title: tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche

abstract:
Extracellular vesicles transfer complex biologic material between cells, whose role in in-vivo organismal physiology is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. Stimulating osteoblastic cells with parathyroid hormone or activating its receptor enhanced extracellular vesicle transfer, myeloid recovery post radiation and improved animal survival from Candida sepsis. The extracellular vesicles contained tiRNAs known to modulate protein translation. 5-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and when transferred to granulocyte macrophage progenitors, increased protein translation, cell proliferation and myeloid differentiation. Therefore, EV-mediated tiRNA transfer provides a stress modulated signaling axis distinct from conventional cytokine-driven stress responses.

One sentence summaryStress regulated tiRNA transfer alters hematopoiesis


title: Bistable host-pathogen interaction explains varied infection outcomes

abstract: Death or survival are the two outcomes of an infection. The development of an infection depends on multiple interplaying factors, including the temporal dynamic of host nutrition, immune response and pathogen proliferation, but how these factors collectively determine the outcome of an infection is less understood. To understand the underlying principle of infection, here, we took a systems approach by developing a simple mathematical model to understand multi-factored host-pathogen interaction. We find that nonlinear interactions feedback between host and pathogens resulting in two distinct trajectories of disease progression. We show that disease progression, measured by the pathogen load, responds in a bistable manner, by either proliferating or eventually being eliminated. We identified key processes accounting for bistability, including hosts resource assimilation, immune response and intrinsic noise. In addition, resource availability, immunity, and pathogen load at the onset of infection are also critical in determining the bistability. We then highlight, by discussing in the light of previous theoretical and experimental work, how our framework provides testable theories for host-pathogen interaction inside the host.

category: ecology


title: RAREsim: A simulation method for very rare genetic variants

abstract: Identification of rare variant associations is crucial to fully characterize the genetic architecture of complex traits and diseases. Essential in this process is the evaluation of novel methods in simulated data that mirrors the distribution of rare variants and haplotype structure in real data. Additionally, importing real variant annotation enables in silico comparison of methods that focus on putative causal variants, such as rare variant association tests, and polygenic scoring methods. Existing simulation methods are either unable to employ real variant annotation or severely under- or over-estimate the number of singletons and doubletons reducing the ability to generalize simulation results to real studies. We present RAREsim, a flexible and accurate rare variant simulation algorithm. Using parameters and haplotypes derived from real sequencing data, RAREsim efficiently simulates the expected variant distribution and enables real variant annotations. We highlight RAREsims utility across various genetic regions, sample sizes, ancestries, and variant classes.

category: genetics


title: Pairing to the microRNA 3' region occurs through two alternative binding modes, with affinity shaped by nucleotide identity as well as pairing position

abstract:
MicroRNAs (miRNAs), in association with Argonaute (AGO) proteins, direct repression by pairing to sites within mRNAs. Compared to pairing preferences of the miRNA seed region (nucleotides 2-8), preferences of the miRNA 3' region are poorly understood, due to the sparsity of measured affinities for the many pairing possibilities. We used RNA bind-n-seq with purified AGO2-miRNA complexes to measure relative affinities of >1,000 3'-pairing architectures for each miRNA. In some cases, optimal 3' pairing increased affinity by >500-fold. Some miRNAs had two high-affinity 3'-pairing modes--one of which included additional nucleotides bridging seed and 3' pairing to enable high-affinity pairing to miRNA nucleotide 11. The affinity of binding and the position of optimal pairing both tracked with the occurrence of G or oligo(G/C) nucleotides within the miRNA. These and other results advance understanding of miRNA targeting, providing insight into how optimal 3' pairing is determined for each miRNA.

HIGHLIGHTSO_LIRNA bind-n-seq reveals relative affinities of >1,000 3'-pairing architectures
C_LIO_LITwo distinct 3'-binding modes can enhance affinity, by >500-fold in some instances
C_LIO_LIG and oligo(G/C) residues help define the miRNA 3' segment most critical for pairing
C_LIO_LISeed mismatch identity can influence the contribution of compensatory 3' pairing
C_LI

category: biochemistry


title: Structure and function of the chromatin remodeler SMARCAD1 with its nucleosome substrate

abstract:
The ATP-dependent chromatin remodeler SMARCAD1 acts on nucleosomes during DNA repair and transcription, but despite its implication in disease, information on its structure and function is scarce. Chromatin remodelers use a variety of ways to engage nucleosomes, and outcomes of the ATP-dependent reactions vary widely. Here we show that SMARCAD1 transfers the entire histone octamer from one DNA segment to another in an ATP-dependent manner but is also capable of de novo nucleosome assembly from histone octamer, due to its ability to bind all histones simultaneously. We describe the cryoEM structure of SMARCAD1 in complex with a nucleosome and show that it engages its substrate unlike any other chromatin remodeler. Our combined data allow us to put forward a testable model for SMARCAD1 mechanism.

One-Sentence SummaryThe single subunit chromatin remodeler SMARCAD1 engages nucleosomes in a unique manner and transfers the entire histone octamer.


title: Towards a comprehensive delineation of white matter tract-related deformation

abstract: Finite element (FE) models of the human head are valuable instruments to explore the mechanobiological pathway from external loading, localized brain response, and resultant injury risks. The injury predictability of these models depends on the use of effective criteria as injury predictors. The FE-derived normal deformation along white matter (WM) fiber tracts (i.e., tract-oriented strain) has recently been suggested as an appropriate predictor for axonal injury. However, the tract-oriented strain only represents a partial depiction of the WM fiber tract deformation. A comprehensive delineation of tract-related deformation may improve the injury predictability of the FE head model by delivering new tract-related criteria as injury predictors. Thus, the present study performed a theoretical strain analysis to comprehensively characterize the WM fiber tract deformation by relating the strain tensor of the WM element to its embedded fiber tract. Three new tract-related strains with exact analytical solutions were proposed, measuring the normal deformation perpendicular to the fiber tracts (i.e., tract-perpendicular strain), and shear deformation along and perpendicular to the fiber tracts (i.e., axial-shear strain and lateral-shear strain, respectively). The injury predictability of these three newly-proposed strain peaks along with the previously-used tract-oriented strain peak and maximum principal strain (MPS) were evaluated by simulating 151 impacts with known outcome (concussion or non-concussion). The results preliminarily showed that four tract-related strain peaks exhibited superior performance than MPS in discriminating concussion and non-concussion cases. This study presents a comprehensive quantification of WM tract-related deformation and advocates the use of orientation-dependent strains as criteria for injury prediction, which may ultimately contribute to an advanced mechanobiological understanding and enhanced computational predictability of brain injury.

category: bioengineering


title: Intercalated Disk Nanoscale Structure Regulates Cardiac Conduction

abstract: The intercalated disk (ID) is a specialized subcellular region that provides electrical and mechanical connections between myocytes in the heart. The ID has a clearly defined passive role in cardiac tissue, transmitting mechanical forces and electrical currents between cells. Recent studies have shown that Na+ channels, the primary current responsible for cardiac excitation, are preferentially localized at the ID, particularly within nanodomains around mechanical and gap junctions, and that perturbations of ID structure alter cardiac conduction. This suggests that the ID may play an important, active role in regulating conduction. However, the structure of the ID and intercellular cleft are not well characterized, and to date, no models have incorporated the influence of ID structure on conduction in cardiac tissue. In this study, we developed an approach to generate realistic finite element model (FEM) meshes replicating ID nanoscale structure, based on experimental measurements from transmission electron microscopy (TEM) images. We then integrated measurements of the intercellular cleft electrical conductivity, derived from the FEM meshes, into a novel cardiac tissue model formulation. FEM-based calculations predict that the distribution of cleft conductances are sensitive to regional changes in ID structure, specifically the intermembrane separation and gap junction distribution. Tissue-scale simulations demonstrated that ID structural heterogeneity leads to significant spatial variation in electrical polarization within the intercellular cleft. Importantly, we find that this heterogeneous cleft polarization regulates conduction by desynchronizing the activation of post-junctional Na+ currents. Additionally, these heterogeneities lead to a weaker dependence of conduction velocity on gap junctional coupling, compared with prior modeling formulations that neglect or simplify ID structure. Further, we find that disruption of local ID nanodomains can lead to either conduction slowing or enhancing, depending on gap junctional coupling strength. Overall, our study demonstrates that ID nanoscale structure can play a significant role in regulating cardiac conduction.


