Spatiotemporal dissection of the cell cycle with single-cell proteogenomics.

The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer1-3. The cell cycle is driven by precise regulation of proteins in time and space, which creates variability between individual proliferating cells. To our knowledge, no systematic investigations of such cell-to-cell proteomic variability exist. Here we present a comprehensive, spatiotemporal map of human proteomic heterogeneity by integrating proteomics at subcellular resolution with single-cell transcriptomics and precise temporal measurements of individual cells in the cell cycle. We show that around one-fifth of the human proteome displays cell-to-cell variability, identify hundreds of proteins with previously unknown associations with mitosis and the cell cycle, and provide evidence that several of these proteins have oncogenic functions. Our results show that cell cycle progression explains less than half of all cell-to-cell variability, and that most cycling proteins are regulated post-translationally, rather than by transcriptomic cycling. These proteins are disproportionately phosphorylated by kinases that regulate cell fate, whereas non-cycling proteins that vary between cells are more likely to be modified by kinases that regulate metabolism. This spatially resolved proteomic map of the cell cycle is integrated into the Human Protein Atlas and will serve as a resource for accelerating molecular studies of the human cell cycle and cell proliferation.

A multi-scale map of cell structure fusing protein images and interactions.

The cell is a multi-scale structure with modular organization across at least four orders of magnitude1. Two central approaches for mapping this structure-protein fluorescent imaging and protein biophysical association-each generate extensive datasets, but of distinct qualities and resolutions that are typically treated separately2,3. Here we integrate immunofluorescence images in the Human Protein Atlas4 with affinity purifications in BioPlex5 to create a unified hierarchical map of human cell architecture. Integration is achieved by configuring each approach as a general measure of protein distance, then calibrating the two measures using machine learning. The map, known as the multi-scale integrated cell (MuSIC 1.0), resolves 69 subcellular systems, of which approximately half are to our knowledge undocumented. Accordingly, we perform 134 additional affinity purifications and validate subunit associations for the majority of systems. The map reveals a pre-ribosomal RNA processing assembly and accessory factors, which we show govern rRNA maturation, and functional roles for SRRM1 and FAM120C in chromatin and RPS3A in splicing. By integration across scales, MuSIC increases the resolution of imaging while giving protein interactions a spatial dimension, paving the way to incorporate diverse types of data in proteome-wide cell maps.

Indigenous plants promote insect biodiversity in urban greenspaces.

The contribution of urban greenspaces to support biodiversity and provide benefits for people is increasingly recognized. However, ongoing management practices favor vegetation oversimplification, often limiting greenspaces to lawns and tree canopy rather than multi-layered vegetation that includes under- and midstorey, and the use of nonnative species. These practices hinder the potential of greenspaces to sustain indigenous biodiversity, particularly for taxa like insects that rely on plants for food and habitat. Yet, little is known about which plant species may maximize positive outcomes for taxonomically and functionally diverse insect communities in greenspaces. Additionally, while cities are expected to experience high rates of introductions, quantitative assessments of the relative occupancy of indigenous vs. introduced insect species in greenspace are rare, hindering understanding of how management may promote indigenous biodiversity while limiting the establishment of introduced insects. Using a hierarchically replicated study design across 15 public parks, we recorded occurrence data from 552 insect species on 133 plant species, differing in planting design element (lawn, midstorey, and tree canopy), midstorey growth form (forbs, lilioids, graminoids, and shrubs) and origin (nonnative, native, and indigenous), to assess (1) the relative contributions of indigenous and introduced insect species and (2) which plant species sustained the highest number of indigenous insects. We found that the insect community was overwhelmingly composed of indigenous rather than introduced species. Our findings further highlight the core role of multi-layered vegetation in sustaining high insect biodiversity in urban areas, with indigenous midstorey and canopy representing key elements to maintain rich and functionally diverse indigenous insect communities. Intriguingly, graminoids supported the highest indigenous insect richness across all studied growth forms by plant origin groups. Our work highlights the opportunity presented by indigenous understory and midstorey plants, particularly indigenous graminoids, in our study area to promote indigenous insect biodiversity in urban greenspaces. Our study provides a blueprint and stimulus for architects, engineers, developers, designers, and planners to incorporate into their practice plant species palettes that foster a larger presence of indigenous over regionally native or nonnative plant species, while incorporating a broader mixture of midstorey growth forms.

Kinematic characteristics of second-order motor planning and performance in 6- and 10-year-old children and adults: Effects of age and task constraints.

