somatosensory Mismatch Responses.

The focus of the current study is on a particular aspect of tactile perception: categorical segmentation on the body surface into discrete body parts. The MMN has been shown to be sensitive to categorical boundaries and language experience in the auditory modality. Here we recorded the somatosensory MMN (sMMN) using two tactile oddball protocols and compared sMMN amplitudes elicited by within- and across-boundary oddball pairs. Both protocols employed the identity MMN method that controls for responsivity at each body location. In the first protocol, we investigated the categorical segmentation of tactile space at the wrist by presenting pairs of tactile oddball stimuli across equal spatial distances, either across the wrist or within the forearm. Amplitude of the sMMN elicited by stimuli presented across the wrist boundary was significantly greater than for stimuli presented within the forearm, suggesting a categorical effect at an early stage of somatosensory processing. The second protocol was designed to investigate the generality of this MMN effect, and involved three digits on one hand. Amplitude of the sMMN elicited by a contrast of the third digit and the thumb was significantly larger than a contrast between the third and fifth digits, suggesting a functional boundary effect that may derive from the way that objects are typically grasped. These findings demonstrate that the sMMN is a useful index of processing of somatosensory spatial discrimination that can be used to study body part categories.

Touching lips and hearing fingers: effector-specific congruency between tactile and auditory stimulation modulates N1 amplitude and alpha desynchronization.

Understanding the interactions between audition and sensorimotor processes is of theoretical importance, particularly in relation to speech processing. Although one current focus in this area is on interactions between auditory perception and the motor system, there has been less research on connections between the auditory and somatosensory modalities. The current study takes a novel approach to this omission by examining specific auditory-tactile interactions in the context of speech and non-speech sound production. Electroencephalography was used to examine brain responses when participants were presented with speech syllables (a bilabial sound /pa/ and a non-labial sound /ka/) or finger-snapping sounds that were simultaneously paired with tactile stimulation of either the lower lip or the right middle finger. Analyses focused on the sensory-evoked N1 in the event-related potential and the extent of alpha band desynchronization elicited by the stimuli. N1 amplitude over fronto-central sites was significantly enhanced when the bilabial /pa/ sound was paired with tactile lip stimulation and when the finger-snapping sound was paired with tactile stimulation of the finger. Post-stimulus alpha desynchronization at central sites was also enhanced when the /pa/ sound was accompanied by tactile stimulation of the lip. These novel findings indicate that neural aspects of somatosensory-auditory interactions are influenced by the congruency between the location of the bodily touch and the bodily origin of a perceived sound.

Infant brain responses to felt and observed touch of hands and feet: an MEG study.

There is growing interest concerning the ways in which the human body, both one's own and that of others, is represented in the developing human brain. In two experiments with 7-month-old infants, we employed advances in infant magnetoencephalography (MEG) brain imaging to address novel questions concerning body representations in early development. Experiment 1 evaluated the spatiotemporal organization of infants' brain responses to being touched. A punctate touch to infants' hands and feet produced significant activation in the hand and foot areas of contralateral primary somatosensory cortex as well as in other parietal and frontal areas. Experiment 2 explored infant brain responses to visually perceiving another person's hand or foot being touched. Results showed significant activation in early visual regions and also in regions thought to be involved in multisensory body and self-other processing. Furthermore, observed touch of the hand and foot activated the infant's own primary somatosensory cortex, although less consistently than felt touch. These findings shed light on aspects of early social cognition, including action imitation, which may build, at least in part, on infant neural representations that map equivalences between the bodies of self and other.

Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS.

ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

Allometric equations for integrating remote sensing imagery into forest monitoring programmes.

Remote sensing is revolutionizing the way we study forests, and recent technological advances mean we are now able - for the first time - to identify and measure the crown dimensions of individual trees from airborne imagery. Yet to make full use of these data for quantifying forest carbon stocks and dynamics, a new generation of allometric tools which have tree height and crown size at their centre are needed. Here, we compile a global database of 108753 trees for which stem diameter, height and crown diameter have all been measured, including 2395 trees harvested to measure aboveground biomass. Using this database, we develop general allometric models for estimating both the diameter and aboveground biomass of trees from attributes which can be remotely sensed - specifically height and crown diameter. We show that tree height and crown diameter jointly quantify the aboveground biomass of individual trees and find that a single equation predicts stem diameter from these two variables across the world's forests. These new allometric models provide an intuitive way of integrating remote sensing imagery into large-scale forest monitoring programmes and will be of key importance for parameterizing the next generation of dynamic vegetation models.