title: CytoSet: Predicting clinical outcomes via set-modeling of cytometry data

abstract: AO_SCPLOWBSTRACTC_SCPLOWSingle-cell flow and mass cytometry technologies are being increasingly applied in clinical settings, as they enable the simultaneous measurement of multiple proteins across millions of cells within a multi-patient cohort. In this work, we introduce CytoSet, a deep learning model that can directly predict a patients clinical outcome from a collection of cells obtained through a blood or tissue sample. Unlike previous work, CytoSet explicitly models the cells profiled in each patient sample as a set, allowing for the use of recently developed permutation invariant architectures. We show that CytoSet achieves state-of-the-art classification performance across a variety of flow and mass cytometry benchmark datasets. The strong classification performance is further complemented by demonstrated robustness to the number of sub-sampled cells per patient and the depth of model, enabling CytoSet to scale adequately to hundreds of patient samples. The strong performance achieved by the set-based architectures used in CytoSet suggests that clinical cytometry data can be appropriately interpreted and studied as sets. The code is publicly available at https://github.com/CompCy-lab/cytoset.

category: bioinformatics


title: Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast

abstract: We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire 4 images from which the phase gradients and quantitative phase image can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be interleaved with single-shot imaging polarimetry.

category: biophysics


title: Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor

abstract: The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. AKT is a relevant target for inhibition, in addition to KIT, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated significant synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to cell cycle arrest and cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

category: cancer biology


title: Identifying planktonic foraminiferal morphospecies: which are the important images?

abstract: Selection of imagery that promotes accurate identification of morphotaxa is viewed as a significant problem in the taxonomy of planktonic foraminifera. Currently, imagery of taxa is sparse, apparently selected by visual judgement, and presented without information about its typicality. What is required are impartially selected images which embrace population variation to serve as training sets for reliable identification of taxa. Outlined here is a simple morphometrically-based solution, applied to the shape of shells in two orientations, in which shape variation is resolved onto three principal component axes. On the premise that the best-adapted shells are the commonest, specimens within 1 standard deviation (sd) of the trivariate mean are recognized as population exemplars suitable for use as trainers. Specimens which project at [≥]2 sd onto at least one axis are mapped as boundary specimens whose identity might be questioned. This procedure is trialled on samples of Truncorotalia crassaformis. Exemplars from the Equatorial Atlantic and Caribbean compare closely; they partially overlap with those from a Holocene Southwest Pacific population provisionally interpreted as a subtaxon, Truncorotalia crassaformis hessi.

category: paleontology


title: Non-clinical evidence supports anti-inflammatories as more effective medication than antihistamines against tarantula local effects envenomation

abstract:
BackgroundTarantulas are the most common invertebrates pets, especially in North America and Europe. The most commercialized genera are from Southern Asia and tropical Americas, represented by Vitalius, found in Southeastern Brazil, and Brachypelma, common in Mexican desert. Bites by these spiders in humans occurs during manipulation and generally result in clinical manifestations such local pain, erythema and oedema, with the possibility of secondary local infection. Hence, the cases are usually treated with prescription free drugs such as antihistamines and anti-inflammatories.

MethodsIn this work, we investigated the post treatment with commercial nonsteroidal and steroidal anti-inflammatories and anti-histamines administered by oral and intraperitoneal routes on rat paw oedema induced by venoms of V. dubius and B. smithi. Hydroplethysmometer standard oedema measurement and Evans blue extravasation were performed. Dose standardization experiments showed that the V. dubius is more potent than B. smithi, and doses were established at 30 g/paw and 60 g/paw respectively.

ResultsThe oral post-administration of ketoprofen (non-selective cyclooxygenase inhibitor) and prednisolone (steroidal anti-inflammatory) markedly reduced a paw oedema evoked by only V. dubius venom, but loratadine (H1-antihistamine) had negligible effect on rat paw oedema induced by both venoms. Intraperitoneal administration, ketoprofen (20 mg Kg-1) and loratadine (5 mg Kg-1) reduced the rat paw oedema induced by V. dubius and B. smithi while methylprednisolone (10 mg Kg-1) only inhibited the oedema induced by V. dubius.

ConclusionThese results suggest that the pos-treatment with nonsteroidal and steroidal anti-inflammatory drugs are more potent than antihistamines in attenuating the local effect induced by V. dubius and B. smithi venoms.

category: pharmacology and toxicology


title: Soft tissue deformations explain most of the mechanical work variations of human walking

abstract:
Humans perform mechanical work during walking, some by leg joints actuated by muscles, and some by passive, dissipative soft tissues. Dissipative losses must be restored by active muscle work, potentially in amounts sufficient to cost substantial metabolic energy. The most dissipative, and therefore costly, walking conditions might be predictable from the pendulum-like dynamics of the legs. If pendulum behavior is systematic, it may also predict the work distribution between active joints and passive soft tissues. We therefore tested whether the overall negative work of walking, and the fraction due to soft tissue dissipation, are both predictable by a pendulum model across a wide range of conditions. The model predicts whole-body negative work from the leading legs impact with ground (termed the Collision), to increase with the squared product of walking speed and step length. We experimentally tested this in humans (N = 9) walking in 26 different combinations of speed (0.7 - 2.0 m{middle dot}s-1) and step length (0.5 - 1.1 m), with recorded motions and ground reaction forces. Whole-body negative Collision work increased as predicted (R2 = 0.73), with a consistent fraction of about 63% (R2 = 0.88) due to soft tissues. Soft tissue dissipation consistently accounted for about 56% of the variation in total whole-body negative work. During typical walking, active work to restore dissipative losses could account for 31% of the net metabolic cost. Soft tissue dissipation, not included in most biomechanical studies, explains most of the variation in negative work of walking, and could account for a substantial fraction of the metabolic cost.

Summary statementSoft tissue deformations dissipate substantial energy during human walking, as predicted by a simple walking model.

category: physiology


title: SUFFICIENT ACTIVITY OF THE UBIQUITIN PROTEASOME SYSTEM IN AGED MICE AND DURING RETINAL DEGENERATION SUPPORTS DHFR-BASED CONDITIONAL CONTROL OF PROTEIN ABUNDANCE IN THE RETINA

abstract: The Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. In the absence of TMP, a DHFR stabilizer, the DD is degraded by the ubiquitin proteasome system (UPS). To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, which may have a compromised UPS, we evaluated the DHFR DD system in aged mice (up to 24 mo), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4-/- mice). Aged, LIRD, and Abca4-/- mice all had similar proteasomal activities and high-molecular weight ubiquitin levels compared to control mice. However, rd2 mice displayed compromised chymotrypsin activity compared to control mice. Nonetheless, the DHFR DD was effectively degraded in all model systems, including rd2 mice. Moreover, TMP increased DHFR DD-dependent retinal bioluminescence in all mouse models, however the fold induction was slightly, albeit significantly, lower in Abca4-/- mice. Thus, the destabilized DHFR DD-based approach allows for efficient control of protein abundance in aged mice and retinal degeneration mouse models, laying the foundation to use this strategy in a wide variety of mice for the conditional control of gene therapies to potentially treat multiple eye diseases.


title: Visual Thalamocortical Mechanisms of Waking State Dependent Activity and Alpha Oscillations

abstract:
The brain exhibits distinct patterns of recurrent activity closely related to the behavioral state of the animal. The neural mechanisms that underlie state-dependent activity in the awake animal are incompletely understood. Here, we demonstrate that two types of state-dependent activity - rapid arousal/movement related signals and a 3-5 Hz alpha-like rhythm - in the primary visual cortex (V1) of mice strongly correlate with activity in the visual thalamus. Inactivation of V1 does not interrupt arousal/movement related signals in most visual thalamic neurons, but it abolishes the 3-5 Hz oscillation. Silencing of the visual thalamus similarly eradicates the alpha-like rhythm and perturbs arousal/movement-related activation in V1. Finally, we observed that whisker movement or locomotion is not required for rapid increases in cortical activation. Our results indicate that thalamocortical interactions together with cell-intrinsic properties of thalamocortical cells play a crucial role in shaping state-dependent activity in V1 of the awake animal.