This study explored age-related differences in motor planning as expressed in arm-hand kinematics during a sequential peg moving task with varying demands on goal insertion complexity (second-order planning). The peg was a vertical cylinder with either a circular or semicircular base. The task was to transport the peg between two positions and rotate it various amounts horizontally before fitting into its final position. The amount of rotation required was either 0°, 90°, 180°, or -90°. The reaching for the peg, the displacement of it, and the way the rotation was accomplished was analyzed. Assessments of end state comfort, goal interpretation errors, and type of grip used were also included. Participants were two groups of typically developing children, one younger (Mage  = 6.7 years) and one older (Mage  = 10.3 years), and one adult group (Mage  = 34.9 years). The children, particularly 6-year-olds, displayed less efficient prehensile movement organization than adults. Related to less efficient motor planning, 6-year-olds, mainly, had shorter reach-to-grasp onset latencies, higher velocities, and shorter time to peak velocities, and longer grasp durations than adults. Importantly, the adults rotated the peg during transport. In contrast, the children made corrective rotations after the hand had arrived at the goal.

The virtual people set: developing computer-generated stimuli for the assessment of pedophilic sexual interest.

The implicit assessment of pedophilic sexual interest through viewing-time methods necessitates visual stimuli. There are grave ethical and legal concerns against using pictures of real children, however. The present report is a summary of findings on a new set of 108 computer-generated stimuli. The images vary in terms of gender (female/male), explicitness (naked/clothed), and physical maturity (prepubescent, pubescent, and adult) of the persons depicted. A series of three studies tested the internal and external validity of the picture set. Studies 1 and 2 yielded good-to-high estimates of observer agreement with regard to stimulus maturity levels by two methods (categorization and paired comparison). Study 3 extended these findings with regard to judgments made by convicted child sexual offenders.

Differentiating sexual preference in men: using dual task rapid serial visual presentation task.

Cognition research suggests that allocating attention resources to evolutionarily relevant stimuli is facilitated suggesting that sexual stimuli interfere with human information processing. In a group of gay (n = 13) and straight men (n = 13) recruited in Finland, Germany and Italy, we investigated if and how sexually relevant visual stimuli affect information processing of both a target one (T1) and a subsequent target two (T2) in a dual target rapid serial visual presentation procedure. We hypothesized that: (1) due to the attentional blink (AB) phenomenon, the accuracy of reporting of T2 would decrease when following accurately identified sexually preferred T1 compared to accurately identified non-sexually preferred T1; 2) due to the pop out effect, the accuracy of reporting of T1 and T2 would be relatively increased when T1 and T2 were sexually preferred by the participants compared to when they were not. Our findings did not support hypothesis 1 but supported hypothesis 2. We further found that the pop out effect had a good capacity to differentiate sexual preference between the groups of gay and straight men. We conclude that dual target rapid serial visual presentation can be used as an attention-based measurement to differentiate sexual preference in men. Limitations and the applicability in the field of measuring sexual preference were discussed.

Development of Motor Imagery in School-Aged Children with Autism Spectrum Disorder: A Longitudinal Study.

Autism spectrum disorder (ASD) is a diagnosis based on social communication deficits and prevalence of repetitive stereotyped behaviors, but sensorimotor disturbances are commonly exhibited. This longitudinal study aimed at exploring the development of the ability to form mental motor representations (motor imagery; MI) in 14 children with ASD and 17 typically developing (TD) children at 7, 8 and 9 years of age. MI was investigated using a hand laterality paradigm from which response times (RT) and error rates were extracted and compared with performance on a visually based mental rotation task (VI). A criterion task was used to ensure that the children could perform the task. The results showed wide performance variability in the ASD group with more failures than TD in the MI criterion task, especially at 7 years. For all age levels and both the MI and VI tasks, the error rates were significantly higher and RTs longer for the ASD group compared with TD. Signs of MI strategies were however noted in the ASD group as biomechanically constrained orientations had longer RTs than less constrained orientations, a RT pattern that differed from the VI task. The presence of MI in the ASD group was most evident at 9 years, but the error rates remained high at all ages, both in the MI and VI task. In comparison, the TD group showed stable MI strategies at all ages. These findings indicate that MI ability is delayed and/or impaired in children with ASD which may be related to difficulties performing required mental rotations.

Motor planning and movement execution during goal-directed sequential manual movements in 6-year-old children with autism spectrum disorder: A kinematic analysis.

