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.