HighlightsO_LIWhisker movements correlate with rapid synaptic activation in V1 and visual thalamus
C_LIO_LISilencing of V1 does not abolish movement related activation in most dLGN or LP cells
C_LIO_LISilencing of visual thalamus strongly reduces movement related activation in V1
C_LIO_LIThalamocortical interactions generate state-dependent alpha frequency oscillation
C_LIO_LIVisual thalamic cells exhibit LTS firing during alpha oscillation in the awake mouse
C_LI

category: neuroscience


title: Adolescent Binge Drinking is Associated with Accelerated Decline of Gray Matter Volume

abstract: The age- and time-dependent effects of binge-drinking on adolescent brain development have not been well characterized even though binge drinking is a health crisis among adolescents. The impact of binge drinking on gray matter volume development was examined using longitudinal data from the National Consortium on Alcohol and NeuroDevelopment in Adolescence (NCANDA). Non-binge drinkers (n=177) were matched to binge drinkers (n=164) on potential confounders. Number of binge drinking episodes in the past year was linked to decreased volumes for total gray matter, frontal, parietal, temporal, and occipital lobes (ps<.001). Interactions of binge drinking episodes and age demonstrated stronger effects in younger subjects for total gray matter, frontal, temporal, and occipital lobes (ps<.001). Subsequent models included binge drinking coded in multiple ways. Models sensitive to number of episodes and temporal proximity to outcomes provided the best fits. Declines in gray matter volume association with binge drinking are potentially related to changes in cognition frequently reported among binge drinking adolescents. Results underscore the potential importance of delaying initiation of binge drinking and provide evidence for a dose-response relationship of binge drinking to gray matter decline. Temporally proximal binge drinking was associated more strongly with gray matter decline, suggesting the potential for recovery.


title: Changes in regional white matter volumetry and microstructure during the post-adolescence period: a cross-sectional study of a cohort of 1,713 university students

abstract: Human brain white matter undergoes a protracted maturation that continues well into adulthood. Recent advances in diffusion-weighted imaging (DWI) methods allow detailed characterizations of the microstructural architecture of white matter, and they are increasingly utilised to study white matter changes during development and ageing. However, relatively little is known about the late maturational changes in the microstructural architecture of white matter during post-adolescence. Here we report on regional changes in white matter volume and microstructure in young adults undergoing university-level education. As part of the MRi-Share multi-modal brain MRI database, multi-shell, high angular resolution DWI data were acquired in a unique sample of 1,713 university students aged 18 to 26. We assessed the age and sex dependence, as well as hemispheric asymmetry of diffusion metrics derived from diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI), in the white matter regions as defined in the John Hopkins University (JHU) white matter labels atlas. We demonstrate that while regional white matter volume is relatively stable over the age range of our sample, the white matter microstructural properties show clear age-related variations. Globally, it is characterised by a robust increase in neurite density index (NDI), and to a lesser extent, orientation dispersion index (ODI). These changes are accompanied by a decrease in diffusivity. In contrast, there is minimal age-related variation in fractional anisotropy. There are regional variations in these microstructural changes: some tracts, most notably cingulum bundles, show a strong age-related increase in NDI coupled with decreases in radial and mean diffusivity, while others, mainly cortico-spinal projection tracts, primarily show an ODI increase and axial diffusivity decrease. These age-related variations are not different between males and females, but males show higher NDI and ODI and lower diffusivity than females across many tracts. We also report a robust hemispheric asymmetry in both the volume and microstructural properties in many regions. These findings emphasise the complexity of changes in white matter structure occurring in this critical period of late maturation in early adulthood.


title: Auditory attention detection with EEG channel attention

abstract: Auditory attention detection (AAD) seeks to detect the attended speech from EEG signals in a multi-talker scenario, i.e. cocktail party. As the EEG channels reflect the activities of different brain areas, a task-oriented channel selection technique improves the performance of brain-computer interface applications. In this study, we propose a soft channel attention mechanism, instead of hard channel selection, that derives an EEG channel mask by optimizing the auditory attention detection task. The neural AAD system consists of a neural channel attention mechanism and a convolutional neural network (CNN) classifier. We evaluate the proposed framework on a publicly available database. We achieve 88.3% and 77.2% for 2-second and 0.1-second decision windows with 64-channel EEG; and 86.1% and 83.9% for 2-second decision windows with 32-channel and 16-channel EEG, respectively. The proposed framework outperforms other competitive models by a large margin in all test cases.


title: Subthalamic nucleus stabilizes movements by reducing neural spike variability in monkey basal ganglia: chemogenetic study

abstract: AbstractsThe subthalamic nucleus (STN) projects to the external pallidum (GPe) and internal pallidum (GPi), the relay and output nuclei of the basal ganglia (BG), respectively, and plays an indispensable role in controlling voluntary movements. To elucidate the neural mechanism by which the STN controls GPe/GPi activity and movements, we utilized a chemogenetic method to reversibly suppress the motor subregion of the STN in three macaque monkeys (Macaca fuscata, both sexes) engaged in reaching tasks. Systemic administration of chemogenetic ligands prolonged movement time and increased spike train variability in the GPe/GPi, but only slightly affected firing rate modulations. Across-trial analyses revealed that the irregular discharge activity in the GPe/GPi coincided with prolonged movement time. STN suppression also induced excessive abnormal movements in the contralateral forelimbs, which was preceded by STN and GPe/GPi phasic activity changes. Our results suggest that the STN stabilizes spike trains in the BG and achieves stable movements.


title: Single-cell immune profiling with TCR clonotype barcoding identifies biomarker signatures that predict response to immune checkpoint blockade

abstract: The identification of predictive biomarkers for patient treatment response is urgently needed to increase the probability of success of existing and novel experimental therapies. Single-cell profiling has provided novel biological insights into drug responses in the tumor microenvironment, but its potential for biomarker discovery has not been fully explored for therapeutic purposes. We describe a novel approach to discover predictive response biomarkers from single-cell data from a small patient cohort using the T cell receptor sequence intrinsic to each T cell to match clonotypes between pre- and post-treatment tumor samples. As a result, we have identified a predictive gene expression signature for immune checkpoint blockade and validated its predictive performance using data from three larger clinical studies. Our results demonstrated that applying clonotyping with single-cell genomic profiling is a promising novel approach for biomarker identification that does not require data collected from large patient cohorts. This could increase success rates, reduce clinical trial size, and significantly impact future clinical developments of immunomodulatory therapeutics.

category: immunology


title: From Structure to Sequence: Identification of polyclonal antibody families using cryoEM

abstract:
One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next generation sequencing of immune repertoires we were able to specifically identify clonal family members, synthesize the monoclonal antibodies and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/439712v2_ufig1.gif" ALT="Figure 1">
View larger version (41K):
org.highwire.dtl.DTLVardef@de0663org.highwire.dtl.DTLVardef@757c30org.highwire.dtl.DTLVardef@12c4709org.highwire.dtl.DTLVardef@1ebd6d7_HPS_FORMAT_FIGEXP  M_FIG C_FIG One Sentence SummaryCryoEM and next generation sequencing were used to identify monoclonal antibodies elicited by HIV Env vaccine candidates.


title: A human factor H-binding protein of Bartonella bacilliformis and potential role in serum resistance

abstract:
Bartonella bacilliformis is a Gram-negative bacterium and etiologic agent of Carrions disease; a potentially life-threatening illness endemic to South America. B. bacilliformis is a facultative parasite that infects human erythrocytes (hemotrophism) and the circulatory system, culminating in a variety of symptoms, including a precipitous drop in hematocrit, angiomatous lesions of the skin (verruga peruana) and persistent bacteremia. Because of its specialized niche, serum complement imposes a continual selective pressure on the pathogen. In this study, we demonstrated the marked serum-resistance phenotype of B. bacilliformis, the role of factor H in serum complement resistance, and binding of host factor H to four membrane-associated polypeptides of [~]131, 119, 60 and 43 kDa by far-western (FW) blots. The [~]119-kDa protein was identified as ABM44634.1 by mass spectrometry; a protein annotated as a 116.5-kDa outer membrane autotransporter (encoded by the BARBAKC583_1133 locus). We designated the protein as factor H-binding protein A (FhbpA). FhbpA possesses three structural motifs common to all autotransporter proteins (i.e., a signal peptide, autotransporter {beta}-barrel domain and passenger domain). Recombinant FhbpA passenger domain, but not the recombinant autotransporter domain, was able to bind human factor H when analyzed by FW blots. Phylogenetic analyses of the passenger domain suggest that it is well-conserved among Bartonella autotransporters, with closest matches from Bartonella schoenbuchensis. Transcriptomic analyses of B. bacilliformis subjected to conditions mimicking the sand fly vector or human host, and infection of human blood or vascular endothelial cells showed maximal expression of fhbpA under human-like conditions and during infection of blood and endothelial cells. Expression during HUVEC infection was significantly higher compared to all other conditions by DESeq2. Surface binding of serum factor H by FhbpA is hypothesized to play a protective role against the alternative pathway of complement fixation during B. bacilliformis infection of the human host.