BACKGROUND: Atypical motor functioning is prevalent in children with autism spectrum disorder (ASD). Knowledge of the underlying kinematic properties of these problems is sparse. AIMS: To investigate characteristics of manual motor planning and performance difficulties/diversity in children with ASD by detailed kinematic measurements. Further, associations between movement parameters and cognitive functions were explored. METHODS AND PROCEDURES: Six-year-old children with ASD (N = 12) and typically developing (TD) peers (N = 12) performed a sequential manual task comprising grasping and fitting a semi-circular peg into a goal-slot. The goal-slot orientation was manipulated to impose different motor planning constraints. Movements were recorded by an optoelectronic system. OUTCOMES AND RESULTS: The ASD-group displayed less efficient motor planning than the TD-group, evident in the reach-to-grasp and transport kinematics and less proactive adjustments of the peg to the goal-slot orientations. The intra-individual variation of movement kinematics was higher in the ASD-group compared to the TD-group. Further, in the ASD-group, movement performance associated negatively with cognitive functions. CONCLUSIONS AND IMPLICATIONS: Planning and execution of sequential manual movements proved challenging for children with ASD, likely contributing to problems in everyday actions. Detailed kinematic investigations contribute to the generation of specific knowledge about the nature of atypical motor performance/diversity in ASD. This is of potential clinical relevance.

A Sample Preparation Protocol for High Throughput Immunofluorescence of Suspension Cells on an Adherent Surface.

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

Slow-release L-Cysteine (Acetium®) Lozenge Is an Effective New Method in Smoking Cessation. A Randomized, Double-blind, Placebo-controlled Intervention.

BACKGROUND/AIM: Because of the major health problems and annual economic burden caused by cigarette smoking, effective new tools for smoking intervention are urgently needed. Our previous randomized controlled trial (RCT) provided promising results on the efficacy of slow-release L-cysteine lozenge in smoking intervention, but the study was not adequately powered. To confirm in an adequately-powered study the results of the previous RCT implicating that effective elimination of acetaldehyde in saliva by slow-release L-cysteine (Acetium® lozenge, Biohit Oyj, Helsinki), would assist in smoking cessation by reducing acetaldehyde-enhanced nicotine addiction. On this matter, we undertook a double-blind, randomized, placebo-controlled trial comparing Acetium® lozenge and placebo in smoking intervention. MATERIALS AND METHODS: A cohort of 1,998 cigarette smokers were randomly allocated to intervention (n=996) and placebo arms (n=1,002). At baseline, smoking history was recorded by a questionnaire, with nicotine dependence testing according to the Fagerström scale (FTND). The subjects used smoking diary recording the daily numbers of cigarettes, lozenges and subjective sensations of smoking. The data were analysed separately for point prevalence of abstinence (PPA) and prolonged abstinence (PA) endpoints. RESULTS: Altogether, 753 study subjects completed the trial per protocol (PP), 944 with violations (mITT), and the rest (n=301) were lost to follow-up (LTF). During the 6-month intervention, 331 subjects stopped smoking; 181 (18.2%) in the intervention arm and 150 (15.0%) in the placebo arm (OR=1.43; 95%CI=1.09-1.88); p=0.010). In the PP group, 170 (45.3%) quitted smoking in the intervention arm compared to 134 (35.4%) in the placebo arm (OR=1.51, 95%CI=1.12-2.02; p=0.006). In multivariate (Poisson regression) model, decreased level of smoking pleasure (p=0.010) and "smoking sensations changed" were powerful independent predictors of quit events (IRR=12.01; 95%CI=1.5-95.6). CONCLUSION: Acetium® lozenge, herein confirmed in an adequately powered study to be an effective means to aid smoking quit, represents a major breakthrough in the development of smoking intervention methods, because slow-release L-cysteine is non-toxic, with no side-effects or limitations of use.

A subcellular map of the human proteome.

Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.

Metastability of Helicobacter pylori bab adhesin genes and dynamics in Lewis b antigen binding.

Heterogeneity among Helicobacter pylori strains in gastric epithelial adherence is postulated to contribute to pathogen fitness in the physiologically diverse human population. H. pylori adherence to ABO and Lewis b (Leb) blood group antigens in the human stomach is mediated by the blood group antigen-binding adhesin BabA. Approximately 70% of Swedish and U.S. H. pylori clinical isolates exhibit Leb binding, but here we show that the babA gene is present in each of 10 Leb-nonbinding strains. Fluorescence microscopy identified occasional bacterial cells with a Leb-binding phenotype in populations of Leb-nonbinding strains. Thus, nonbinding seemed to be a metastable phenotype. To model metastable transition into the virulence-associated Leb-binding mode, Leb-binding clones were isolated from nonadherent strains by panning with Leb-magnetic beads and characterized. Strain 17875 has two babA genes, babA1 (silent) and babA2 (expressed). We found that a babA2-cam derivative of strain 17875 regained Leb binding by recombination of the formerly silent babA1 gene into the expressed and partially homologous babB locus. The chimeric BabB/A adhesin binds Leb with an affinity similar to that of wild-type BabA adhesin, but its expression level was lower and was subject to phase variation through slipped-strand mispairing. Equivalent results were obtained with strain NCTC11638. We propose that adhesin metastability and heterogeneity contributes to bacterial fitness and results in some clones having potential for periodic activation and deactivation of virulence appropriate for intensity of the host response to infection.

Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.