Author SummaryB. bacilliformis is a bacterial pathogen that colonizes the circulatory system of humans, where it can cause a life-threatening illness unless treated. Serum complement is a major effector of innate humoral immunity and a significant obstacle that must be evaded for successful survival and colonization by pathogens, especially those residing in the vasculature. In this study, we examined the serum complement resistance phenotype of B. bacilliformis and identified four membrane-associated proteins that bind serum factor H; a protein used by the host to protect its own tissues from complement activation. One of the proteins was identified by mass spectrometry, characterized, and designated factor H-binding protein A (FhbpA). FhbpA is a predicted autotransporter, and we determined that the translocated " passenger" domain of the protein is responsible for binding factor H. We also determined that expression of the fhbpA gene was highest during infection of human blood and especially vascular endothelial cells or under conditions that simulate the human host. The results suggest that FhbpA binding of host serum factor H protects the bacterium against complement activation during infection.

category: microbiology


title: Identification of beneficial and detrimental bacteria that impact sorghum responses to drought using multi-scale and multi-system microbiome comparisons

abstract:
BackgroundDrought is a major abiotic stress that limits agricultural productivity. Previous field-level experiments have demonstrated that drought decreases microbiome diversity in the root and rhizosphere and may lead to enrichment of specific groups of microbes, such as Actinobacteria. How these changes ultimately affect plant health is not well understood. In parallel, model systems have been used to tease apart the specific interactions between plants and single, or small groups of microbes. However, translating this work into crop species and achieving increased crop yields within noisy field settings remains a challenge. Thus, the next scientific leap forward in microbiome research must cross the great lab-to-field divide. Toward this end, we combined reductionist, transitional and ecological approaches, applied to the staple cereal crop sorghum to identify key beneficial and detrimental, root associated microbes that robustly affect drought stressed plant phenotypes.

ResultsFifty-three bacterial strains, originally characterized for association with Arabidopsis, were applied to sorghum seeds and their effect on root growth was monitored for seven days. Two Arthrobacter strains, members of the Actinobacteria phylum, caused root growth inhibition (RGI) in Arabidopsis and sorghum. In the context of synthetic communities, strains of Variovorax were able to protect both Arabidopsis and sorghum from the RGI caused by Arthrobacter. As a transitional system, we tested the synthetic communities through a 24-day high-throughput sorghum phenotyping assay and found that during drought stress, plants colonized by Arthrobacter were significantly smaller and had reduced leaf water content as compared to control plants. However, plants colonized by both Arthrobacter and Variovorax performed as well or better than control plants. In parallel, we performed a field trial wherein sorghum was evaluated across well-watered and drought conditions. Drought responsive microbes were identified, including an enrichment in Actinobacteria, consistent with previous findings. By incorporating data on soil properties into the microbiome analysis, we accounted for experimental noise with a newly developed method and were then able to observe that the abundance of Arthrobacter strains negatively correlated with plant growth. Having validated this approach, we cross-referenced datasets from the high-throughput phenotyping and field experiments and report a list of high confidence bacterial taxa that positively associated with plant growth under drought stress.

ConclusionsA three-tiered experimental system connected reductionist and ecological approaches and identified beneficial and deleterious bacterial strains for sorghum under drought stress.


title: The RNA-binding protein RBP42 regulates cellular energy metabolism in mammalian-infective Trypanosoma brucei

abstract: RNA-binding proteins are key players in coordinated post-transcriptional regulation of functionally related genes, defined as RNA regulons. RNA regulons play particularly critical roles in parasitic trypanosomes, which exhibit unregulated co-transcription of long arrays of unrelated genes. In this report, we present a systematic analysis of an essential RNA-binding protein, RBP42, in the mammalian-infective slender bloodstream form of African trypanosome, and we show that RBP42 is a key regulator of parasites central carbon and energy metabolism. Using individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify genome-wide RBP42-RNA interactions, we show that RBP42 preferentially binds within the coding region of mRNAs encoding core metabolic enzymes. Using global quantitative transcriptomic and proteomic analyses, we also show that loss of RBP42 reduces the abundance of target mRNA-encoded proteins, but not target mRNA, suggesting a plausible role of RBP42 as a positive regulator of target mRNA translation. Analysis reveals significant changes in central carbon metabolic intermediates following loss of RBP42, further supporting its critical role in cellular energy metabolism.

category: molecular biology


title: Quantification and image-derived phenotyping of retinal ganglion cell nuclei in the nee mouse model of congenital glaucoma

abstract: The nee mouse model exhibits characteristic features of congenital glaucoma, a common cause of childhood blindness. The current study of nee mice had two components. First, the time course of neurodegeneration in nee retinal flat-mounts was studied over time using a retinal ganglion cell (RGC)-marker, BRN3A; a pan-nuclear marker, TO-PRO-3; and H&E staining. Based on segmentation of nuclei using ImageJ and RetFM-J, this analysis identified a rapid loss of BRN3A+ nuclei from 4-15 weeks of age, with the first statistically significant difference in average density compared to age-matched controls detected in 8-week-old cohorts (49% reduction in nee). Consistent with a model of glaucoma, no reductions in BRN3A- nuclei were detected, but the combined analysis indicated that some RGCs lost BRN3A marker expression prior to actual cell loss. These results have a practical application in the design of experiments using nee mice to study mechanisms or potential therapies for congenital glaucoma. The second component of the study pertains to a discovery-based analysis of the large amount of image data with 748,782 segmented retinal nuclei. Using the automatedly collected region of interest feature data captured by ImageJ, we tested whether RGC density of glaucomatous mice was significantly correlated to average nuclear area, perimeter, Feret diameter, or MinFeret diameter. These results pointed to two events influencing nuclear size. For variations in RGC density above approximately 3,000 nuclei/mm2 apparent spreading was observed, in which BRN3A- nuclei--regardless of genotype--became slightly larger as RGC density decreased. This same spreading occurred in BRN3A+ nuclei of wild-type mice. For variation in RGC density below 3,000 nuclei/mm2, which only occurred in glaucomatous nee mutants, BRN3A+ nuclei became smaller as disease was progressively severe. These observations have relevance to defining RGCs of relatively higher sensitivity to glaucomatous cell death and the nuclear dynamics occurring during their demise.


title: Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay

abstract: Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins, transmembrane proteins, and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

category: cell biology


title: Arf6 is necessary for high level Wingless signalling during Drosophila wing development

abstract: Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of {beta}-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, we analyse the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/{beta}-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling.

category: developmental biology


title: Bleeding in patients and mice with Alagille syndrome: sex differences and risk factors

abstract: Spontaneous lethal bleeds are major cause of death in the pediatric liver disease Alagille syndrome (ALGS), yet risk factors and screening methods have not been established. We performed a systematic review and identified significantly more female than male patients with idiopathic intracranial hemorrhage (10:1). We investigated bleeding and vasculature in patients and a mouse model for ALGS (Jag1Ndr/Ndr mice) and asked whether phenotypes identified in mice could be detected in patients non-invasively. Jag1Ndr/Ndr mice bled spontaneously, exhibiting a thin skull and vascular defects including artery-vein crossings, tortuous vessels, capillary breakdown and CADASIL-like sparse vascular smooth muscle cell coverage which was aggravated by hypertension. Retinographs from patients confirmed tortuous blood vessels and artery-vein crossings in ALGS. In conclusion, Jag1Ndr/Ndr mice could be used to develop interventions for vascular defects in ALGS, and retinography could provide a non-invasive method for vascular analysis in these pediatric patients.


title: Sex differences and risk factors for bleeding in Alagille syndrome


title: Facilitation between ecosystem engineers, salt marsh grass and mussels, produces pattern formation on salt marsh shorelines

abstract: Interspecific facilitation between ecosystem engineers, such as salt marsh grass and mussel aggregations, is a key process that structures communities and enhances biodiversity. Scale-dependent pattern formation via self-organization is ubiquitous in terrestrial, aquatic and marine ecosystems. Despite their prevalence and ecological importance, these two phenomena have rarely been linked. We provide empirical evidence that the facilitative interaction in salt marshes between smooth cordgrass Spartina alterniflora and the ribbed mussel Geukensia demissa produces distinct spatial patterns along marsh shorelines. These findings advance our understanding of linkages between facilitation and pattern formation in nature, and are particularly relevant to conservation and restoration of salt marshes threatened by climate change and sea-level rise.


title: Optimising biodiversity protection through artificial intelligence

abstract: Over a million species face extinction, carrying with them untold options for food, medicine, fibre, shelter, ecological resilience, aesthetic and cultural values. There is therefore an urgent need to design conservation policies that maximise the protection of biodiversity and its contributions to people, within the constraints of limited budgets. Here we present a novel framework for spatial conservation prioritisation that combines simulation models, reinforcement learning and ground validation to identify optimal policies. Our methodology, CAPTAIN (Conservation Area Prioritisation Through Artificial Intelligence Networks), quantifies the trade-off between the costs and benefits of area and biodiversity protection, allowing the exploration of multiple biodiversity metrics. Under a fixed budget, our model protects substantially more species from extinction than the random or naively targeted protection of areas. CAPTAIN also outperforms the most widely used software for spatial conservation prioritisation (Marxan) in 97% of cases and reduces species loss by an average of 40% under simulations, besides yielding prioritisation maps at substantially higher spatial resolution using empirical data. We find that regular biodiversity monitoring, even if simple and with a degree of inaccuracy - characteristic of citizen science surveys - substantially improves biodiversity outcomes. Given the complexity of people-nature interactions and wealth of associated data, artificial intelligence holds great promise for improving the conservation of biological and ecosystem values in a rapidly changing and resource-limited world.


title: Changing microbial activities during low salinity acclimation in the brown alga Ectocarpus subulatus

abstract:
Ectocarpus subulatus is one of the few brown algae found in river habitats. Its ability to tolerate freshwater is due, in part, to its uncultivated microbiome. We investigated this phenomenon by modifying the microbiome of laboratory-grown E. subulatus using mild antibiotic treatments, which affected its ability to grow in low salinity. The acclimation to low salinity of fresh water-tolerant and intolerant holobionts was then compared. Salinity had a significant impact on bacterial gene expression as well as the expression of algae- and bacteria-associated viruses in all holobionts, albeit in different ways for each holobiont. On the other hand, gene expression of the algal host and metabolite profiles were affected almost exclusively in the fresh water intolerant holobiont. We found no evidence of bacterial protein production that would directly improve algal stress tolerance. However, we identified vitamin K synthesis as one possible bacterial service missing specifically in the fresh water-intolerant holobiont in low salinity.

We also noticed an increase in bacterial transcriptomic activity and the induction of microbial genes involved in the biosynthesis of the autoinducer AI-1, a compound that regulates quorum sensing. This could have caused a shift in bacterial behavior in the intolerant holobiont, resulting in virulence or dysbiosis.

Originality-Significance StatementThe importance of symbiotic microbes for the health and stress resistance of multicellular eukaryotes is widely acknowledged, but understanding the mechanisms underlying these interactions is challenging. They are especially difficult to separate in systems with one or more uncultivable components. We bridge the gap between fully controlled, cultivable model systems and purely environmental studies through the use of a multi-omics approach and metabolic models on experimentally modified "holobiont" systems. This allows us to generate two promising working hypotheses on the mechanisms by which uncultivated bacteria influence their brown algal hosts fresh water tolerance.


title: Interacting Phenotypes and the Coevolutionary Process

abstract: Coevolution occurs when species interact to influence one anothers fitness, resulting in reciprocal evolutionary change. In many coevolving lineages, trait expression in one species is modified by the genotypes and phenotypes of the other, forming feedback loops reminiscent of models of intraspecific social evolution. Here, we adapt the theory of within-species social evolution, characterized by indirect genetic effects and social selection imposed by interacting individuals, to the case of interspecific interactions. In a trait-based model, we derive general expressions for multivariate evolutionary change in two species and the expected between-species covariance in evolutionary change when selection varies across space. We show that reciprocal interspecific indirect genetic effects can dominate the coevolutionary process and drive patterns of correlated evolution beyond what is expected from direct selection alone. In extreme cases, interspecific indirect genetic effects can lead to coevolution when selection does not covary between species or even when one species lacks genetic variance. Moreover, our model indicates that interspecific indirect genetic effects may interact in complex ways with cross-species selection to determine the course of coevolution. Importantly, our model makes empirically testable predictions for how different forms of reciprocal interactions contribute to the coevolutionary process.

category: evolutionary biology


title: Interacting phenotypes and the coevolutionary process: Interspecific indirect genetic effects alter coevolutionary dynamics


title: Genome stability is in the eye of the beholder: recent retrotransposon activity varies significantly across avian diversity

abstract:
Since the sequencing of the zebra finch genome it has become clear the avian genome, while largely stable in terms of chromosome number and gene synteny, is more dynamic at an intrachromosomal level. A multitude of intrachromosomal rearrangements and significant variation in transposable element content have been noted across the avian tree. Transposable elements (TEs) are a source of genome plasticity, because their high similarity enables chromosomal rearrangements through non-allelic homologous recombination, and they have potential for exaptation as regulatory and coding sequences. Previous studies have investigated the activity of the dominant TE in birds, CR1 retrotransposons, either focusing on their expansion within single orders, or comparing passerines to non-passerines. Here we comprehensively investigate and compare the activity of CR1 expansion across orders of birds, finding levels of CR1 activity vary significantly both between and with orders. We describe high levels of TE expansion in genera which have speciated in the last 10 million years including kiwis, geese and Amazon parrots; low levels of TE expansion in songbirds across their diversification, and near inactivity of TEs in the cassowary and emu for millions of years. CR1s have remained active over long periods of time across most orders of neognaths, with activity at any one time dominated by one or two families of CR1s. Our findings of higher TE activity in species-rich clades and dominant families of TEs within lineages mirror past findings in mammals.

Author SummaryTransposable elements (TEs) are mobile, self replicating DNA sequences within a species genome, and are ubiquitous sources of mutation. The dominant group of TEs within birds are chicken repeat 1 (CR1) retrotransposons, making up 7-10% of the typical avian genome. Because past research has examined the recent inactivity of CR1s within model birds such as the chicken and the zebra finch, this has fostered an erroneous view that all birds have low or no TE activity on recent timescales. Our analysis of numerous high quality avian genomes across multiple orders identified both similarities and significant differences in how CR1s expanded. Our results challenge the established view that TEs in birds are largely inactive and instead suggest that their variation in recent activity may contribute to lineage-specific changes in genome structure. Many of the patterns we identify in birds have previously been seen in mammals, highlighting parallels between the evolution of birds and mammals.


title: The TRRAP transcription cofactor represses interferon-stimulated genes in colorectal cancer cells

abstract: Transcription is essential for cells to respond to signaling cues and involves factors with multiple distinct activities. One such factor, TRRAP, functions as part of two large complexes, SAGA and TIP60, which have crucial roles during transcription activation. Structurally, TRRAP belongs to the PIKK family but is the only member classified as a pseudokinase. Recent studies established that a dedicated HSP90 co-chaperone, the TTT complex, is essential for PIKK stabilization and activity. Here, using endogenous auxin- inducible degron alleles, we show that the TTT subunit TELO2 promotes TRRAP assembly into SAGA and TIP60 in human colorectal cancer cells (CRC). Transcriptomic analysis revealed that TELO2 contributes to TRRAP regulatory roles in CRC cells, most notably of MYC target genes. Surprisingly, TELO2 and TRRAP depletion also induced the expression of type I interferon genes. Using a combination of nascent RNA, antibody-targeted chromatin profiling (CUT&RUN) and kinetic analyses, we show that TRRAP directly represses the expression of IRF9, which is a master regulator of interferon stimulated genes. We have therefore uncovered a new, unexpected transcriptional repressor role for TRRAP, which may contribute to its tumorigenic activity.


title: Custom long non-coding RNA capture enhances detection sensitivity in different human sample types

abstract: Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.

category: genomics


title: Computational and biochemical analysis of type IV Pilus dynamics and stability

abstract: Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend, retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating Nuclear Magnetic Resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability and function, we performed a total of 108 s all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH and ionic strength was complemented by targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments revealed a conserved set of residues defining a novel calcium-binding site at the interface between three pilin subunits. Calcium binding enhanced T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.


title: Creation of X-linked Alport Syndrome Rat Model with Col4a5 Deficiency

abstract: Alport syndrome is an inherited chronic human kidney disease, characterized by glomerular basement membrane abnormalities. This disease is caused by mutations in COL4A3, COL4A4, or COL4A5 gene. The knockout mice for Col43, Col44, and Col45 are developed and well characterized for the study of Alport syndrome. However, disease progression and effects of pharmacological therapy depend on the genetic variability. This model is reliable only to mice. Therefore in this study, we created a novel Alport syndrome rat model utilizing rGONAD technology. Col45 deficient rats showed hematuria, proteinuria, high levels of BUN, Cre, and then died at 18 to 28 weeks of age (Hemizygous mutant males). Histological and ultrastructural analyses displayed the abnormalities including parietal cell hyperplasia, mesangial sclerosis, and interstitial fibrosis. Then, we demonstrated that 3/4/5 (IV) and 5/5/6 (IV) chains of type IV collagen disrupted in the Col45 deficient rats. Moreover, immunofluorescence analyses revealed that some glomeruli of Col45 mutant rats were found to be disrupted from postnatal day 0. Thus, Col45 mutant rat is a reliable candidate for Alport syndrome model for underlying the mechanism of renal diseases and further identifying potential therapeutic targets for human renal diseases.


title: A Platform for Oncogenomic Reporting and Interpretation

abstract: Manual interpretation of variants remains rate limiting in precision oncology. The increasing scale and complexity of molecular data generated from comprehensive sequencing of cancer samples requires advanced interpretative platforms as precision oncology expands beyond individual patients to entire populations. To address this unmet need, we created the Platform for Oncogenomic Reporting and Interpretation (PORI), comprising an analytic framework created to facilitate the interpretation and reporting of somatic variants in cancer. PORI is unique in its integration of reporting and graph knowledge base tools combined with support for manual curation at the reporting stage. PORI represents one of the first open-source platform alternatives to commercial reporting solutions suitable for comprehensive genomic data sets in precision oncology. We demonstrate the utility of PORI by matching 9,961 TCGA tumours to the graph knowledge base, revealing that 88.2% have at least one potentially targetable alteration, and making available reports describing select individual samples.


title: Interpretable, scalable, and transferrable functional projection of large-scale transcriptome data using constrained matrix decomposition

abstract: Large-scale transcriptome data, such as single-cell RNA-sequencing data, have provided unprecedented resources for studying biological processes at the systems level. Numerous dimensionality reduction methods have been developed to visualize and analyze these transcriptome data. In addition, several existing methods allow inference of functional variations among samples using gene sets with known biological functions. However, it remains challenging to analyze transcriptomes with reduced dimensions that are interpretable in terms of dimensions directionalities, transferrable to new data, and directly expose the contribution of individual genes. In this study, we used gene set non-negative principal component analysis (gsPCA) and non-negative matrix factorization (gsNMF) to analyze large-scale transcriptome datasets. We found that these methods provide low-dimensional information about the progression of biological processes in a quantitative manner, and their performances are comparable to existing functional variation analysis methods in terms of distinguishing multiple cell states and samples from multiple conditions. Remarkably, upon training with a subset of data, these methods allow predictions of locations in the functional space using data from experimental conditions that are not exposed to the models. Specifically, our models predicted the extent of progression and reversion for cells in the epithelial-mesenchymal transition (EMT) continuum. These methods revealed conserved EMT program among multiple types of single cells and tumor samples. Finally, we demonstrate this approach is broadly applicable to data and gene sets beyond EMT and provide several recommendations on the choice between the two linear methods and the optimal algorithmic parameters. Our methods show that simple constrained matrix decomposition can produce to low-dimensional information in functionally interpretable and transferrable space, and can be widely useful for analyzing large-scale transcriptome data.


title: Limitations of the ABEGO representation: ambiguity between αα-corner and αα-hairpin

abstract: ABEGO is a coarse-grained representation for polypeptide backbone dihedral angles. The Ramachandran map is divided into four segments denoted as A, B, E, and G to represent the local conformation of polypeptide chains in the character strings. Although the ABEGO representation is widely used in structural informatics and protein design, it cannot capture minor differences in backbone dihedral angles, which potentially leads to ambiguity between two structurally distinct fragments. Here, we show a nontrivial example of two local motifs that could not be distinguished by their ABEGO representations. We found that two well-known local motifs -hairpins and -corners are both represented as -GBB- and thus indistinguishable in the ABEGO representation, although they show distinct arrangements of the flanking -helices. We also found that -GBB- motifs caused a loss of efficiency in the ABEGO-based fragment-assembly simulations for protein backbone design. Nevertheless, we designed amino-acid sequences that were predicted to fold into the target topologies that contained these -GBB- motifs. Our finding that certain local motifs bottleneck the ABEGO-based fragment-assembly simulations for construction of backbone structures suggests that finer representations of backbone torsion angles are required for efficiently generating diverse topologies containing such indistinguishable local motifs.


title: Cryptic SARS-CoV2-spike-with-sugar interactions revealed by 'universal' saturation transfer analysis

abstract:
Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present  universal saturation transfer analysis (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an  end on manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and  end on mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems.

Extended AbstractThe surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of  saturation transfer protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this  universal saturation transfer analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins - SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants - by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer  end on-binding to sialoside sugars was revealed contrasting with  extended surface-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.


title: Cryptic pathogen-sugar interactions revealed by universal saturation transfer analysis


title: The allosteric activation of α7 nAChR by α-conotoxin MrIC is modified by mutations at the vestibular site

abstract:
-conotoxins are 13-19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. -conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets 7 nAChR. Although MrIC has no activating effect on 7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates 7 nAChR response by recovering it from a desensitized state. Lack of standalone activity despite activation upon co-application with a positive allosteric modulator was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate 7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations to gather evidence in support of 7 nAChR activation by MrIC through the AA site. The experiments with the wild-type 7 nAChR supported an allosteric mode of action, which was confirmed by the increased MrIC + PNU-120596 responses of three 7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on allosteric activation of 7 nAChR by MrIC and suggest involvement of the AA site.

Significance Statement-conotoxin MrIC (MrIC) is an allosteric agonist of the 7 nicotinic acetylcholine receptor (nAChR). This mode of action is unique among -conotoxins since these peptides typically act as orthosteric antagonists of nAChR. However, the mechanism of 7 nAChR activation by MrIC has been elusive so far. This work demonstrates that activation by MrIC is independent of the 7 nAChR orthosteric site and is related to a vestibular allosteric activation site at the extracellular domain of the receptor. Our experimental and computational studies identified the residues that play a role in allosteric activation and confirmed the utility of ensemble docking methods in understanding peptide - nAChR interactions, thus providing a basis for the design of peptides for allosteric modulation of nAChR.


title: Investigating the two regimes of fibrin clot lysis: an experimental and computational approach

abstract:
It has been observed in vitro that complete clot lysis is generally preceded by a period of latency during which the degradation seems to be inefficient. However, this latency was merely notified but not yet quantitatively discussed. In our experiments we observed that the lysis ubiquitously occurred in two distinct regimes, a slow and a fast lysis regime. We quantified extensively the duration of these regimes for a wide spectrum of experimental conditions and found that on average the slow regime lasts longer than the fast one, meaning that during most of the process the lysis is ineffective. We proposed a computational model in which the two regimes result from a spatially constrained kinetic of clot lysis: first the biochemical reactions take place at the outer core of the fibrin fibers composing the clot, then in the bulk resulting in the observed fast lysis regime. This simple hypothesis appeared to be sufficient to reproduce with a great accuracy the lysis profiles obtained experimentally. Our results shed light on new insights regarding the dynamical aspects of the lysis of fibrin rich clots in a context where the timing is so critical for patient treatment and outcome.

SignificanceWhile the interplay between the main components of the fibrinolytic system is well understood, some dynamical aspects of the fibrinolysis remain unclear. Notably, we observe that in vitro fibrin rich clots undergo a slow and inefficient phase of degradation when subject to endogenous fibrinolysis. In fact, it turns out that a large part of the lysis process operates in this slow regime. To explain this observation, we proposed a computational model in which the properties of the binding of the proteins change during the lysis. First plasminogen and tissue plasminogen activator bind at the surface of the fibers, resulting in a slow lysis, then in the bulk of the fibers thus speeding up the degradation of the clot..


title: Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity

abstract: Understanding the functional role of protein excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in {beta}-lactamases and particularly a subset of states containing a cryptic pocket which forms under the {Omega}-loop. Given the known importance of the {Omega}-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol labeling assays to probe {Omega}-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across {beta}-lactamase homologs, those with the {Omega}-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM {beta}-lactamase variants with greater open {Omega}-loop pocket populations. We further investigate the open population using a combination of NMR CEST experiments and molecular dynamics simulations. To test our understanding of the {Omega}-loop pockets functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.


title: Visualizing reactive astrogliosis extends survival in glioblastoma patients

abstract: Glioblastoma multiforme (GBM) is a devastating brain tumor with dismal prognosis of only 15-month survival regardless of surgical resection. Here, we report an advanced neuroimaging technique combining 11C-acetate PET and MRI (AcePET), visualizing the boundary beyond the MRI-defined tumor. Targeted biopsy of the regions with increased 11C-acetate uptake revealed the presence of reactive astrocytes with enhanced acetate-transporter MCT1, along with cancer stem cells. Reactive astrogliosis and MCT1-dependent 11C-acetate-uptake were recapitulated in U87MG-orthotopic models. Mechanistically, glycolytic tumor cells release excessive acetate causing reactive astrogliosis, leading to the release of aberrant astrocytic GABA and H2O2, which further down-regulate the neuronal glucose uptake through GLUT3. Clincally, AcePET-guided surgery allows complete tumor resection of infiltrating cancer stem cells and extends the overall survival of patients by 5.25 months compared to conventional MRI-guided surgery. We established a new concept of the metabolic interactions between GBM cells and neighboring neurons through reactive astrocytes and developed AcePET-guided surgery to fight against GBM.


title: Visualizing cancer-originated acetate uptake through MCT1 in reactive astrocytes demarcates tumor border and extends survival in glioblastoma patients


title: Disruption of the surfactant protein A receptor SP-R210L (CD245α/MYO18Aα) alters respiratory function and iron sequestration in alveolar macrophages of aged mice

abstract: Previous studies demonstrated that the host defense collectins, surfactant protein A and complement component 1q, modulate tissue-dependent macrophage activation, pathogen clearance, and regulatory macrophage functions through the receptor SP-R210, which consists of two isoforms SP-R210L and SP-R210S. These isoforms are encoded by alternatively spliced mRNAs of the Myo18a MYO18A gene in mice and humans. The present study in conditional transgenic mice revealed novel age-related functions of the SP-R210L isoform in modulating pulmonary mechanics, iron sequestration in alveolar macrophages (AMs), and life-long maintenance of the alveolar macrophage population. Our findings support the novel idea that SP-R210L-deficient AMs undergo bi-directional epigenetic adaptation that results in chronic dysregulation of broncho-alveolar function, immune homeostasis, and maintenance of oncotic balance at the airway-capillary interface. Disruption of SP-R210L increases the risk for development of severe interstitial lung disease during development and aging.


title: Stress related network activity in the intact adrenal medulla.

abstract:
The adrenal medulla has long been recognized as playing a critical role in mammalian homeostasis and the stress response. The adrenal medulla is populated by clustered chromaffin cells that secrete epinephrine or norepinephrine along with other peptides into the general bloodstream affecting multiple distant target organs. Although the sympatho-adrenal pathway has been heavily studied, detailed knowledge on the central control and in-situ spatiotemporal responsiveness remains poorly understood. For this work we implemented electrophysiological techniques originally developed to elucidate CNS circuitry to characterize the functional micro-architecture of the adrenal medulla. To achieve this, we continuously monitored the electrical activity inside the adrenal medulla in the living anesthetized rat under basal conditions and under physiological stress. Under basal conditions, chromaffin cells fired action potentials with frequencies between [~]0.2 and 4 Hz. Activity was exclusively driven by sympathetic inputs coming through the splanchnic nerve. Furthermore, chromaffin cells were organized into arrays of independent local networks in which cells fire in a specific order, with latencies from hundreds of microseconds to few milliseconds. Electrical stimulation of the splanchnic nerve evoked the exact same spatiotemporal firing patterns that occurred spontaneously. Induction of hypoglycemic stress by administration of insulin resulted in an increase in the activity of a subset of the chromaffin cell networks. In contrast, respiratory arrest induced by anesthesia overdose resulted in an increase in the activity of the entire adrenal medulla before cessation of all activity when the animal died. The results suggest the differential activation of specific networks inside the adrenal gland depending on the stressor. These results revealed a surprisingly complex electrical organization and circuitry of the adrenal medulla that likely reflects the dynamic nature of its neuroendocrine output during basal conditions and during different types of physiological stress. To our knowledge, these experiments are the first to use multi-electrode arrays in vivo to examine the electrical and functional architecture of any endocrine gland.

Significance StatementStress from extrinsic (environmental, psychological) and intrinsic (biological) challenges plays a critical role in disturbing the homeostatic balance. While the bodys responses to stress are designed to ameliorate these imbalances, prolonged and dysregulated stress often drives adverse health consequences in many chronic illnesses. The better understanding of the sympatho-adrenal stress response, will potentially impact and improve the treatment of several stress related illnesses. This work focusses on the study of the functional architecture of the adrenal medulla, a key component in neuronal stress response.


title: Investigating the correlation of muscle function tests and sarcomere organization in C. elegans

abstract:
BackgroundCaenorhabditis elegans has been widely used as a model to study muscle structure and function due to many genes having human homologs. Its body wall muscle is functionally and structurally similar to vertebrate skeletal muscle with conserved molecular pathways contributing to sarcomere structure, and muscle function. However, a systematic investigation of the relationship between muscle force and sarcomere organization is lacking. Here, we investigate the contribution of various sarcomere proteins and membrane attachment components to muscle structure and function to introduce C. elegans as a model organism to study the genetic basis of muscle strength.

MethodsWe employ two recently developed assays that involve exertion of muscle forces to investigate the correlation of muscle function to sarcomere organization. We utilized a microfluidic pillar-based platform called NemaFlex that quantifies the maximum exertable force and a burrowing assay that challenges the animals to move in three dimensions under a chemical stimulus. We selected 20 mutants with known defects in various substructures of sarcomeres and compared the physiological function of muscle proteins required for force generation and transmission. We also characterized the degree of sarcomere disorganization using immunostaining approaches.

ResultsWe find that mutants with genetic defects in thin filaments, thick filaments and M-lines are generally weaker, and our assays are successful in detecting the functional changes in response to each sarcomere location tested. We find that the NemaFlex and burrowing assays are functionally distinct informing on different aspects of muscle physiology. Specifically, the burrowing assay has a larger bandwidth in phenotyping muscle mutants, because it could pick ten additional mutants impaired while exerting normal muscle force in NemaFlex. This enabled us to combine their readouts to develop an integrated muscle function score that was found to correlate with the score for muscle structure disorganization.

ConclusionsOur results highlight the suitability of NemaFlex and burrowing assays for evaluating muscle physiology of C. elegans. Using these approaches, we discuss the importance of the studied sarcomere proteins for muscle function and structure. The scoring methodology we have developed lays the foundation for investigating the contribution of conserved sarcomere proteins and membrane attachment components to human muscle function and strength.


title: Investigating the Effect of Flow Compensation and Quantitative Susceptibility Mapping Method on the Accuracy of Venous Susceptibility Measurement

abstract:
Quantitative susceptibility mapping (QSM) is a promising non-invasive method for obtaining information relating to the oxygen metabolism. However, the optimal acquisition sequence and QSM reconstruction method for reliable venous susceptibility measurements are unknown. Full flow compensation is generally recommended to correct for the influence of venous blood flow, although the effect of flow compensation on the accuracy of venous susceptibility values has not been systematically evaluated. In this study, we investigated the effect of different acquisition sequences, including different flow compensation schemes, and different QSM reconstruction methods on venous susceptibilities.

Ten healthy subjects were scanned with five or six distinct QSM sequence designs implementing different flow compensation schemes. All data sets were processed using six different QSM pipelines and venous blood susceptibility was evaluated in whole-brain segmentations of the venous vasculature and single veins. The quality of vein segmentations and the accuracy of venous susceptibility values were analyzed and compared between all combinations of sequences and QSM methods.

The influence of the QSM method on average venous susceptibility values was found to be 2.7 - 11.6 times greater than the influence of the acquisition sequence, including flow compensation. The majority of the investigated QSM reconstruction methods tended to underestimate venous susceptibility values in the vein segmentations that were obtained.

Using multi-echo gradient-echo acquisitions with monopolar readout gradients, we found that sequences without full flow compensation yielded venous susceptibility values comparable to sequences with full flow compensation. However, the QSM method had a great influence on susceptibility values and thus needs to be considered carefully for accurate venous QSM.


title: DENTATE GRANULE CELLS ARE HYPEREXCITABLE IN THE TGF344-AD RAT MODEL OF ALZHEIMER'S DISEASE

abstract: The dentate gyrus is both a critical gatekeeper for hippocampal signal processing and one of the first regions to dysfunction in Alzheimers disease (AD). Accordingly, the appropriate balance of excitation and inhibition through the dentate is a compelling target for mechanistic investigation and therapeutic intervention of early AD. Previously, we reported increased LTP magnitude at medial perforant path-dentate granule cell (MPP-DGC) synapses in slices from TgF344-AD rats compared to Wt as early as 6 months. We next determined that the enhanced LTP magnitude was due to heightened function of {beta}-ARs leading us to ask if dentate granule cells (DGCs) also have modified passive or active membrane properties which may also contribute to hyperexcitability or aberrant hippocampal processing. Although there was no detectable difference in spine density and presynaptic release probability, dentate granule cells (DGCs) themselves might have increased electrical response to synaptic input during LTP induction, which was measured as a significant increase in charge transfer during high-frequency stimulation. In this study, we found passive membrane properties and active membrane properties are altered, leading to increased TgF344-AD DGC excitability. Specifically, TgF344-AD DGCs have an increased input resistance and decreased rheobase, decreased sag, and increased action potential (AP) spike accommodation. Importantly, we find that the voltage response increased in DGCs from TgF344-AD compared to Wt accompanied by decreased delay to fire the first AP, indicating that for the same amount of depolarizing current injection TgF344-AD DGCs membranes are more excitable. Taken together, DGCs of TgF344-AD rats are more excitable, but due to heightened accommodation, may be unable to discharge at high frequency for longer durations of time, compared to their Wt littermates.


title: Inhibition of Heat Shock Protein 90 in the Spinal Cord Improves the Therapeutic Index of Morphine

abstract: Opioid drugs are the gold standard for treating acute and chronic pain, but are limited by negative side effects, including tolerance, constipation, and addiction. New approaches are thus greatly needed to improve opioid therapy and decrease side effects. Our investigations have revealed Heat shock protein 90 (Hsp90) as a critical regulator of opioid receptor signaling, and that inhibition of spinal cord Hsp90 boosts opioid anti-nociception. These findings led us to hypothesize that spinal Hsp90 inhibitor treatment could improve the therapeutic index of opioids, boosting anti-nociception while improving or not altering side effects, enabling a dose-reduction strategy. To test this hypothesis, we injected the Hsp90 inhibitor KU-32 by the intrathecal route into male and female CD-1 mice. We then tested these mice for their response to morphine in a battery of anti-nociception and side effect models. We found that spinal KU-32 treatment increased the anti-nociceptive potency of morphine by 2-4 fold in the tail flick, post-surgical paw incision, and HIV peripheral neuropathy pain models. At the same time, morphine anti-nociceptive tolerance was reduced from 21 fold to 2.9 fold by KU-32 treatment, and established morphine tolerance could be rescued by KU-32 injection. Reward as measured by conditioned place preference and constipation were both unchanged. These results support our hypothesis, and suggest that Hsp90 inhibitors could be a novel approach to improve the therapeutic index of opioids, enable a dose-reduction strategy, and lead to fewer side effects during pain therapy.


title: Towards a causal role of Broca's area in language: A TMS-EEG study on syntactic prediction

abstract: Categorical predictions have been proposed as the key mechanism supporting the fast pace of syntactic composition in human language. Accordingly, grammar-based expectations facilitate the analysis of incoming syntactic information--e.g., hearing the determiner "the" enhances the prediction of a noun--which is then checked against a single or few other word categories. Previous functional neuroimaging studies point towards Brocas area in the left inferior frontal gyrus (IFG) as one fundamental cortical region involved in categorical prediction during on-line language processing. Causal evidence for this hypothesis is however still missing. In this study, we combined Electroencephalography (EEG) and Transcranial Magnetic Stimulation (TMS) to test whether Brocas area is functionally relevant in predictive mechanisms for language. Specifically, we transiently perturbed Brocas area during the categorical prediction phase in two-word constructions, while simultaneously measuring the Event-Related Potential (ERP) correlates of syntactic composition. We reasoned that if Brocas area is involved in predictive mechanisms for syntax, disruptive TMS during the processing of the first word (determiner/pronoun) would mitigate the difference in ERP responses for predicted and unpredicted categories when composing basic phrases and sentences. Contrary to our hypothesis, perturbation of Brocas area at the predictive stage did not affect the ERP correlates of basic composition. The correlation strength between the electrical field induced by TMS and the magnitude of the EEG response on the scalp further confirmed this pattern. We discuss the present results in light of an alternative account of the role of Brocas area in syntactic composition, namely the bottom-up integration of words into constituents.


title: Laminar perfusion imaging with zoomed arterial spin labeling at 7 Tesla

abstract:
Laminar fMRI based on BOLD and CBV contrast at ultrahigh magnetic fields has been applied for studying the dynamics of mesoscopic brain networks. However, the quantitative interpretations of BOLD/CBV fMRI results are confounded by different baseline physiology across cortical layers. Here we introduce a novel 3D zoomed pseudo-continuous arterial spin labeling technique at 7T that offers the unique capability for quantitative measurements of laminar cerebral blood flow (CBF) both at rest and during task activation with high spatial specificity and sensitivity. We found arterial transit time in superficial layers is [~]100 msec shorter than in middle/deep layers revealing the dynamics of labeled blood flowing from pial arteries to downstream microvasculature. Resting state CBF peaked in the middle layers which is highly consistent with microvascular density measured from human cortex specimens. Finger tapping induced a robust two-peak laminar profile of CBF increases in the superficial (somatosensory and premotor input) and deep (spinal output) layers of M1, while finger brushing task induced a weaker CBF increase in superficial layers (somatosensory input). We further demonstrated that top-down attention induced a predominant CBF increase in deep layers and a smaller CBF increase on top of the lower baseline CBF in superficial layers of V1 (feedback cortical input), while bottom-up stimulus driven activity peaked in the middle layers (feedforward thalamic input). These quantitative laminar profiles of perfusion activity suggest an important role of M1 superficial layers for the computation of finger movements, and that visual attention may amplify deep layer output to the subcortex.

Significance StatementCBF or microvascular perfusion measured by arterial spin labeling (ASL) is a key parameter for in vivo assessment of neurovascular function. Compared to BOLD or VASO fMRI, ASL perfusion contrast offers the unique capability for quantitative CBF measurements both at baseline and during task activation, which is critical for quantitative estimation of metabolic activities tightly related to neuronal activation. We proposed a zoomed 3D ASL technique at 7T for laminar perfusion imaging with high spatial specificity and sensitivity. This technique is able to differentiate and quantify the input/output and feedforward/feedback activities of human motor and visual cortex, thereby providing an important tool for quantitative assessment of neurovascular function and metabolic activities of neural circuits across cortical layers.


title: Fixational Eye Movements Depend on Task and Target

abstract: Human fixational eye movements are so small and precise that they require high-speed, accurate tools to fully reveal their properties and functional roles. Where the fixated image lands on the retina and how it moves for different levels of visually demanding tasks is the subject of the current study. An Adaptive Optics Scanning Laser Ophthalmoscope (AOSLO) was used to image, track and present Maltese cross, disk, concentric circles, Vernier and tumbling-E letter fixation targets to healthy subjects. During these different passive (static) or active (discriminating) fixation tasks under natural eye motion, the landing position of the target on the retina was tracked in space and time over the retinal image directly. We computed both the eye motion and the exact trajectory of the fixated targets motion over the retina. We confirmed that compared to passive fixation, active tasks elicited a partial inhibition of microsaccades, leading to longer drifts periods compensated by larger corrective saccades. Consequently the fixation stability during active tasks was larger overall than during passive tasks. The preferred retinal locus of fixation was the same for each task and did not coincide with the location of the peak cone density.


title: Visual search for upright bigrams predicts reading fluency in children

abstract: Fluent reading is an important milestone in education, but we lack a clear understanding of why children vary so widely in attaining this milestone. Language-related factors such as rapid automatized naming (RAN) and phonological awareness have been identified as important factors that influence reading fluency. Of theoretical interest is also, however, whether aspects of visual processing influence reading fluency. To investigate this issue, we tested primary school children (n = 68) on four tasks: two reading fluency tasks (word reading and passage reading), a RAN task to measure naming speed, and a visual search task using letters and bigrams to measure visual processing. As expected, the RAN score was strongly correlated with reading fluency. In addition, visual processing of bigrams was correlated with reading fluency. Importantly, this association was specific to upright but not inverted bigrams, and to bigrams with normal but not large letter spacing. Thus, reading fluency in children is accompanied by specialized changes in upright bigram processing. We propose that bigram processing during visual search could complement existing measures of language processing to understand individual differences in reading fluency.


title: A Paradigm Free Regularization Approach to Recover Brain Activation from Functional MRI Data

abstract:
ContextFunctional MRI is a non-invasive imaging technique that provides an indirect view into brain activity, via the blood-oxygen-level-dependent (BOLD) response. In particular, resting-state fMRI poses challenges to the recovery of brain activity without prior knowledge on the experimental paradigm, as it is the case for task-fMRI. Conventional methods to infer brain activity from the fMRI signals, for example the general linear model (GLM), require the knowledge of the experimental paradigm to define regressors and estimate the contribution of each voxels time course to the task. To overcome this limitation, approaches to deconvolve the BOLD response and recover the underlying neural activation without a priori information on the task have been proposed. State-of-the-art techniques, and in particular the Total Activation (TA), formulates the deconvolution as an optimization problem with decoupled spatial and temporal regularization terms. This increases the number of hyperparameters to be set and requires an optimization strategy that alternates between the constraints.

ApproachIn this work, we propose a paradigm-free regularization algorithm named Paradigm-Free fMRI (PF-fMRI) that is applied on the 4-D fMRI image, acting simultaneously in the 3-D space and 1-D time dimensions. Based on the idea that large image variations should be preserved as they occur during brain activation, whereas small variations considered as noise should be removed, the PF-fMRI applies an anisotropic regularization, thus recovering the location and the duration of brain activation.

ResultsUsing the experimental paradigm as ground truth, the PF-fMRI is validated on synthetic and real task-fMRI data from 51 subjets, and its performance is compared to the TA. Results show higher correlations of the recovered time-courses with the ground truth compared to the TA and lower computational times. In addition, we show that the PF-fMRI recovers activity that agrees with the GLM, without requiring or using any knowledge of the experimental paradigm.